Two-partner secretion system binding compounds

Abstract

The invention relates to peptides and compounds that bind a two partner secretion (TPS) system protein. In some embodiments of the invention, the peptide or compound binds to a translocator pore protein (TpsB) thereby reducing translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane.

Description

[0001] TWO-PARTNER SECRETION SYSTEM BINDING COMPOUNDS

[0002] RELATED APPLICATIONS

[0003] This application claims priority to United States Provisional Patent Application No. 63 / 688673, filed on 29 August 2024, the entire contents of which are hereby incorporated by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] Preceding applications contained a sequence listing which was originally submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. Said ST.26 copy, created on 28 August 2024, is named ‘P0067823 AU ST26 Sequence Listing’ and is 107,687 bytes in size. The instant application contains a sequence listing which has been submitted electronically as an XML document in the ST.26 format and is hereby incorporated by reference in its entirety. Said XML copy, created on 25 August 2025, is named “P0067823PCT Sequence Listing” and is 192,829 bytes in size.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under Grant Numbers AI180112 and All 53160 awarded by the National Institutes of Health. The government of the United States of America has certain rights in the invention.

[0008] FIELD OF THE INVENTION

[0009] The present invention relates to the secretion of bacterial proteins. Primarily, the invention relates to the two-partner secretion system and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

[0010] BACKGROUND ART

[0011] The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of the common general knowledge in the field. The threat of antimicrobial resistant (AMR) bacteria, or “superbugs” accounts for ~ 5 million deaths annually and is largely driven by Gram-negative bacteria, namely Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Furthermore, it is estimated that the global increase in healthcare costs related to superbug infections may range from US$300 billion to more than US$1 trillion annually by 2050. In 2017, the WHO released a list of priority pathogens, a large majority of which are Gramnegative bacteria (A. baumannii, P. Aeruginosa, Enterobacteriaceae, H. pylori, Campylobacter spp., Salmonellae, N. gonorrhoeae, H. influenzae, and Shigella spp.), and highlighted the need for novel antibiotics to target these pathogens. An increasing number of emerging / re-emerging Gram-negative pathogens are developing resistance towards commonly prescribed first-line antibiotics. For instance, patients infected by Bordetella pertussis (Bp) are usually prescribed with macrolides as a first-line antibiotic. However, macrolide-resistant p strains have been isolated globally in countries including Iran, China, and neighbouring countries in the Asia-Pacific region.

[0012] The two-partner secretion (TPS) system is characterised by two genes organised in an operon that encodes for a ~65 kDa translocator pore protein “TpsB” that is embedded in the bacterial outer membrane, and a partner (100 - 500 kDa) exoprotein “TpsA” that is secreted to the bacterial cell surface by the TpsB. These TPS systems are found in most Gramnegative pathogens. TpsB proteins serve to export and secrete their respective TpsA exoprotein partners, although some TpsB are found to secrete more than one TpsA exoprotein. The TpsA exoproteins act as virulence factors that have diverse functions as cytolysins / hemolysins, proteases, adhesins, acquiring iron, and contact-dependent growth inhibitors which inhibit growth of neighbouring cells / bacteria.

[0013] The TpsB protein family possess a 16-stranded C-terminal membrane-embedded P- barrel domain. The P-barrel is preceded by an N-terminal a-helix (Hl), an unstructured linker, and two periplasmic polypeptide transport-associated (POTRA) domains (“POTRA1” and “POTRA2”). At rest, Hl blocks the pore of the P-barrel. However, once the “TPS domain” of a TpsA exoprotein substrate is recognised by the POTRA domains, it initiates conformational changes that pulls Hl out from the P-barrel pore and into the periplasm, leaving the pore open for TpsA secretion.

[0014] For example, in Bordetella pertussis, the TPS system comprises the TpsB protein “FhaC” and the TpsA exoprotein “FhaB”. FhaC is a 63.3 kDa protein, possessing the characteristic C-terminal P-barrel, two N-terminal POTRA domains, and Hl observed in other TpsB proteins. Highly-conserved features commonly found in TpsB superfamily members are also observed such as the extracellular loops 3 (L3) and 4 (L4) formed by extended P-strands 5 / 6 and 7 / 8, respectively. Meanwhile, the FhaB exoprotein is a large, 375 kDa protein that folds into a P-helical shaft.

[0015] At resting state, the Hl domain plugs the barrel pore of FhaC and secretion is initiated once FhaC recognises the characteristic N-terminus TPS domain of FhaB. This interaction then leads to a large conformational change which pulls the Hl -linker domain out from the P- barrel lumen and opens a pore in the barrel to translocate FhaB across the outer membrane. While the N-terminus of FhaB remains anchored to the POTRA domain, a significant proportion of FhaB gets secreted in the N-to C-terminal direction and folded on the L3 / 4 platform into a P-helical structure topped by the mature C-terminal domain (MCD). The remaining C-terminal 1,200 amino acids (the “prodomain”) is retained in the periplasm by the prodomain N-terminus (PNT) domain that blocks further translocation, anchoring FhaB to FhaC. Eventually, the entire FhaB is translocated across the OM. FhaB secretion by FhaC is essential for Bordetella spp adherence to the lower respiratory tract to cause infection in laboratory animals, humans, and livestock.

[0016] It is an object of the present invention to overcome or ameliorate one or more the disadvantages of the prior art, or at least to provide a useful alternative.

[0017] It is an object of at least one preferred embodiment of the present invention to provide a peptide or compound that binds a TPS system protein.

[0018] SUMMARY OF THE INVENTION

[0019] In a first aspect, the invention provides a compound for inhibiting a two-partner secretion system, wherein the compound comprises an amino acid sequence selected from the group consisting of:

[0020] VAAMAA (SEQ ID NO: 1);

[0021] VAAMAAEASARARAEFAAR (SEQ ID NO: 2); VAAMAAEASARAEAEFAAR (SEQ ID NO: 3);

[0022] V A AM A AE AS AR AR ARFA AR (SEQ ID NO: 4);

[0023] V A AM A AE AS ARAE ARFA AR (SEQ ID NO: 5); YVLCTLRAKRFYALFLSG (SEQ ID NO: 6); YRKHWPDWYRTHYVVCG (SEQ ID NO: 7); YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8); VAAMAAEASARARAE (SEQ ID NO: 64); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences; and an amino acid sequence with at least 70% sequence identity to any one of SEQ

[0024] ID Nos: 1-8, and 64, and wherein the compound binds to a translocator pore protein (TpsB) thereby reducing translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane.

[0025] In a second aspect, the invention provides a pharmaceutical composition comprising the compound according to the first aspect and one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0026] In a third aspect, the invention provides a method of treating or preventing a bacterial infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound according to the first aspect or the pharmaceutical composition according to the second aspect.

[0027] In a fourth aspect, the invention provides use of the compound according to the first aspect in the manufacture of a medicament for treating or preventing a bacterial infection in a subject.

[0028] In a fifth aspect, the invention provides a compound according to the first aspect or a pharmaceutical composition according to the second aspect for use in the treatment or prevention of a bacterial infection in a subject.

[0029] In a sixth aspect, the invention provides a method of screening for inhibitors of TpsA secretion, the method comprising: providing a bacterial strain comprising: a nucleotide sequence encoding a TpsB protein; and a nucleotide sequence encoding a TpsA protein; exposing the bacterial strain to a candidate inhibitor; and measuring the amount of TpsA protein or TpsB protein in a fraction isolated from the bacterial strain.

[0030] In a seventh aspect, the invention provides a method of screening for inhibitors of TpsA secretion, the method comprising: providing a bacterial strain comprising: a nucleotide sequence encoding a TpsB protein; a nucleotide sequence encoding a TpsA protein; and a nucleotide sequence encoding a signal peptide and a candidate inhibitor, and measuring the amount of TpsA protein or TpsB protein in a fraction isolated from the bacterial strain.

[0031] In an eighth aspect, the invention provides a method of treating or preventing a plant disease, the method comprising applying the compound according to the first aspect to a plant in an amount effective to treat the plant disease.

[0032] In a ninth aspect, the invention provides a method of preventing or reducing bacterial replication, the method comprising exposing the bacteria to the compound according to the first aspect.

[0033] In a tenth aspect, the invention provides a compound for binding a translocator pore protein (TpsB), wherein the compound comprises an amino acid sequence selected from the group consisting of: VAAMAA (SEQ ID NO: 1); VAAMAAEASARARAEFAAR (SEQ ID NO: 2); VAAMAAEASARAEAEFAAR (SEQ ID NO: 3); VAAMAAEASARARARFAAR (SEQ ID NO: 4); VAAMAAEASARAEARFAAR (SEQ ID NO: 5); YVLCTLRAKRFYALFLSG (SEQ ID NO: 6); YRKHWPDWYRTHYVVCG (SEQ ID NO: 7);

[0034] YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8); YRKHWPEWYRTHYVVCG (SEQ ID NO: 11); YRKHWPDWYRTHYVACG (SEQ ID NO: 12); YRKHWPDWYRTHYVICG (SEQ ID NO: 13); YHILYYKVLYYLYCG (SEQ ID NO: 14); YRIICWYLICG (SEQ ID NO: 15); YRYPTWYSTYYNQCG (SEQ ID NO: 16); YYWYYLFWSCG (SEQ ID NO: 17);

[0035] YAIVVSPKYLFIRVCG (SEQ ID NO: 18); YHYYLYQSKFIWECG (SEQ ID NO: 19); YGGGKDRKSTRLNSSH (SEQ ID NO: 20); YIVSTIPSNVICLICG (SEQ ID NO: 21); YEEHTSELQSLGIISYAVFC (SEQ ID NO: 22); YLKISTCTWVLSISFCG (SEQ ID NO: 23); YFILKLLLVCG (SEQ ID NO: 24); YFVLTLVYIIVIHAVCG (SEQ ID NO: 25); YYVVYFDYLFWIVVGCG (SEQ ID NO: 26); YFLIWWLLLCG (SEQ ID NO: 27);

[0036] YLILVFDSSEVWVVLICG (SEQ ID NO: 28); YAVAYWSRNFVLVYTICG (SEQ ID NO: 29); YLIVTRVNERWHFSHVCG (SEQ ID NO: 30); YVFLYEKGFLIVFTICG (SEQ ID NO: 31); YTWLYVLLVCG (SEQ ID NO: 32); YWQKRHICFEIVLCG (SEQ ID NO: 33); YRLTIVRNHIGWTLYVCG (SEQ ID NO: 34); YINVIWFWYCG (SEQ ID NO: 35);

[0037] YLCLLFVFLTLCIIICG (SEQ ID NO: 36); YEEHTSELQSRTPISYAVFC (SEQ ID NO: 37); YVLCTLRAKRFYALFLCG (SEQ ID NO: 63); VAAMAAEASARARAE (SEQ ID NO: 64); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences; and an amino acid sequence with at least 70% sequence identity to any one of SEQ ID Nos: 1-8, 11- 37, 63, and 64. In an eleventh aspect, the invention provides a peptide comprising an amino acid sequence as set forth in any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93; an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93; or an amino acid sequence with at least 70% sequence identity to any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93.

[0038] In a twelfth aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the peptide according to the eleventh aspect.

[0039] In a thirteen aspect, the invention provides a kit comprising the compound according to the first or tenth aspect, or the peptide according to the eleventh aspect.

[0040] The skilled person will appreciate that the present invention may provide one or more significant advantages and improvements in the field and / or in view of the prior art. For example, these advantages include:

[0041] (i) A novel class of TPS inhibitors that bind to the TpsB protein in a conserved location in the POTRA domains and potently inhibit the secretion of toxic or virulence TpsA exoproteins. Alternatively, a novel class of TPS inhibitors that may bind to the TpsB protein P-barrel domain and potently inhibit the secretion of toxic or virulence TpsA exoproteins.

[0042] (ii) A class of TPS inhibitors which target a different bacterial pathway than conventional antibiotics. Without wishing to be bound by theory, it is hypothesized that the disclosed inhibitors will have a reduced impact on commensal bacteria in a host that is treated with the inhibitors.

[0043] (iii) A class of TPS inhibitors which targets essential virulence factors of a pathogen instead of an essential central growth pathway; thereby not being bacteriostatic or bactericidal. Without wishing to be bound by theory, it is hypothesised that a strategy targeting virulence factors instead of bacterial growth may maintain efficacy longer than traditional strategies due to reduced selection pressure.

[0044] (iv) The ability to use the TPS inhibitors for diverse applications, including to: (1) treat or prevent bacterial infections in humans; (2) treat or prevent bacterial infections in non-human animals, including livestock animals (e.g., pigs); (3) treat or prevent bacterial disease in a plant; and (4) kill bacteria or prevent or reduce bacterial replication on a surface (e.g., medical devices). BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The aspects described above, as well as other apparent aspects, advantages, and objectives of the present invention are apparent from the detailed description below in combination with the drawings as described below.

[0046] Figure 1 shows purification of FhaCAHl. (A) Model of FhaC in vivo and purified FhaCAHl solubilised into DDM micelles. (B) Coomassie-stained gel of purification fractions. S / N = Supernatant, Post Ultra 1 = After first ultracentrifuge spin, WMP = Whole membrane pellet post-DDM treatment. (C) Same samples as (B) probed by Western Blot to detect FhaCAHl (anti-His).

[0047] Figure 2 shows that pure FhaCAHl target is natively folded. (A) AlphaFold model of FhaCAHl with aromatic residues shown (Purple - Trp, Grey - Phe / Tyr). (B) Purified FhaCAHl from Fig. 1 probed by barrel heat-modifiability assay. (C) Tryptophan fluorescence spectrum of purified FhaCAHl. F = Folded (322 nm maxima), UF = Unfolded (340 nm maxima). (D) Left, chymotrypsin digestion of purified FhaCAHl and detection of FhaCAHl with Western Blotting (anti-His). “a” and “b” denote FhaCAHl chymotrypsin fragments. CT = Chymotrypsin. Right, percentage of stable FhaCAHl band remaining.

[0048] Figure 3 shows sequences of macrocyclic peptides obtained from mRNA display that bind to FhaC. Peptides are cyclized between purple-coloured residues, with either D- (top) or L- (bottom) isomer of chloroacetylated Tyrosine (Y*) readily reacts with the first Cysteine residue. Residues marked with grey are uncyclized Cys.

[0049] Figure 4 shows predicted binding site of the Pl peptide (VAAMAAEASARARAEFAAR [SEQ ID NO: 2]) and its derivatives. Pl is predicted to bind to the POTRA1 domain of FhaC.

[0050] Figure 5 shows a novel FhaB secretion assay as a measure of the activity of FhaC. (A) Scheme of FhaBsec secretion. Secretion strain was grown at 37 °C to log phase before supplementation with 0.2% rhamnose and growth for 1 hour (Secretion strain releases FhaBsec by the activity of FhaC when induced by rhamnose). .R = Promoter rhamnose. (B) Coomassie-stained gel of the growth medium supernatant. (C) Same supernatant sample probed by Western blot to detect FhaBsec (anti-Strep antibody), * = Unrelated endogenous secreted protein.

[0051] Figure 6 shows a screen of peptide activities using the FhaBsec secretion assay. (A) Assay was conducted as in Fig. 5, but with peptides added immediately before adding rhamnose. Representative Coomassie gel from n = 3. (B) Quantitation of secreted FhaBsec from (A). Line denotes the mean percentage value of FhaBsec band intensity (relative to respective vehicle controls). Ordinary one-way ANOVA (with Dunnett’s multiple comparisons post-hoc tests) was conducted, **P < 0.01, 0.0001.

[0052] Figure 7 shows the 2D chemical structure of macrocyclic peptides “RD-4” (SEQ ID NO: 8), “RD-6” (SEQ ID NO: 7), “RD-7” (SEQ ID NO: 6).

[0053] Figure 8 shows that peptides Pl (SEQ ID NO: 2) and Pl-CPP (SEQ ID NO: 10) inhibit FhaC from secreting FhaB from E. coli in a concentration-dependent manner. (A) Representative Coomassie-stained gels of samples treated with titrated Pl and Pl-CPP (n = 3). Assay was conducted as in Fig. 6. Cone. = Concentration. (B and C) Quantitation of percentage FhaBsec secreted in samples treated with titrated Pl, and Pl-CPP, respectively (relative to vehicle control). Ordinary two-way ANOVA (with Dunnett’s multiple comparisons tests) were conducted, compared against the vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0054] Figure 9 shows that the inhibition of FhaBsec secretion by peptide Pl-CPP causes retention and accumulation of unprocessed FhaBsec inside the bacterial cells. Assay was conducted as in Fig. 6, but cell pellet samples were taken instead of the supernatant. (Left) Samples were probed by Western Blot to detect FhaBsec (anti-Strep) n = 3. (Right) Quantitation of FhaBsec retention from (Left). Line denotes the mean value of FhaBsec band intensity. Unpaired, two-tailed student’s t-test were conducted, compared against the vehicle control, *P < 0.05.

[0055] Figure 10 shows preliminary Isothermal Calorimetry (ITC) assay data that the Pl- CPP peptide directly binds to FhaC. Assay was conducted by titrating 20pM Pl-CPP to 400pM FhaC-POTRAl-2 domains. (A) Titration of Pl-CPP to POTRA1-2 in lx PBS buffer. Raw heat changes peaks (inset) were subtracted with the average value from the Pl-CPP to lx PBS control (to account for heat of dilution) and fitted according to a one set binding site model. Resulting binding curve is logarithmic. (B) Binding behaviour signature plot showing that Pl-CPP binding to POTRA1-2 has favourable hydrogen bonds and hydrophobic interactions. (C) Thermodynamic parameters of Pl-CPP:POTRAl-2 binding.

[0056] Figure 11 shows a variant of the secretion assay method (from Figure 6) where FhaC and FhaBsec is produced together with the peptide Pl also encoded on the same operon on the same plasmid and containing an N-terminal signal peptide to facilitate translocation across the inner membrane by the Sec translocon (EP1). (A) Assay was conducted as in Fig. 6, but with only rhamnose added (no peptide). (B) Representative Coomassie gel from n = 3, samples express reduced FhaBsec when the Pl insert (EP1) is present. (C) Quantitation of percentage secreted FhaBsec (relative to no Pl insert (EP1) control) from (A). Line denotes the mean value. Unpaired, two-tailed student’s t-test was conducted, ***P < 0.001.

[0057] Figure 12 shows screening of an N-terminally truncated Pltmnc-CPP variant (PIANI- CPP). (A) Sequence comparison of Pl-CPP and its N-terminally truncated variant (PIANI- CPP) and representative Coomassie gel from n = 3. Assay was conducted as in Fig. 6 (B) Quantitation of percentage secreted FhaBsec (relative to mock control) from (A). Line denotes the mean. Ordinary one-way ANOVA (with Dunnett’s multiple comparisons post- hoc tests) was conducted, ****p < 0.0001. (C) Bottom view of the hydrophobic cleft of the POTRA1 domain of FhaC (Left, red square). The Pl-CPP peptide is predicted to be bound to this area strongly (Middle), and truncation of the first 6 N-terminal residues of Pl-CPP (Pltrunc-CPP, i.e. PIANI-CPP) leaves the cleft open (Right).

[0058] Figure 13 shows testing of roles of charged residues in Pl activity. (A) Predicted model of Pl binding to FhaC (Left) and POTRA1 residues involved in forming salt bridge interactions (Inset). (B) Representative Coomassie gel from n = 3. Assay was conducted as in Fig. 6. (C) Quantitation of percentage secreted FhaBsec (relative to mock control) from (B). Line denotes the mean. Ordinary one-way ANOVA (with Dunnett’s multiple comparisons post-hoc tests) was conducted, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0059] Figure 14 shows that peptides PIRBE-CPP, PIEISR-CPP and P1RI3E,EI5R-CPP inhibit the secretion of FhaB in a concentration-dependent manner. (A) Representative Coomassie- stained gels of samples treated with titrated PIRBE-CPP, PIEISR-CPP and P1RI3E,EI5R-CPP (n = 3). Assay was conducted as in Fig. 6. Cone. = Concentration. (B, C and D) Quantitation of percentage FhaBsec secreted in samples treated with titrated PIRBE-CPP, PIEISR-CPP and P1RI3E,EI5R-CPP, respectively (relative to vehicle control). Ordinary two-way ANOVA (with Dunnett’s multiple comparisons tests) were conducted, *P < 0.05, **P < 0.01, ***p < 0.001, ****P < 0.0001.

[0060] Figure 15 shows the experimental setup for testing inhibition of FhaB secretion by B. bronchiseptica RB50 FhaC. (Bottom) Western blot of supernatant fractions derived from B. bronchiseptica treated with Pl-CPP peptides (ACT = adenylate cyclase toxin; FHA = FhaB exoprotein).

[0061] Figure 16 shows whole cell lysates of B. bronchiseptica RB50 and a fhaC knock-out strain post treatment and probed by western immunoblot with either anti-FhaB or anti-DegP antisera. Pl-CPP and PIEISR-CPP treatment of B. bronchiseptica RB50 results in cellular FhaB levels being reduced. DegP serves as a control. Figure 17 shows in vitro infection of Chinese Hamster Ovary (CHO) with B. bronchiseptica to observe bacterial adhesion. Bacteria were either mock-treated (top) or treated with 10pm of Pl-CPP for either 15 min or 30 min prior to infection (bottom panels). Pl-CPP treatment reduced specific binding to the CHO cells and caused bacteria to non- specifically aggregate (red arrows).

[0062] Figure 18 shows Pl is stably bound to FhaC for 50ns. (A) Time point 0 of a 50 ns molecular dynamics simulation with Pl binding to FhaC POTRA1. (B) Root Mean Square Deviation (RMSD) values of the simulation trajectory relative to the 1st frame of the simulation.

[0063] Figure 19 shows a variant of the secretion assay method (from Figure 6) where FhaC and FhaBsec are produced together with the peptide Pl, also encoded on the same operon on the same plasmid and containing an N-terminal signal peptide to facilitate translocation across the inner membrane by the Sec translocon (EP 1 ). (A) Coomassie gels from n = 3, samples expressingEP1 releases reduced amounts of FhaBsec. Deletion of the signal sequence from theEP1 insert (EP1ASS) recovers secretion slightly. (B) Quantitation of percentage secreted FhaBsec (relative to noEP1 insert control) from (A). Line denotes the mean value. Ordinary two-way ANOVA (with Dunnett’s multiple comparisons tests) were conducted, **P < 0.01, ***p < 0.001.

[0064] Figure 20 shows that treatment of cells with the Pl-CPP inhibitor does not result in a change in the abundance of the target FhaC transporter. (A) A representative blot of samples encoding FhaBsec (or not) treated with Pl-CPP (or mock) (n = 3). Assay was conducted as in Fig. 6, but cell pellet samples were taken instead of the supernatant. Samples were probed by Western Blot to detect FhaC (aHis). (B) Quantitation of FhaC expression relative to untreated samples from (A). Line denotes the mean value of relative FhaC expression.

[0065] Figure 21 shows that Pl inhibits FhaC by stabilizing the a-Helix (Hl) of the FhaC within the barrel of the transporter. (A) Scheme of a novel helix mobility assay.HlsFhaC- expressing strain was grown at 37 °C for 2 hours before being supplemented with rhamnose (to express FhaC) and Pl-CPP. Samples were incubated for 30 min, and then 100 pL aliquots were taken and incubated with 1° Ab (anti -His). Samples are washed and then incubated with fluorescent 2° Ab and scanned for fluorescence count. Inhibitor-induced stabilisation of the inactive state (helix-in / surface exposed) will increase probability of antibody binding. (B) Quantitation of fluorescence intensity (normalised to endpoint ODeoo) relative to vehicle mock (Left) and endpoint ODeoo (Right). Line denotes the mean relative normalised fluorescence. The assay was conducted as in (A). Ordinary one-way ANOVA (with Sidak’s multiple comparisons post-hoc tests) was conducted. *P < 0.05, **P < 0.01. (C) Primary structures of theHlsFhaC constructs used in the experiment in (D). The location of protein segments [P-barrel, POTRA2, POTRA1 (pink), Linker (black), Hl (blue), and His-tag (red)] are shown. (D) Quantitation of relative FhaBsec secretion (normalised to mock-treated controls). Constructs from (C) were used. Line denotes the mean relative FhaBsec secretion. Assay was conducted as in Fig. 6. Ordinary one-way ANOVA (with Dunnett’s multiple comparisons post-hoc tests) was conducted, *P < 0.05.

[0066] Figure 22 shows screening of an N-terminally and C-terminally truncated Pl -CPP variant (PIANI-CPP and PIACI-CPP). (A) Sequence comparison of Pl-CPP and its N- terminally and C-terminally truncated variant (PIANI-CPP and PIACI-CPP). (B) Representative Coomassie gel from n = 3. (C) Quantitation of FhaBsec secreted when treated with Pl-CPP, its truncated derivatives (N-terminal truncation: PIANI-CPP; C-terminal truncation: PIACI- CPP), or mock-treated. PIAQ-CPP and Pl have equivalent activity. Line denotes the mean value of FhaBsec band intensity (relative to mock). Ordinary one-way ANOVA (with Dunnett’s multiple comparisons post-hoc tests) was conducted, ****p < 0.0001.

[0067] Figure 23 shows testing of roles of charged residues in Pl activity. (A) Bottom view of the top-ranked predicted structure of FhaC-Pl coloured by electrostatic potential as seen on the colour key. Red signifies the most negatively charged surface potential (-10) while blue signifies the most positively charged surface potential (+10). (Left). Residues matched in colour denote salt bridge-forming pairs. Distances between green atom pairs were monitored. (B) Distance between green atom pairs as in (A). Dotted line denotes the maximum distance to form a stable salt bridge interaction. (C) Quantitation of FhaBsec secreted in samples (relative to mock) treated with titrated PIRHE-CPP (pink), PIRBE-CPP (purple), and PIEISR-CPP (brown). Assay was conducted as in Fig. 8. Plots denote the mean. IC50 values were obtained from the plot - PIRHE-CPP (13.0 pM), PIRBE-CPP (1.29 pM), and PIEISR-CPP (0.59 pM). (D) Quantitation of FhaBsec secreted in samples (relative to mock) treated with titrated P1RHE,RI3E,EI5R-CPP (army green). Assay was conducted as in Fig. 8. Plots denote the mean. IC50 values were obtained from the plot - P1RHE,RI3E,EI5R-CPP (5.35 pM).

[0068] Figure 24 shows that reversing the charges of salt-bridge forming residues in Pl alters inhibition activity. Assay was conducted as in Fig. 8. Representative Coomassie gel from n = 3. Figure 25 shows PIEISR-CPP prevents B. bronchiseptica from adhering to lung cells. Quantitation of recovered, adherent B. bronchiseptica in cell lysates recovered from infected A549 lung cells. Samples were treated with PIEISR-CPP or mock-treated. As negative adherence controls, mock-treated B. bronchiseptica strains lacking FhaB (AfhaB and FhaC (Afha ) were also used. Line denotes the mean value of recovered, adherent B. bronchiseptica from lung cells (relative to mock). Ordinary one-way ANOVA (with Dunnett’s multiple comparisons post-hoc tests) was conducted, ****p < 0.0001.

[0069] Figure 26 shows that Pl inhibits secretion of FHA (FhaB exoprotein) from clinical isolates of B. pertussis (strains L1423, L1756, or L2228). (A) Pl-CPP inhibits the secretion of FHA into the culture media and causes FHA accumulation in cells. Samples were probed with Western Blot (aFHA). A prn- (L1756) and fha- (L2228) clinical isolate strain was also used as controls. S / N = supernatant, WCL = whole cell lysate (B) Endpoint ODeoo values of B. pertussis clinical isolates treated with Pl-CPP (relative to the mock-treated control for each strain). Delayed growth was observed in fha+ isolates.

[0070] Figure 27 shows predicted binding model of macrocycles RD-4, RD-6, and RD-7 bound to FhaC. All 3 macrocycles were predicted to bind to the lumen of FhaC. Structure of FhaC was sliced to reveal the barrel pore and the macrocycles bound inside the barrel.

[0071] Figure 28 shows that macrocycles RD-4 (SEQ ID NO: 8) and RD-7 (SEQ ID NO: 6) inhibit FhaC from secreting FhaB from E. coli in a concentration-dependent manner. (Top) Representative Coomassie-stained gels of samples treated with titrated RD-4 (left) and RD-7 (right) from n = 3. Assay was conducted as in Fig. 8. (pM) = Concentration. (Bottom) Quantitation of percentage FhaB sec secreted in samples treated with titrated RD-4 (left), and RD-7 (right), respectively (relative to vehicle control). Ordinary two-way ANOVA (with Dunnett’s multiple comparisons tests) were conducted, compared against the vehicle control, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0072] Figure 29 shows that residues R11 and E7 interchangeably bind to FhaC N121. (Top) Model showing hydrogen bonds formed between POTRA1 and Pl. Residues matched in colour denote hydrogen bond interacting pairs. P 1E7 <=> PIRII (teal), FhaC 2i O P1E7 (dark grey), and FhaC 2i O PIRII (yellow). Distances between green atom pairs were monitored. (Bottom) Distance between green atom pairs as in (Top). Dotted line denotes the maximum distance to form a stable hydrogen bond. Abbreviations: R1 = Replicate 1, R2 = Replicate 2, R3 = Replicate 3. Figure 30 shows that Pl binds the conserved hydrophobic pocket of FhaC POTRA1.

[0073] (A) ConSurf Web Server-generated structure and map of FhaC (PDB: 4QKY). Colour depicts the conservation level of each residue across homologues of FhaC from different diderm species. Dotted box denotes the location of the conserved hydrophobic pocket of POTRA1.

[0074] (B) Sequence of FhaC (residues 51-150) coloured by conservation level per residue. Asterisks denote residues that interact with Pl. Asterisks are labelled by the type of interaction observed: red = salt bridge interaction (SB), brown = contact or Van der Waals interactions (CoV), blue = hydrogen bonds (H), purple = cation-7t interactions (C), black = hydrogen backbone interactions (Hbb).

[0075] Figure 31 shows that PIEISR-CPP prevents fha+ B. pertussis clinical isolate L1423 from adhering to both lung and macrophage cells. Quantitation of fluorescent, adherent B. pertussis relative to the number of cells from slides of infected A549 lung cells and THP-1 macrophage-induced cells (n = 3). Values for each replicate were taken as the average reading from 5 slides. Samples were treated with PIEISR-CPP or mock-treated (DMSO). Line denotes the mean value of fluorescent, adherent B. pertussis from lung cells and macrophage cells (relative to vehicle mock control). Unpaired, two-tailed Student’s t-test was conducted, compared against the vehicle control, ***P < 0.001, 0.0001.

[0076] DEFINITIONS

[0077] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications and patents referred to herein are incorporated by reference herein in their entireties.

[0078] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0079] Unless the context clearly requires otherwise, throughout the description and the claims, the terms “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method. The transitional phrase “consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0080] The transitional phrase “consisting essentially of’ is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characterise cfs) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0081] Where the applicant has defined an invention or a portion thereof with an open-ended term such as “comprising”, it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms “consisting essentially of’ or “consisting of.” In other words, with respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of’ or, alternatively, by “consisting essentially of’.

[0082] While reference may be made in this disclosure to the invention comprising a combination of a plurality of elements, it is also understood that this invention is regarded to comprise combinations which omit or exclude one or more of such elements, even if this omission or exclusion of an element or elements is not expressly stated herein, unless it is expressly stated herein that an element is essential to the applicant’s combination and cannot be omitted. It is further understood that the related prior art may include elements from which this invention may be distinguished by negative claim limitations, even without any express statement of such negative limitations herein. It is to be understood, between the positive statements of applicant’s invention expressly stated herein, and the prior art and knowledge of the prior art by those of ordinary skill which is incorporated herein even if not expressly reproduced here for reasons of economy, that any and all such negative claim limitations supported by the prior art are also considered to be within the scope of this disclosure and its associated claims, even absent any express statement herein about any particular negative claim limitations.

[0083] As used herein, with reference to numbers in a range of numerals, the terms “about,” “approximately” and “substantially” are understood to refer to the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably - 1% to + 1% of the referenced number, most preferably -0.1% to +0.1 % of the referenced number. Moreover, with reference to numerical ranges, these terms should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from 8 to 10, and so forth.

[0084] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0085] Unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or”. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0086] The term “and / or” used in the context of “X and / or Y” should be interpreted as “X,” or “ Y,” or “X and Y.” Similarly, “at least one of X or Y” should be interpreted as “X,” or “Y,” or “both X and Y .”

[0087] The indefinite articles “a” and “an” preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular

[0088] It will be understood that use of the term “between” herein when referring to a range of numerical values encompasses the numerical values at each endpoint of the range. For example, a range of between 10 and 100 is inclusive of 10 and 100.

[0089] Various features of the embodiments of the invention disclosed herein are, for brevity, described in the context of a single embodiment, but may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the illustrative embodiments disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0090] No limitation applies in relation to the mode of administration of the peptides or compounds in the methods and uses of the present invention. The mode of administration of the peptides or compounds described herein may be any suitable route that delivers the peptide or compound to the subject, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous and / or subcutaneous; pulmonary; transmucosal (e.g., oral, intranasal, intravaginal and / or rectal); using a formulation in a tablet, capsule, solution, suspension, powder, gel and / or particle; and contained in a syringe, an implanted device, osmotic pump, cartridge and / or micropump; or other means appreciated by the skilled person as well known in the art.

[0091] As used herein, and unless otherwise specified, the terms “therapeutically effective amount” and “effective amount” of a peptide or compound means an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A “therapeutically effective amount” and “effective amount” of a peptide or compound means an amount of therapeutic agent, alone or in combination with one or more other compound(s) or agent(s), which provides a therapeutic benefit in the treatment or management of the disease or disorder. The terms “therapeutically effective amount” and “effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic compound or agent.

[0092] As used herein, and unless otherwise specified, the term “preventing or reducing bacterial replication” means at least partially reducing replication of bacteria after the bacteria are exposed to a peptide or compound. It will be appreciated, although not precluded, that preventing or reducing bacterial replication does not require that bacterial replication be completely stopped. As used herein, preventing or reducing bacterial replication encompasses killing bacteria. In some embodiments, reducing bacterial replication may refer to a reduction in replication of bacteria to: about 0%, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 75% as compared to replication of bacteria in a reference or control condition. In some embodiments reducing bacterial replication may refer to a reduction in replication of bacteria to between: about 0% and about 5%, about 0% and about 10%, about 0% and about 20%, about 0% and about 30%, about 0% and about 40%, about 0% and about 50%, about 0% and about 60%, about 0% and about 75%, about 5% and about 75%, or about 10% and about 75% as compared to replication of bacteria in a reference or control condition.

[0093] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder / disease and / or symptoms associated therewith. It will be appreciated, although not precluded, that treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0094] As used herein, the term “prevent”, “preventing”, “prevention”, and the like, when used in relation to a disorder / disease and / or symptom, refer to reducing the occurrence of a disorder / disease and / or symptoms associated therewith. It will be appreciated, although not precluded, that preventing a disorder or condition does not require that the disorder, condition, or symptoms associated therewith are completely removed in every subject that is administered treatment.

[0095] As used herein, the term “composition” are intended to encompass compositions comprising the specified ingredients, as well as any product or products which result, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0096] As used herein, the terms “pharmaceutical” or “pharmaceutically acceptable” mean that any diluent(s), excipient(s) or carrier(s) in the composition are compatible with the other ingredient(s) and not deleterious to the recipient thereof.

[0097] As used herein, the terms “inhibition” or “inhibiting” when used in terms of a transport pathway refers to reducing the function of the transport pathway, i.e., reducing the transport of a molecule through the pathway. The term “inhibition” or “inhibiting” does not necessarily mean that the transport pathway is completely halted, i.e., transport of a molecule through the pathway may still occur albeit to a reduced extent. In some embodiments, inhibition refers to a reduction in transport of a molecule to: about 0%, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, about 95%, or about 99% as compared to transport of the molecule in a control or reference condition. In some embodiments, inhibition refers to a reduction in transport of a molecule to between: about 0% and about 5%, about 0% and about 10%, about 0% and about 20%, about 0% and about 30%, about 0% and about 40%, about 0% and about 50%, about 0% and about 60%, about 0% and about 75%, about 5% and about 75%, about 10% and about 75%, about 0% and about 70%, about 0% and about 80%, about 0% and about 90%, about 0% and about 95%, or about 0% and about 99%, as compared to transport of the molecule in a control or reference condition.

[0098] As used here, the phrase “reducing translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane” does not necessarily mean that translocation of a TpsA across a lipid membrane is completely halted, i.e., translocation across a lipid membrane may still occur albeit to a reduced extent. In some embodiments, “reducing” refers to a reduction in translocation of the TpsA to: about 0%, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, about 95%, or about 99% as compared to translocation of the TpsA in a control or reference condition. In some embodiments, “reducing” refers to a reduction in translocation of the TpsA to between: about 0% and about 5%, about 0% and about 10%, about 0% and about 20%, about 0% and about 30%, about 0% and about 40%, about 0% and about 50%, about 0% and about 60%, about 0% and about 75%, about 5% and about 75%, about 10% and about 75%, about 0% and about 70%, about 0% and about 80%, about 0% and about 90%, about 0% and about 95%, or about 0% and about 99% as compared to translocation of the TpsA in a control or reference condition.

[0099] As used herein, the term “subject” refers to any animal, including, but not limited to: humans, non-human primates, and non-human animals such as livestock (e.g., cows, horses, sheep, goats, pigs, and chickens), fish, amphibians, reptiles, birds, canines, felines, and rodents.

[0100] As used herein, the term “two-partner secretion system” or “TPS” refers to the bacterial system which comprises two genes in an operon, wherein one gene encodes an outer membrane embedded translocator pore protein of “TpsB” family and the other gene encodes a partner exoprotein (also referred to as “TpsA”). The TpsB exports the TpsA to the bacterial cell surface, thereby secreting the TpsA.

[0101] As used herein, the terms “bind”, “binds to” and “binding” refer to an interaction between a peptide or compound and a biological molecule. In certain embodiments the interaction is mediated by one or more ionic bond, covalent bond, hydrogen bond, or combinations thereof. In certain embodiments the interaction is mediated by Van der Waals forces and / or dipole-dipole interactions.

[0102] As used herein, the term “TpsB” or “TpsB protein” refers to the translocator pore protein (also referred to as an “outer membrane-integrated subunit”) in the two-partner secretion system. As used herein, the term “TpsA” or “TpsA protein” refers to the partner exoprotein in the two-partner secretion system.

[0103] As used herein, the term “FhaB” refers to the TpsA (partner exoprotein) in Bordetella spp.

[0104] As used herein, the term “FhaC” refers to the TpsB (translocator pore protein or outer membrane-integrated subunit) in Bordetella spp.

[0105] As used herein, the terms “adenylate cyclase toxin” and “ACT” refer to a virulence factor produced by Bordetella spp. The terms “adenylate cyclase toxin” and “ACT” are used interchangeably in this disclosure.

[0106] As used herein, the term “lipid membrane” refers to a membrane in a cell. The term "lipid membrane” may refer to the outer membrane in a Gram-negative bacterial species.

[0107] As used herein, the terms “encode,” “encodes,” and “encoding” refer to a nucleotide sequence that, when serving as a template for transcription and subsequent translation, is capable of producing a specified amino acid sequence (e.g., a peptide or protein).

[0108] As used herein, the term “biofilm” refers to a population of bacteria that adhere to a surface and form a film with an extracellular matrix comprising one or more of polysaccharides, proteins, lipids and DNA. A “biofilm” often constitutes a distinct microenvironment and may comprise multiple bacterial species.

[0109] As used herein, the term “signal peptide” refers to an amino acid sequence that directs a protein, peptide, or other cargo to cross a lipid membrane. These “signal peptides” are attached to many proteins made by a cell and, in some embodiments, are required in order for the protein to be exported across a lipid membrane. In certain embodiments, the signal peptide allows a protein to be transported across the inner membrane of a bacterial cell. In further embodiments, the signal peptide is located at the N-terminus (amino-terminus) of a protein or peptide. Signal peptides are well characterised, and a skilled person will be familiar with the amino acid sequences which can serve as a signal peptide. Non-limited examples of an amino acid sequence of a signal peptide are as follows: MAMKKLLIASLLFSSATVYGA (SEQ ID NO: 69) from BamA or MKKHKRILALCFLGLLQSSYSFA (SEQ ID NO: 70) from EspP.

[0110] As used herein, the term “fraction” or “isolated fraction” refers to a sample derived from a starting sample after being subjected to a separating step. For example, a “fraction” or “isolated fraction” may refer to a pellet or supernatant fraction produced by subjecting a starting sample to a centrifugation or ultra-centrifugation step. A “fraction” or “isolated fraction” may also refer to a discrete layer produced by subjecting a starting sample to a centrifugation or ultra-centrifugation step. The centrifugation or ultra-centrifugation step may also include the use of a density gradient. In some embodiments, a “fraction” or “isolated fraction” refers to a supernatant fraction or a pellet fraction isolated from bacterial cell culture media. In other embodiments, a “fraction” or “isolated fraction” refers to a supernatant fraction or a pellet fraction which is further isolated from a bacterial cell pellet.

[0111] DETAILED DESCRIPTION

[0112] The following detailed description conveys exemplary embodiments of the present invention in sufficient detail to enable those of ordinary skill in the art to practice the present invention. Features or limitations of the various embodiments described do not necessarily limit other embodiments of the present invention, or the present invention as a whole. It will be appreciated by persons of ordinary skill in the art that numerous variations and / or modifications can be made to the present invention as disclosed in the specific embodiments without departing from the spirit or scope of the present invention as broadly described. Hence, the following detailed description does not limit the scope of the present invention, which is defined only the claims. The skilled addressee will understand that the invention comprises the embodiments and features disclosed herein as well as all combinations and / or permutations of the disclosed embodiments and features. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0113] The present invention relates to targeting surface-exposed factors that are found on the bacterial outer membrane. In targeting surface-exposed factors, there is no need for the peptide, compound or agent to cross the entire bacterial envelope. The present invention also targets essential virulence factors of a pathogen instead of essential central growth pathway; thereby not being bacteriostatic or bactericidal. Without wishing to be bound by theory, the Inventors hypothesise that a strategy targeting virulence factors instead of bacterial growth may maintain efficacy longer than traditional strategies due to reduced selection pressure. Furthermore, the Inventors also hypothesise that because the strategy of the present invention specifically targets virulence factors, they are unlikely to disturb the host’s normal microbiome, thereby providing a clear advantage over traditional antibiotics that disrupt important commensal bacteria and can lead to adverse consequences to a subject after antibiotic treatment.

[0114] In an embodiment, the invention provides a peptide comprising an amino acid sequence as set forth in any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93; an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93; or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93. In some embodiments, the peptide consists of, or consists essentially of, an amino acid sequence as set forth in any one of SEQ ID Nos: 1-8, 10-37, 58- 60, 63-68, and 89-93. In some embodiments, the peptide is isolated. In some embodiments, the peptide binds to a translocator pore protein (TpsB). In a further embodiment, the peptide reduces translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane.

[0115] In an embodiment, the invention provides a compound which binds to a translocator pore protein (TpsB). In a further embodiment, the compound reduces translocation of a two- partner secretion exoprotein (TpsA) across a lipid membrane.

[0116] In an embodiment, the invention provides a compound for inhibiting a two-partner secretion system, wherein the compound binds to a translocator pore protein (TpsB), thereby reducing translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane.

[0117] In certain embodiments of the invention the compound comprises an amino acid sequence selected from the group consisting of: VAAMAA (SEQ ID NO: 1); VAAMAAEASARARAEFAAR (SEQ ID NO: 2); VAAMAAEASARAEAEFAAR (SEQ ID NO: 3); VAAMAAEASARARARFAAR (SEQ ID NO: 4); VAAMAAEASARAEARFAAR (SEQ ID NO: 5); YVLCTLRAKRFYALFLSG (SEQ ID NO: 6);

[0118] YRKHWPDWYRTHYVVCG (SEQ ID NO: 7); YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8); YRKHWPEWYRTHYVVCG (SEQ ID NO: 11); YRKHWPDWYRTHYVACG (SEQ ID NO: 12); YRKHWPDWYRTHYVICG (SEQ ID NO: 13); YHILYYKVLYYLYCG (SEQ ID NO: 14); YRIICWYLICG (SEQ ID NO: 15); YRYPTWYSTYYNQCG (SEQ ID NO: 16); YYWYYLFWSCG (SEQ ID NO: 17); YAIVVSPKYLFIRVCG (SEQ ID NO: 18); YHYYLYQSKFIWECG (SEQ ID NO: 19); YGGGKDRKSTRLNSSH (SEQ ID NO: 20); YIVSTIPSNVICLICG (SEQ ID NO: 21); YEEHTSELQSLGIISYAVFC (SEQ ID NO: 22); YLKISTCTWVLSISFCG (SEQ ID NO: 23); YFILKLLLVCG (SEQ ID NO: 24);

[0119] YFVLTLVYIIVIHAVCG (SEQ ID NO: 25); YYVVYFDYLFWIVVGCG (SEQ ID NO: 26); YFLIWWLLLCG (SEQ ID NO: 27); YLILVFDSSEVWVVLICG (SEQ ID NO: 28); YAVAYWSRNFVLVYTICG (SEQ ID NO: 29); YLIVTRVNERWHFSHVCG (SEQ ID NO: 30); YVFLYEKGFLIVFTICG (SEQ ID NO: 31); YTWLYVLLVCG (SEQ ID NO: 32); YWQKRHICFEIVLCG (SEQ ID NO: 33); YRLTIVRNHIGWTLYVCG (SEQ ID NO: 34); YINVIWFWYCG (SEQ ID NO: 35); YLCLLFVFLTLCIIICG (SEQ ID NO: 36); YEEHTSELQSRTPISYAVFC (SEQ ID NO: 37); YVLCTLRAKRFYALFLCG (SEQ ID NO: 63); VAAMAAEASARARAE (SEQ ID NO: 64); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences (SEQ ID Nos: 1-8, 11-37, 63, and 64); and an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 1-8, 11-37, 63, and 64. In some embodiments, the compound consists of, or consists essentially of, an amino acid sequence as set forth in any one of SEQ ID Nos: 1-8, 11-37, 63, and 64.

[0120] In preferred embodiments, the peptide or compound comprises an amino acid sequence selected from the group consisting of: VAAMAA (SEQ ID NO: 1); VAAMAAEASARARAEFAAR (SEQ ID NO: 2); VAAMAAEASARAEAEFAAR (SEQ ID NO: 3); VAAMAAEASARARARFAAR (SEQ ID NO: 4); VAAMAAEASARAEARFAAR; (SEQ ID NO: 5); YVLCTLRAKRFYALFLSG (SEQ ID NO: 6);

[0121] YRKHWPDWYRTHYVVCG (SEQ ID NO: 7); YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8); VAAMAAEASARARAE (SEQ ID NO: 64); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences (SEQ ID Nos: 1-8 and 64); and an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 1-8 and 64.

[0122] In some embodiments the amino acid sequence further comprises a cell penetrating peptide amino acid sequence. Cell penetrating peptides are a well-known class of peptides which facilitate translocation of cargo across a lipid membrane. In certain embodiments the cell penetrating peptide amino acid sequence comprises the amino acid sequence: KFFKFFKFFK (SEQ ID NO: 9). Without wishing to be bound by theory, usage of a cell penetrating peptide is expected to allow the peptide or compound to cross the outer membrane of a gram-negative bacterial cell and reside in the periplasm to exert its effects by binding to the periplasmically-located features of the TpsB protein. In other words, the cell penetrating peptide may allow the peptide or compound to cross the bacterial cell envelope partially. The cell penetrating peptide may also allow the peptide or compound to cross the bacterial cell envelope completely.

[0123] In a specific embodiment the peptide or compound comprises the amino acid sequence: KFFKFFKFFKGVAAMAAEASARARAEFAAR (SEQ ID NO: 10).

[0124] In some embodiments the peptide or compound comprises a cyclized amino acid sequence. In certain embodiments the amino acid sequence is cyclized between a tyrosine (Y) residue and a cysteine (C) residue. In some embodiments, the cyclized amino acid sequence comprises an amino acid sequence represented by any one of SEQ ID NOs: 6-8 and 11-37, an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of SEQ ID NOs: 6-8 and 11-37, or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 6-8 and 11-37. In certain embodiments the cyclised amino acid sequence comprises the amino acid sequence: YVLCTLRAKRFYALFLSG (SEQ ID NO: 6); YRKHWPDWYRTHYVVCG (SEQ ID NO: 7), YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8), an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences (SEQ ID NOs: 6-8), or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 6-8. In further embodiments the cyclized amino acid sequence is formed when the D-isomer or the L-isomer of a chloroacetylated tyrosine residue reacts with the cysteine residue. In some embodiments, the peptide or compound comprises an amino acid sequence with a two-dimensional structure selected from the group consisting of: In some embodiments the peptide or compound comprises an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, and / or substitutions as compared to any one of the abovementioned amino acid sequences (SEQ ID Nos: 1-37, 58- 60, 63-68, and 89-93). In some embodiments the peptide or compound comprises an amino acid sequence with between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, between 1 and 2, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 2 and 4, between 2 and 3, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, or between 3 and 4 amino acid deletions, additions, and / or substitutions as compared to any one of the abovementioned amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93). In certain embodiments, the peptide or compound comprises an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93). In some embodiments the peptide or compound comprising the amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, and / or substitutions substantially retains the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93). For example, the peptide or compound comprising the amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, and / or substitutions has: about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63- 68, and 89-93). In other embodiments the peptide or compound comprising the amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, and / or substitutions has between: about 70% and about 100%, about 75% and about 100%, about 80% and about 100%, about 85% and about 100%, about 90% and about 100%, or about 95% and about 100% of the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93). In further embodiments, the peptide or compound comprising the amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, and / or substitutions has more than 100% of the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93).

[0125] In some embodiments the peptide or compound comprises an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of the abovementioned amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93). In some embodiments the peptide or compound comprising the amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity substantially retains the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93). For example, the peptide or compound comprising the amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity has: about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93). In other embodiments the peptide or compound comprising the amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity has between: about 70% and about 100%, about 75% and about 100%, about 80% and about 100%, about 85% and about 100%, about 90% and about 100%, or about 95% and about 100% of the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93). In further embodiments, the peptide or compound comprising the amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity has more than 100% of the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93).

[0126] In the context of the present invention, an amino acid sequence “having a sequence identity” of at least, for example, 95% to a query amino acid sequence of the present invention, is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence except that the subject amino acid sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain an amino acid sequence having a sequence of at least 95% identity to a query amino acid sequence, up to 5% (i.e., 5 of 100) of the amino acids in the subject sequence may be inserted or substituted with another amino acid or deleted. Methods for comparing the identity and homology of two or more sequences are well known in the art. The percentage to which two sequences are identical can for example be determined using a mathematical algorithm such as those integrated into the BLAST family of programs accessible through the home page of the NCBI (ncbi.nlm.nih.gov). In the context of the present invention, where the amino acid sequence of the peptide or compound includes one or more substitutions and / or additions as compared to a reference / query sequence, it should be understood that such substitutions and / or additions may include canonical amino acids, non-canonical amino acids, and / or any synthetic derivatives thereof.

[0127] The skilled person will appreciate that the amino acid sequences of the peptides and compounds disclosed herein can by cyclised or bi-cyclised without departing from the invention. Such a modification of the amino acid sequence may improve membrane permeability of the peptide or compound. In some embodiments one or more residues in the amino acid sequence may be substituted with non-canonical amino acids to facilitate cyclising the sequence. In specific embodiments solvent-exposed, non-interacting residues of the amino acid sequence are substituted with non-canonical amino acids.

[0128] Generally, substitutions for one or more amino acids present in the referenced amino acid sequence should be made conservatively. As an example, a substitution which retains or enhances the biochemical properties of the original sequence (e.g., hydrophobicity, electrostatic charge, aromatic features, or similar) may be considered conservative substitutions. Additionally, the substitution of L-amino acids with chemically similar non- canonical amino acids (e.g. D-amino acids, beta- substituted amino acids, etc), may also be considered conservative substitutions. In some embodiments, making a conservative substitution will retain between: about 70% and about 100%, about 75% and about 100%, about 80% and about 100%, about 85% and about 100%, about 90% and about 100%, about 95% and about 100%, or above 100% of the inhibiting activity or binding activity of any one of the above amino acid sequences (SEQ ID Nos: 1-37, 58-60, 63-68, and 89-93).

[0129] Binding of a peptide or compound disclosed herein to the translocator pore protein (TpsB) can be measured by any suitable method. In some embodiments binding of a peptide or compound can be screened using mRNA Display as described herein. In some embodiments, binding can be measured using quantitative methods such as, but not limited to, isothermal calorimetry (ITC), biolayer interferometry (BLI) and / or surface plasmon resonance (SPR).

[0130] In this disclosure, inhibition of the two-partner secretion system refers to reducing translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane. In some embodiments, the translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane may be toward an extracellular space. This reduction in translocation of TpsA across a lipid membrane can be measured by any suitable method. In certain embodiments the reduction in translocation of TpsA across a lipid membrane can be measured using the FhaB Secretion Assay described herein. In other embodiments, the reduction in translocation of TpsA across a lipid membrane can be measured in wild type isolates by exposing the isolate to a candidate inhibitor, centrifuging the isolate, and measuring the amount of TpsA in the supernatant, which is then compared to a control (see, for example, Figure 15). A peptide or compound of the present invention may reduce translocation of TpsA across a lipid membrane to: about 0%, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, about 95%, or about 99% as compared to translocation of TpsA across a lipid membrane when no inhibitor is present. In some embodiments a peptide or compound of the present invention reduces translocation of TpsA across a lipid membrane to between: about 0% and about 5%, about 0% and about 10%, about 0% and about 20%, about 0% and about 30%, about 0% and about 40%, about 0% and about 50%, about 0% and about 60%, about 0% and about 75%, about 5% and about 75%, about 10% and about 75%, about 0% and about 70%, about 0% and about 80%, about 0% and about 90%, about 0% and about 95%, about 0% and about 99% as compared to translocation of TpsA across a lipid membrane when no inhibitor is present.

[0131] In other embodiments, the reduction in translocation of TpsA across a lipid membrane can be assessed by measuring the effects of the TpsA on a cell or system. In a specific embodiment, binding of bacteria to eukaryotic cells may be disrupted by the TPS inhibitor and aggregation of the bacteria may indicate a reduced translocation of TpsA (see, for example, Figure 17). In a further specific embodiment, the TPS inhibitor may be assessed by evaluating the toxic effects of the TpsA, with a reduction in toxic effects (e.g., cell death) being indicative of reduced translocation of the TpsA. In another specific embodiment the TPS inhibitor may be assessed by evaluating the enzymatic properties of the TpsA with a reduction of enzymatic processing being indicative of reduced translocation of the TpsA.

[0132] In some embodiments the two-partner secretion system to be inhibited or bound by the peptide or compound disclosed herein is from one of the more than 6000 species that contain a TpsB family protein such as Pseudomonas spp., Salmonella spp., Bordetella spp., Klebsiella spp., Escherichia spp, Enterobacter spp., Citrobacter spp., Shigella spp., Photorhabdus spp., Providencia spp., Xenorhabdus spp., Proteus spp., Morganella spp., Yersinia spp., Serratia spp., Pantoea spp., Erwinia spp., Pectobacterium spp., Stenotrophomonas spp., Xanthomonas spp., Haemophilus spp., Acinetobacter spp., Moraxella spp., Vibrio spp., Burkholderia spp., Paraburkholderia spp., Ralstonia spp., Cupriavidus spp., Pandoraea spp., Caballer onia spp., Variovorax spp., Janthinobacterium spp., Achromobacter spp., Bordetella spp., Neisseria spp., Bradyrhizobium spp., Rhizobium spp., Bartonella spp., Sphingomonas spp., Novosphingobium spp., Symploca spp., Moorena spp., Nostoc spp., Selenomonas spp., Campylobacter spp., Helicobacter spp., Fusobacterium spp., Nitrospira spp., or any other species that contains a TpsB protein.

[0133] In certain embodiments the Bordetella spp. is Bordetella pertussis or Bordetella bronchiseptica.

[0134] In some embodiments the TpsB is one of at least 1700 TpsB family proteins found in the above species including FhaC, HMW1B, LspBl, CdiB, ShlB, HpmB, HecB, CdrB or EthB. In some embodiments the TpsA is FhaB, HMW1A, LspAl, LspA2, CdiA, ShlA, HpmA, HecA, CdrA or EthA.

[0135] In some embodiments the peptide or compound inhibits translocation of adenylate cyclase toxin (ACT) across a lipid membrane. In certain embodiments the peptide or compound inhibits translocation of a TpsA and ACT across a lipid membrane.

[0136] In some embodiments the invention provides a composition comprising the peptide or compound described above.

[0137] In some embodiments the invention provides a pharmaceutical composition comprising the peptide or compound described above and one or more pharmaceutically acceptable diluent, excipient, or carrier. A person skilled in the art will appreciate that various diluents, excipients, and / or carriers can be used to prepare a pharmaceutical composition with desired characteristics. For example, a skilled person will be able to use diluents, excipients, and / or carriers to alter the solubility, stability, pharmacokinetic profile, or pharmacodynamic properties of a peptide or compound in a pharmaceutical composition.

[0138] In some embodiments the invention provides methods of treating or preventing a bacterial infection in a subject using the peptide or compound or pharmaceutical composition described above. In other embodiments the invention provides use of the peptide or compound or pharmaceutical composition described above for treating or preventing a bacterial infection in a subject. The invention also relates, in embodiments, to use of the peptide or compound described above in the manufacture of a medicament to treat or prevent a bacterial infection in a subject. In one preferred embodiment the subject is a human. In other embodiments the subject is a non-human primate or a non-human animal (e.g., a nonhuman mammal). In some embodiments the non-human mammal is a livestock animal such a cow, horse, sheep, goat, pig, or chicken. In a preferred embodiment, the subject is a pig. In some embodiments the bacterial infection to be treated or prevented is caused by one or more of the abovementioned bacterial species. In certain embodiments the bacterial infection is caused by Bordetella pertussis, Bordetella parapertussis or Bordetella bronchiseptica. In further non-limiting embodiments, the bacterial infection is associated with whooping cough, cystic fibrosis, a lung infection, a wound infection, diabetes, a gastrointestinal disease, a urinary tract infection, bacterial meningitis, vaginosis, pneumonia, chancroid, diarrhea, septicaemia, an eye infection, an ear infection, or a surgery such as an organ transplant or joint replacement.

[0139] In some embodiments, the invention provides a method of screening for inhibitors of TpsA secretion. In such embodiments the method comprises providing a bacterial strain comprising a nucleotide sequence encoding a TpsB protein and a nucleotide sequence encoding a TpsA protein. In certain embodiments, the method further comprises exposing the bacterial strain to a candidate inhibitor and measuring the amount of TpsA protein or TpsB protein in a fraction isolated from the bacterial strain. For example, by measuring the amount of TpsA protein, one can then evaluate whether a candidate inhibitor successfully inhibits TpsA secretion. In another embodiment the bacterial strain comprises a nucleotide sequence encoding a TpsB protein, a nucleotide sequence encoding a TpsA protein, and a nucleotide sequence encoding a signal peptide and a candidate inhibitor. In some embodiments, the signal peptide is an N-terminal (amino-terminal) signal peptide. In such an embodiment the method further comprises measuring the amount of TpsA protein or TpsB protein in a fraction isolated from the bacterial strain. In certain embodiments of the above screening methods, the TpsA protein has an increased ability to secrete from the TpsB protein when compared to a wildtype TpsA protein. In an embodiment, the TpsA protein with an increased ability to secrete from the TpsB protein comprises an amino acid sequence as set forth in SEQ ID NO: 56, or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 56. In further embodiments, the nucleotide sequences encoding the TpsA protein, TpsB protein, and / or candidate inhibitor and a cell penetrating peptide may be present on one or more constructs which the bacterial strain is transformed with. In some embodiments, the TpsB is FhaC, HMW1B, LspBl, CdiB, ShlB, HpmB, HecB, CdrB or EthB. In some embodiments the TpsA is FhaB, HMW1A, LspAl, LspA2, CdiA, ShlA, HpmA, HecA, CdrA or EthA. In certain embodiments the TpsB is FhaC and the TpsA is FhaB. In some embodiments the fraction isolated from the bacterial strain is a supernatant fraction or a pellet fraction isolated after centrifugation or ultracentrifugation. In other embodiments the fraction is a discrete layer in the sample after centrifugation or ultracentrifugation.

[0140] In some embodiments, the invention provides a method of treating or preventing plant disease by applying the peptide or compound described above to a plant in an amount effective to treat or prevent the plant disease. A person skilled in the art will appreciate that use of the peptide or compound to treat or prevent the plant disease does not necessarily mean, although it is not precluded, that the plant disease is completely eradicated. In some embodiments the plant is a vegetable. In certain embodiments the vegetables are cabbage, lettuce, Brassica spp., cucurbit, tomatoes, capsicum, potatoes, sweet potatoes, or carrots. In some embodiments the plant disease is bacterial soft rot. In some embodiments the plant is a fruit. In certain embodiments the fruits are lime, lemons, oranges, grapefruit, or other citrus fruits. In some embodiments the plant disease is citrus canker. In some embodiments the plant disease is caused by the bacteria Xanthomonas axopodis pv. citri.

[0141] In some embodiments, the invention provides a method of preventing or reducing bacterial replication, the method comprising exposing the bacteria to the peptide or compound described above. In some embodiments the bacteria are in a biofilm. In some embodiments the bacteria are on a biological surface or a non-biological surface. In certain embodiments the biological surface is a skin surface on a subject. In some embodiments the non-biological surface is a hospital or household surface. In certain embodiments the non- biological surface is a surface on a medical device, surgical implant, or ventilator.

[0142] In another embodiment, the invention provides a compound for binding a translocator pore protein (TpsB), wherein the compound comprises an amino acid sequence selected from the group consisting of: VAAMAA (SEQ ID NO: 1); VAAMAAEASARARAEFAAR (SEQ ID NO: 2); VAAMAAEASARAEAEFAAR (SEQ ID NO: 3);

[0143] V A AM A AE AS AR AR ARFA AR (SEQ ID NO: 4); V A AM A AE AS ARAE ARFA AR (SEQ ID NO: 5); YVLCTLRAKRFYALFLSG (SEQ ID NO: 6); YRKHWPDWYRTHYVVCG (SEQ ID NO: 7); YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8); YRKHWPEWYRTHYVVCG (SEQ ID NO: 11); YRKHWPDWYRTHYVACG (SEQ ID NO: 12);

[0144] YRKHWPDWYRTHYVICG (SEQ ID NO: 13); YHILYYKVLYYLYCG (SEQ ID NO: 14); YRIICWYLICG (SEQ ID NO: 15); YRYPTWYSTYYNQCG (SEQ ID NO: 16);

[0145] YYWYYLFWSCG (SEQ ID NO: 17); YAIVVSPKYLFIRVCG (SEQ ID NO: 18); YHYYLYQSKFIWECG (SEQ ID NO: 19); YGGGKDRKSTRLNSSH (SEQ ID NO: 20); YIVSTIPSNVICLICG (SEQ ID NO: 21); YEEHTSELQSLGIISYAVFC (SEQ ID NO: 22); YLKISTCTWVLSISFCG (SEQ ID NO: 23); YFILKLLLVCG (SEQ ID NO: 24);

[0146] YFVLTLVYIIVIHAVCG (SEQ ID NO: 25); YYVVYFDYLFWIVVGCG (SEQ ID NO: 26); YFLIWWLLLCG (SEQ ID NO: 27); YLILVFDSSEVWVVLICG (SEQ ID NO: 28); YAVAYWSRNFVLVYTICG (SEQ ID NO: 29); YLIVTRVNERWHFSHVCG (SEQ ID NO: 30); YVFLYEKGFLIVFTICG (SEQ ID NO: 31); YTWLYVLLVCG (SEQ ID NO: 32); YWQKRHICFEIVLCG (SEQ ID NO: 33); YRLTIVRNHIGWTLYVCG (SEQ ID NO: 34); YINVIWFWYCG (SEQ ID NO: 35); YLCLLFVFLTLCIIICG (SEQ ID NO: 36); YEEHTSELQSRTPISYAVFC (SEQ ID NO: 37); YVLCTLRAKRFYALFLCG (SEQ ID NO: 63); VAAMAAEASARARAE (SEQ ID NO: 64); VAAMAAEASAEARAEFAAR (SEQ ID NO: 65); VAAMAAEASAEAEAEFAAR (SEQ ID NO: 66);

[0147] VAAMAAEASAEARARFAAR (SEQ ID NO: 67); VAAMAAEASAEAEARFAAR (SEQ ID NO: 68); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences (SEQ ID Nos: 1-8, 11-37, 63, and 64-68); and an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 1-8, 11-37, 63, and 64-68. In a further embodiment, the amino acid sequence further comprises the amino acid sequence: KFFKFFKFFK (SEQ ID NO: 9). In further embodiments, the peptide or compound further comprises a fluorophore, radio ligand, colorimetric label, histidine tag (e.g., 6x His or lOx His), streptavidin tag, avidin tag, or any other suitable label / marker. A person skilled in the art will appreciate that any suitable label / marker may be conjugated to the peptide or compound of the present invention in order for the peptide or compound to be detected by various means (e.g., fluorescent microscopy, light microscopy, radiography, western blot, ELISA, or similar). It is envisioned that the peptides or compounds of the present invention may be used to detect the presence of a translocator pore protein (TpsB) or the location of a TpsB in a sample or subject.

[0148] In another embodiment, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a peptide as disclosed herein. In an embodiment, the nucleic acid molecule is isolated. In a further embodiment, the invention provides a vector comprising a nucleotide sequence encoding a peptide disclosed herein. The invention also relates, in an embodiment, to a cell comprising: a nucleotide sequence encoding the peptide, the nucleic acid molecule, or the vector as disclosed herein. In a specific embodiment, the cell is a bacterial cell. In another specific embodiment, the cell is isolated.

[0149] In another embodiment, the invention provides a kit comprising the compound or the peptide disclosed herein. In a further embodiment, the kit is for use in a method as disclosed herein.

[0150] EXAMPLES The present invention will now be described with reference to the following examples which should be considered in all respects as illustrative and non-restrictive.

[0151] EXAMPLE 1

[0152] Materials and Methods

[0153] Growth Media and Bacterial Strains

[0154] Liquid Broth Media

[0155] All strains were grown in lysogeny broth (LB). Concentration of bacteria was measured by optical absorbance at 600 nm (ODeoo). Solid Growth Media

[0156] All spread, streak, and patch plates of all strains were grown on LB agar plates. Selection and Additives

[0157] For selection, kanamycin (50 pg / mL), trimethoprim (50 pg / mL), tetracycline (50 pg / mL) and / or ampicillin (100 pg / mL) were used as selection agents at their appropriate concentrations. For constructs controlled by a Prha promoter, expression was induced with 0.2% L-rhamnose (w / v) and repressed with 0.2% glucose (w / v). For constructs controlled by a PTK promoter, expression was induced with 0.4 mM of IPTG and repressed with 0.2% glucose (w / v).

[0158] Storage of Strains

[0159] Strains were grown on solid LB agar as a lawn, harvested by a sterile loop, and stored in storage solution at -80 °C. All plasmids were stored in either E. coli K-12 strain NEB-5a or E. coli strain BL21(DE3).

[0160] Overnight Culture Conditions

[0161] Cells were grown from a single colony in LB supplemented with appropriate concentrations of selection agents at 37 °C (unless otherwise stated) with orbital shaking at 250 rpm overnight before being pelleted (3,220 x g, 10 min, 25 °C), washed, and resuspended with one overnight culture volume of LB before further subculturing.

[0162] DNA Techniques

[0163] Plasmid Isolation

[0164] Plasmids used are listed below (Table 1). Plasmids were obtained from overnight liquid cultures (LB, 10 mL) and purified with the QIAprep® Spin Miniprep kit (Qiagen) following manufacturer’s protocols.

[0165] Table 1. Plasmids.

[0166] AmpR= Ampicillin-resistant,

[0167] TrimR= Trimethoprim-resistant,

[0168] KanR= Kanamycin-resistant,

[0169] PNT = Prodomain N-terminus, MBP = Maltose / Maltodextrin binding protein,

[0170] His = His tag,

[0171] Hisio = His x 10 tag,

[0172] Avi = AviTag™,

[0173] TS = TwinStrepII tag. Pl = Pl peptide insert (codons encoding amino acid sequence VAAMAAEASARARAEFAAR [SEQ ID NO: 2]),EP1

[0174] DNA Quantitation

[0175] Measurement of plasmid concentrations were done using Nanodrop ND- 1000 Spectrophotometer (Thermo Scientific) to measure absorbance at 260 nm (A260).

[0176] Concentration is measured in ng / pL as follows: A260X 50 ng / pL (for dsDNA); A260 x 33 ng / pL (for ssDNA).

[0177] Restriction Enzyme Digestions Restriction enzymes were used following manufacturer’s protocols and heat- inactivated following digestion.

[0178] Oligonucleotides

[0179] Oligonucleotides used are listed below (Table 2). Oligonucleotides (obtained from Integrated DNA Technologies (IDT)) were resuspended in UltraPure water to make a storage stock (at a concentration of 100 pM), and stored at -20 °C.

[0180] Table 2. Single-stranded oligonucleotides. DNA Gel Electrophoresis

[0181] 1% agarose TAE gels were supplemented with 10,000x GelGreen® Nucleic Acid Gel Stain (according to manufacturer’s protocols). Samples of DNA were mixed with 6x purple loading dye [Volume, pL = DNA sample, pL / 6] and loaded onto the gel. Size marker used was the 1 kb DNA ladder. Gels were run at 100 V for 50 minutes and visualised using a GelDoc XR+ System.

[0182] Gel DNA Extraction DNA fragments were excised following electrophoretic separation and the QIAquick® Gel Extraction kit was used following manufacturer’s protocol.

[0183] DNA Sequencing

[0184] Samples containing 40 ng of DNA sample with 3.2 pmol oligonucleotide primer were processed using Sanger sequencing according to standard protocols.

[0185] DNA Ligation

[0186] Linear DNA fragments were ligated using T4 DNA Ligase according to manufacturer’s protocols. Ligation reactions were then incubated at either 25 °C (for 2 hours) or 16 °C (overnight). Construction of a FhaC AHI -Expressing Vector

[0187] Gene synthesis of an E. coli codon-optimized FhaC gene from B. pertussis was conducted by IDT (Table 3).

[0188] Table 3. Codon-optimized FhaC gene from B. pertussis

[0189]

[0190] SS = signal sequence, His = His x 8 tag, Avi = AviTagTM peptide tag

[0191] The sequence C'fhacAH P') was engineered such that the FhaC signal sequence, Hl helix, and linker were deleted and replaced with a BamA signal sequence, a His-tag, and an Avi-tag attached in frame at the N-terminus of the POTRA domain. The jhacAHl gene flanked by Ndel / SphI sites was inserted into a generic vector pUCIDT(Kan), resulting in the plasmid pMTDS33. To subclone fhaCAHl to the expression vector pSCRhaB2 (Cardona and Valvano, 2005), pMTDS33 was digested with restriction enzymes Ndel and Sphl-HF (see Restriction Enzyme Digestions section above), the / Ar / cG / TV-containing fragment was isolated by gel extraction (see DNA Gel Electrophoresis and Gel DNA Extraction sections above), and were ligated to similarly digested pSCRhaB2, forming pMTDS39.

[0192] Construction of an Endogenous Pl peptide Producing Secretion Assay Vector

[0193] Gene synthesis of an E.coli codon-optimized gene encoding the Pl peptide was constructed by IDT (Table 4).

[0194] Table 4. Codon-optimized Pl peptide gene

[0195] SS = signal sequence

[0196] The sequence was engineered by inserting an Pl -encoding synthetic gene (containing a bamA signal sequence) downstream of the FhaBsec ORF to create a synthetic operon for the endogenous production of FhaC, FhaBsec, and Pl altogether. The pl gene flanked by Xbal / Hindlll sites was inserted into a generic vector, pUCIDT(Kan), resulting in the plasmid pMTDS246. To subclone pl to the secretion assay vector (pRJ72), pMTDS246 was digested with restriction enzymes Xbal and Hindlll (see Restriction Enzyme Digestions section above), the pl containing fragment was isolated by gel extraction (see DNA Gel Electrophoresis and Gel DNA Extraction sections above), and were ligated to similarly digested pRJ72, forming pMTDS263.

[0197] Bacterial Transformations

[0198] 50 pL aliquots of chemically-competent E. coli were obtained from NEB and were thawed on ice. 1 pL purified plasmid (or 5 pL of a ligation reaction) was then added to the bacteria and incubated (for 30 minutes). Bacteria was then heat-shocked at 42 °C for 30 sec and incubated on ice 5 minutes. 950 pL of recovery media (either SOC outgrowth medium or LB supplemented with 0.2 % (w / v) glucose) was then added and mixture was left to incubate at 37 °C for 30 min (for ampicillin-resistance plasmids) or 60 mins before being plated by the spread-plate method.

[0199] General Protein Techniques

[0200] TCA Precipitation

[0201] Bacterial samples were centrifuged (3,220 x g, 4 °C, 10 minutes) and resuspended in 500 pL lx PBS before being precipitated. Bacteria were precipitated by adding phenylmethylsulfonylfluoride (PMSF) at a final concentration of 0.4 mM PMSF and trichloroacetic acid (TCA) at a final concentration of 10% (v / v), and incubated on ice for 10 minutes. Precipitates were then pelleted (20,817 x g, 4 °C, 10 minutes), washed twice with 600 pL acetone, and air-dried (for 15 minutes). For culture supernatant samples, the same concentration of PMSF / TCA was used and incubated for 1 hour before pelleting (5,000 x g, 4 °C, 10 minutes), and being washed 2x with acetone as above.

[0202] Separation of Protein Samples by SDS-PAGE

[0203] Unless otherwise stated, TCA-precipitated protein samples were resuspended in 2x SDS protein gel loading solution using normalised volumes based on the final ODeoo reading of the subculture where the sample was taken from [volume, pL = ODeoo x 200], Samples were then boiled (at 99 °C) for 15 minutes with shaking (1200 rpm). Protein samples were then resolved by SDS-PAGE which was conducted using an 8-16% Tris-Glycine gradient protein gel and lx Tris / Glycine / SDS as a running buffer. Size marker used was Chameleon® Duo Pre-stained ladder. Gels were run at 25 °C (unless otherwise stated) at a constant voltage of 150 V for the required amount of time (typically 1 - 2 h). Coomassie Staining

[0204] SDS-PAGE gels were immediately immersed in Coomassie stain overnight. Stained gels were then immersed in destaining agent for as long as required, and rehydrated with MQ water to its full size before being scanned. All Coomassie-stained gels were imaged with an Odyssey infrared imager (Licor, model 9120) using maximum quality and resolution settings and a 700 nm laser. Western Blots

[0205] Western blot was conducted as previously described according to standard protocols (Doyle and Bernstein, 2019). Briefly, after SDS-PAGE, gels were immediately transferred to nitrocellulose membranes using iBlotll transfer devices. Immunoblotting buffer (Odyssey Blocking Buffer (Li-Cor) diluted at a 1 : 1 ratio with PBS and supplemented with 0.01% Tween-20) was used for all blocking and antibody incubations, PBS supplemented with 0.01% Tween-20 (PBS-T) for initial washes, and PBS for final washes. Membranes were blocked overnight, incubated with 1° antibody (1 :5,000-10,1000 dilution) for 4 h, washed (3 x 1 min with PBS-T), incubated with 2° antibody (1 :5,000 dilution) for 2 h, before finally washing (2x with PBS-T, 3x with PBS), and being air-dried. Otherwise, membranes for double-antibody immunoblots were blocked for 1 h, then incubated overnight at room temperature with 1° antibody and 2 h with 2° antibody before final washes (with PBS-T (x 2) and PBS (x 3)). Membranes were then air-dried. Mouse monoclonal anti -His was obtained from Genscript. Mouse monoclonal anti-Strep was obtained from Qiagen. Goat anti-mouse Ig 800CW IRDye and Goat anti-rabbit (2° Antibody) were both obtained from Licor. All other Rabbit polyclonal antisera were raised by us. All dried membranes were imaged with an Odyssey infrared imager (Licor, model 9120) using maximum quality and resolution settings. Pixel intensities were measured using Fiji software (v2.9.0).

[0206] Purification of FhaCAHl

[0207] Large-Scale Culture

[0208] E. coli B strain BL21(DE3) transformed with pMTDS39 was grown overnight at 25 °C with shaking (250 rpm) in LB media supplemented with 50 pg / mL trimethoprim. Four Ultra Yield® flasks containing 1 L LB each and supplemented with 0.2% L-rhamnose were seeded with bacteria to a starting ODeoo of 0.05. These subcultures were then incubated at 25 °C for 24 hours.

[0209] Bacterial Cell Harvesting, Lysis, and Membrane Collection

[0210] Cells were harvested by centrifugation (5,000 x g, 4 °C, 10 minutes), washed in 20 mL ice-cold PBS, and resuspended in 100 mL ice-cold PBS containing 1 mM EDTA-free SigmaFast protease inhibitors (Sigma Aldrich). Cell suspensions were then lysed using a Constant Systems cell disruptor at a pressure of 30 kpsi. Lysate was then collected and centrifuged (15,000 x g, 10 min, 4 °C) to pellet undisrupted cells. The supernatant was then aliquoted and centrifuged (60,000 rpm, 4 °C, 40 minutes) to isolate the inner and outer membranes (“whole membrane”).

[0211] Solubilisation and Purification of FhaCAHl

[0212] Whole membrane pellets (WMP) were gently washed with 20 mL ice-cold PBS and then homogenised into 40 mL DDM solubilisation buffer supplemented with EDTA-free SigmaFast™ protease inhibitors using a Dounce homogeniser. WMP were incubated at 4 °C for 4 hours with constant rotation. The solution was then centrifuged (70,000 rpm, 4 °C, 30 minutes) to remove insoluble material and the supernatant was transferred to a tube containing 1 mL nickel-nitrilotriacetic acid (Ni-NTA) agarose beads equilibrated in IMAC / DDM wash buffer, and the mixture was rotated at 4 °C overnight.

[0213] Beads were collected in a gravity flow column, and washed with 3 x 10 mL IMAC / DDM wash buffer. FhaCAHl was then eluted in 2.5 mL of imidazole elution buffer. Imidazole was removed from FhaCAHl and exchanged into TNiow-DDM buffer using a PD- 10 desalting column following manufacturer’s protocol. Proteins were then concentrated using an Amicon Ultra-0.5 Centrifugal Filter Unit, 10K MWCO. Concentrated proteins were then aliquoted, frozen in liquid nitrogen, and stored at -80 °C. Absorbance at 280 nm (A280) of the purified sample was taken using a Nanodrop ND- 1000 (Thermo Scientific) and concentration was calculated by the predicted extinction coefficient (s).

[0214] Heat-Modifiable Gel Mobility Shift Assay To test if the P-barrel domain of purified FhaCAHl exhibits resistance to unfolding by SDS, the assay was conducted as in previous works (Hanson et al., 2023). Briefly, FhaCAHl was diluted 1 : 10 in ice-cold TNiow-DDM buffer, and then aliquots were further diluted 1 :9 in modified loading buffer on ice. Aliquots were then incubated at 0, 20, 40, 60, 80, or 99 °C for 10 minutes. Proteins were then immediately resolved by cold SDS-PAGE (gel tank embedded in packed ice, ran at 150 V, in a 4 °C cold room). Proteins were detected by Coomassie or western blotted.

[0215] Tryptophan Fluorescence Assay

[0216] To probe FhaCAHl folding via tryptophan fluorescence, assays were conducted as previous (Hanson et al., 2023). Briefly, purified FhaCAHl was diluted in 4 M G-PBS-DDM to a final concentration of 0.75 pM, and incubated at 25 °C for 24 h. The protein was then aliquoted into a clear-bottomed, black 96-well tray (200 pL / well) alongside freshly prepared solutions of 0.75 pM protein in PBS-DDM. The tray was then equilibrated at 25 °C for 1 hour and then read using a FlexStation® 3 multi-mode microplate reader using an excitation wavelength of 295 nm (5 nm bandwidth) and an emission range of 313-400 nm. Chymotrypsin Digestion

[0217] To monitor the resistance of the purified protein to digestion by protease due to folded states. Chymotrypsin was added to FhaCAHl at either 1 pg / mL : 150 pg / mL; or 1 pg / mL : 15 pg / mL (protease : protein) in TNiow-DDM buffer (at a final volume of 30 pL) and incubated for 0, 2, 10, and 30 minutes. At each timepoint, corresponding samples were stopped by TCA precipitation. Precipitates were resuspended in 10 pL of 2x SDS protein loading dye, and boiled at 99 °C (with 1,200 rpm shaking). Samples were then separated by SDS-PAGE and Coomassie-stained or western blotted. After image analysis (see Image Analysis section below), FhaCAHl remaining was calculated in GraphPad Prism vl0.0.2 (232) with the equation

[0218] Screening for Peptide Binders Using mRNA-Display

[0219] 100 ng of FhaCAHl was used to conduct mRNA-display to discover macrocyclic peptide binders and subsequently synthesise them as previously described (Passioura and Suga, 2017, Norman et al., 2021, Tsiamantas et al., 2001).

[0220] Design and Synthesis of Peptide Binders

[0221] A peptide Pl was also designed to bind to FhaC POTRA domain 1 based on the crystal structures of FhaC. All peptides, including Pl and peptides derived from Pl, were synthesised by Genscript or in-house using widely used protocols, for example, as described in Norman et al., 2021.

[0222] FhaB Secretion Assay in an E. coli Model

[0223] To test whether candidate peptides can inhibit the secretion function of FhaC, a FhaB secretion assay was developed. The plasmid pRJ72 was transformed into E. coli B strain BL21(DE3), forming the strain MTDS110. The E. coli strain MTDS110 was cultured at 37 °C in LB media overnight. Bacteria were then subcultured to an ODeoo of 0.05 and grown for 2 hours before adding rhamnose (at 0.2% final concentration) to induce FhaBsec and FhaC expression. At the same time as rhamnose addition, peptide binders were also added at the required final concentration. Subcultures were then grown for another hour after rhamnose and peptide addition. An ODeoo reading was then taken. The remaining bacterial cultures were centrifuged (3220 x g, 25 °C, 10 mins), the supernatants collected, filter-sterilised (with a 0.22 pm filter) and aliquoted (5 mL). Aliquots were TCA-precipitated and air-dried precipitates were then resuspended in 40 pL of 2x SDS Protein Loading Dye, boiled (1200 rpm, 99 °C, 15 min), and separated by SDS-PAGE. Gels were then Coomassie-stained. After image analysis, within-lane intensity values of the band corresponding to FhaBsec were quantified and statistical tests were conducted in GraphPad Prism vl0.0.2 (232) by comparing band intensity of each treatment groups to their respective vehicle control values.

[0224] For the time-course version of this assay, 25 mL samples were used. Samples were instead taken at 0, 15, 30 and 60 minutes after adding 0.2% rhamnose and peptide (at required concentrations). Samples were processed as above, then supernatants were taken and standardised to 2 mL each before being TCA-precipitated (as above).

[0225] For the endogenous Pl -inserted variant of this assay, the assay was conducted as above but only with the addition of rhamnose (at 0.2% final concentration) to induce FhaBsec, FhaC, and Pl expression together (no externally added peptide added). Samples were processed as above.

[0226] Chinese Hamster Ovary (CHO) cell infection assay

[0227] FhaB is required for Bordetella to adhere to eukaryotic cells. It was hypothesized that treatment with Pl-CPP inhibits FhaB secretion by FhaC and would reduce the ability of B. bronchiseptica to adhere to eukaryotic cells in vitro.

[0228] Chinese hamster ovary cells (CHO-K1, ATCC # CCL-61) were seeded at 3xl05cells per well on sterile #1.5 coverslips in 6 well tissue culture plates for 18 hours.

[0229] Wildtype Bordetella bronchiseptica RB50 (Cotter and Miller, 1994) and the isogenic EfhaB strain were grown overnight in Stainer Scholte (SS) media (Stainer and Scholte, 1970; Hulbert and Cotter, 2009) containing 50 mM MgSC which inactivates the BvgAS two component regulatory system and prevents expression of fhaB, fhaC, and all other known Bordetella virulence proteins. We included the EfhaB strain as a control for bacteria with adherence defects. The bacteria were washed twice with sterile DPBS to remove MgSC and resuspended to an OD of 0.02 OD600 in SS broth without MgSC (BvgAS activating conditions) to allow expression of fhaB and fhaC. Wildtype bacteria were treated with either 10 pM of the Pl-CPP in DMSO or the same volume of pure DMSO as a vehicle control.

[0230] Bacteria were treated at 37 °C for 15 or 30 minutes before beginning the adherence assay. Bacteria were diluted into CHO cell media (Ham’s F12 + 10% FBS) such that 100 colony forming units (cfus) were added for every CHO cell in the well (Multiplicity of Infection 100). The CHO cells were inoculated in the 6 well plates and the plates were spun for 5 minutes at 500 xg to synchronize bacterial interactions with the CHO cells. After 30 minutes of incubation at 37 °C 5% CO2 the inoculation media was removed and the infected cells were washed three times with 2mL of DPS to remove non-adherent bacteria.

[0231] The cells were fixed with ice cold methanol and stained with Giemsa stain following manufacturer instructions (Sigma Aldrich) and stained cells on coverslips were mounted onto slides using prolong diamond antifade mounting media and imaged using an inverted fluorescence microscope (Keyence).

[0232] Image Analysis

[0233] Pixel intensities were measured using Fiji software (v2.9.0).

[0234] Results

[0235] Purification of FhaC

[0236] To discover inhibitors against FhaC, a pure FhaC sample was needed as a target for screening potential binders. A derivative of FhaC was designed that has its linker and a-helix 1 (Hl) replaced with an N-terminal His-tag attached in-frame to the POTRA1 domain, forming “FhaCAHl” (Fig. 1A). Without wishing to be bound by theory, the Inventors hypothesised that removing the Hl -linker domain increases the probability of peptide binders to bind inside the lumen of FhaC, which is the path of FhaB translocation. Furthermore, Hl exits the barrel pore and moves toward the periplasm when secretion is initiated. The Inventors hypothesised that strong lumen binders will occupy the pore in the barrel domain of FhaC and thus block the secretion pathway.

[0237] The gene that encodes FhaCAHl was codon-optimised for A. coli (Table 3) and ligated into a rhamnose-inducible expression vector pSCRhaB2 (Cardona and Valvano, 2005), which was then transformed into E. coli B strain BL21(DE3). The strain was then grown in 4 x 1 L cultures of LB supplemented with rhamnose overnight. Whole membrane pellet (WMP) was isolated (see Bacterial Cell Harvesting, Lysis, and Membrane Collection section above), solubilised in DDM detergent, and subjected to immobilised-metal ion affinity chromatography (IMAC) to isolate FhaCAHl. This process yielded a total of 219.6 pg of FhaCAHl (at a final concentration of 15.6 mM) from 4 L of culture.

[0238] A Coomassie staining was conducted to evaluate purity of the purification fractions and the final product (Fig. IB and C). A single band was present in the elution fraction which corresponds to the expected size of FhaCAHl - 58.8 kDa (Fig. IB). The concentrated sample indicates that the final FhaCAHl product was -99% pure. The same band was observed when the same sample was probed by western immunoblot using an anti-His antibody (Fig. 1C).

[0239] Assessing that FhaCAHl is Folded

[0240] To confirm whether FhaCAHl is correctly folded, a heat-modifiability assay was conducted which exploits the unique feature of P-barrel domain proteins (Fig. 2A) which intrinsically resist unfolding by SDS when properly folded and therefore migrate faster on SDS-PAGE. However, when unfolding is induced by heating, they migrate slower in SDS- PAGE. As expected, a faster migrating band was observed at -38 kDa (which corresponds to folded FhaCAHl) below the expected FhaCAHl band at - 58 kDa (which corresponds to unfolded FhaCAHl) in unheated samples (0, 20 °C) (Fig. 2B). However, as samples were heated to higher temperatures (40, 60 °C) we observed reduced intensity on the folded band, and an increased intensity on the unfolded band of FhaCAHl. Interestingly, bands that correspond to unfolded and folded FhaCAHl were observed at a roughly equal ratio in unheated samples. This was expected and it pertains to the variability in the degree of intrinsic resistance to SDS in different P-barrel proteins. These results suggested that the barrel domain of the purified FhaCAHl is properly folded.

[0241] To further assess folding of the purified FhaCAHl, a tryptophan fluorescence assays was conducted (Hanson et al., 2023). The assay measures the environment of the protein’s tryptophan residues (e.g., solvent exposure if unfolded). The FhaCAHl construct possesses 7 tryptophan residues (Fig. 2A), which, when the protein is folded properly, will be in nonpolar environments. In each of these cases, when these tryptophan residues are excited with a 295 nm laser, they produce a significantly different emission spectra. As expected, the purified protein sample produced a strong emission spectrum with a maxima at 332 nm (i.e., the maxima when properly folded) (Fig. 2C). Furthermore, a weaker emission spectrum with a “red-shifted” maxima was produced when the purified protein sample was deliberately unfolded with guanidine (Fig. 2C). These results also suggest that the purified protein is folded correctly.

[0242] To further validate whether FhaCAHl was correctly folded, the resistance of the purified FhaCAHl towards digestion by chymotrypsin (CT) was investigated. The assay is based on the concept that chymotrypsin is a protease that digests proteins specifically at their aromatic residues. Aromatic residues are typically protected, enclosed within the folded protein’s hydrophobic core or by outer membrane lipids. In concept, the FhaCAHl protein should be encased by DDM micelles (Fig. 1A) which shields these aromatic residues from chymotrypsin digestion. When samples were western blotted with anti-His, it revealed that the band that corresponds to FhaCAHl decreased in intensity and numerous His-tagged lower molecular weight bands (corresponding to FhaCAHl fragments) increased in intensity overtime in both 15: 1 and 150: 1 ratio (FhaCAHl :CT) (Fig. 2D). Two bands that corresponds to FhaCAHl fragments (denoted as fragment “a” and “b”) were also prominently found at molecular weight sizes ~30 and ~25 kDa, respectively. Both fragments carry a His-tag and, based on the sizes, the Inventors hypothesise that this would contain a partially-digested part of the P-barrel given its size and most likely was digested at the extracellular loops given its exposure and abundance on the extracellular loops (Fig. 2A). Nevertheless, the appearance of two strong and otherwise CT-resistant fragments strongly suggested that FhaCAHl was properly folded.

[0243] Discovery of Peptide Binders of FhaC

[0244] Peptides Obtained from mRNA-Display on FhaCAHl

[0245] To discover macrocyclic peptide binders of FhaC, a sample of the purified FhaCAHl was used as a target for mRNA-display screens. mRNA-display is a method to screen a library of 1012peptides for binders to purified targets. Multiple sequence alignment of 31 macrocyclic peptide binders of FhaCAHl is shown in Fig. 3.

[0246] Further Peptide Binders

[0247] A candidate peptide Pl was also identified (Table 5) which binds to the POTRA1 domain (Fig. 4).

[0248] Table 5. Peptide Sequences

[0249] The peptide is designed to occupy the hydrophobic substrate binding grooves of the TpsB P0TRA1 domain. However, given the periplasmic location of the POTRA domains, these two peptides would need to be able to cross the outer membrane. Therefore, a second version was synthesised with a cell penetrating peptide (CPP) (Yamamoto et al., 2022), forming Pl -CPP (Table 5).

[0250] Development of a FhaB Secretion Assay

[0251] A novel secretion assay was then developed to test whether the peptide binders can inhibit the secretion function of FhaC. A strain of E. coli carrying the genes encoding FhaC and a truncated hyper-secreting form of FhaB (FhaBsec) under the control of a rhamnose- inducible promoter was constructed (Fig. 5A).

[0252] The sequence of the truncated hyper-secreting form of FhaB (FhaBsec) is represented by SEQ ID NO: 56 below:

[0253] MNTNLYRLVFSHVRGMLVPVSEHCTVGNTSCGRTRGQARSGARATSL SVAPNALAWALMLACAGLPLVTHAQGLVPQGQTQVLQGGNKVPVV NIANPNSGGVSHNKFQQFNVANPGVVFNNGLTDGVSRIGGALTKNPN LTRQASAILAEVTGTSPSRLAGTLEVYGKGADLIIANPNGISVNGLSTL NASNLTLTTGRPSVNGGRIGLDVQQGTVTIERGGVNVTGLGYFDVVA RLVKLQGAVSSEQGKPLADIAVVAGANRYDHATRRATPIAAGARDAA AGAYAIDGTAAGAMYGKHITL VS SDSGLGVRQLGSLS SPS AITVS SQG EIALGDATVQRGPLSLKGAGAVSAGKLASGGAVRVAGGGAVKIASAS SVGNLAVQGGGKVQATLLNAGGTLQVSGRQAVQLGTASSRQVLSVN AGGALKADQLSATGRLEVDGKQAVTLGSAASRNALSVRAGGALGA WSHPQFEKGGGSGGGSGGSAWSHPQFEK

[0254] The sequence of wildtype FhaC is represented by SEQ ID NO: 61 and the wildtype sequence of FhaB is represented by SEQ ID NO: 62. As shown in Fig. 5B, when the secretion of FhaBsec by FhaC was induced by the addition of rhamnose, a strong band corresponding to FhaBsec could be easily detected in culture supernatants by Coomassie staining (Fig. 5B) and via anti-StrepII western blot (Fig. 5C) (as the FhaBsec has a C-terminal StrepII tag). This shows that the assay is a viable way of testing FhaC activity.

[0255] Screening Peptides for FhaC Inhibition Activity

[0256] The above assay was then used to test whether the identified peptide binders are bioactive and inhibit the secretion function of FhaC. All putative anti -FhaC linear peptides were first screened (Pl, Pl-CPP). For negative inhibition controls, an expression vector control, vehicle control (DMSO and H2O), and a CPP -treated culture (to account for CPP activity) were used.

[0257] At a concentration of 10 pM the peptides Pl and Pl-CPP were able to significantly inhibit FhaC and reduced FhaBsec secretion (Fig. 6A and B). Pl and Pl-CPP were both designed to target the POTRA1 domain and, without wishing to be bound by theory, the Inventors theorise that the strong inhibition of FhaC’s secretion function is due to Pl and Pl- CPP blocking the recognition site where FhaB and FhaC initially interact. The fact that Pl was able to inhibit FhaC’s secretion function in the absence of a terminal CPP sequence, strongly suggests that Pl can intrinsically cross the outer membrane. The pl of Pl is 9.5 and has high hydrophobicity which supports the hypothesis.

[0258] Additionally, it is also observed that Pl-CPP treatment negatively impacts the bacterial cell, where at lOpM it causes proteins to leak into the supernatant (Fig. 6A), indicative of cell lysis or membrane disruptions.

[0259] The macrocyclic peptides derived from mRNA-display were then tested to see whether they are bioactive and can inhibit FhaC’s secretion function. Peptides RD-4 (SEQ ID NO: 8), RD-6 (SEQ ID NO: 7), and RD-7 (SEQ ID NO: 6) were able to reduce FhaBsec secretion by ~ 50-75% when compared to their vehicle control (DMSO) (Fig. 6A and B). RD-4 and RD-6 are macrocycle that possesses several hydrophobic residues (Fig. 7). Meanwhile, RD-7 is a linear peptide with a small, macrocyclic N-terminal head and carries mainly positively-charged residues and hydrophobic residues (Fig. 7). Without wishing to be bound by theory, the Inventors theorise that these peptides can cross the bacterial outer membrane if they are POTRA-acting or, alternatively, the peptides might act by binding from the cell surface onto the surface loops of FhaC or by obstructing the lumen of FhaC. To determine the minimum concentrations where P1 / P1-CPP were active, the abovementioned secretion assay was used to test different concentrations of each peptide. Pl was able to significantly inhibit FhaBsec secretion down to a final concentration of 10 pM (p

[0260] < 0.01) (Fig. 8A (Left) and B). Pl-CPP was able to significantly inhibit secretion down to a concentration of 0.8 pM (p < 0.05) (Fig. 8A (Right) and C). These data points were well fit by a sigmoidal function allowing us to calculate the IC50 of peptides Pl and Pl-CPP as 11.2 pM and 0.69 pM, respectively.

[0261] To determine whether reduction in FhaBsec secretion by Pl-CPP treatment also causes accumulation of FhaBsec in the bacterial cell, the same secretion assay was conducted but the whole cell pellet sample was taken instead of the supernatant. Pl-CPP treatment significantly increased levels of FhaBsec associated within the cell pellet (Fig. 9; p < 0.05).

[0262] Isothermal Calorimetry Data

[0263] The isothermal calorimetry (ITC) assay was used to confirm direct binding of peptide Pl-CPP to the POTRA domains of FhaC. For negative controls, a peptide-only control was conducted where an equivalent amount of Pl-CPP is injected to the cell (buffer without protein).

[0264] The assay shows that each injection of Pl-CPP into the cell (containing FhaC POTRA1-2) produces exothermic heat (negative peaks; Fig. 10A, Right). Next, the PEAQ- ITC Analysis Software was used to fit the raw heat change data to the “one site” model, producing a logarithmic binding curve. From the curve, thermodynamic parameters where extracted using the software (Fig. 10B and C). Data shows that the Pl-CPP:POTRAl-2 binding event occurs spontaneously (AG < 0) and has favourable hydrogen and hydrophobic bonds (AH < 0, -TAS < 0).

[0265] Furthermore, binding event is largely dominated by hydrophobic interactions (A-TAS

[0266] < AH; Fig. 10B and C) which also correlates with the hypothesis that the Pl-CPP peptide binds to the hydrophobic domains of the POTRA1 (Fig. 4).

[0267] Development of a Peptide Activity Probing Secretion Assay

[0268] The above assay was used to test whether peptide Pl can be expressed by the test strain itself and is bioactive. For negative inhibition controls, the original secretion assay construct (Fig. 5A) was used. The endogenously produced Pl (Fig. 11 A) was able to inhibit FhaC and significantly reduced FhaBsec secretion (Fig. 11B and C, Fig. 19). This finding further solidifies the evidence that Pl can inhibit FhaC’s secretion function, in the absence of a terminal CPP sequence, and when present at 1 : 1 peptide:target stoichiometry. This construct can be used to screen for Pl derivatives with improved activity (through site-directed or random mutagenesis of the Pl sequence within the expression vector).

[0269] Screening Peptides to Probe the Pharmacophore of Pl

[0270] To probe for the regions required for Pl’s inhibition activity, several Pl-CPP variants was tested on the original secretion assay (as in Fig. 5). All variants of the Pl-CPP peptide were screened (Table 6). For negative inhibition controls, a vehicle control (DMSO) was used. For positive inhibition controls, a Pl-CPP -treated culture was used.

[0271] Table 6. Peptide Sequence and Properties of Pl-CPP Compared to Variants Designed

[0272] The inventors theorise that the strong inhibition of FhaC’s secretion function by Pl- CPP is due to its binding to the hydrophobic cleft of FhaC P0TRA1 (Fig. 12C, Left and Middle) and that the removal of the first 6 hydrophobic residues would remove its activity (Fig. 12C, Right). Pltmnc-CPP (P I M-CPP) has the first 6 residues (VAAMAA) of the Pl peptide removed (i.e., the N-terminal residues) (Fig. 12A) which has no change on the charge (pl) of the peptide but does increase its hydrophilicity (Table 6; GRAVY score 0.213 to - 0.287). Pltrunc-CPP (PI NI-CPP) lost inhibition activity at 2 pM (Fig. 12A and B). This also strongly corroborates the ITC data where binding is predicted to be largely driven by hydrophobic interactions (Fig. 10B and C).

[0273] Next, several charge-reversed variants of Pl were designed (PIRBE-CPP, PIEISR-CPP, P1R13E,E15R- CPP) to test the effects of charge on the activity of Pl (Fig. 13A). Pl RBE-CPP had a reduced activity compared to Pl-CPP, although still reduced FhaBsec secretion by -30% compared to untreated (Fig. 13B and C). Both PIEISR-CPP and P1RI3E,EI5R-CPP retained inhibition at 2 pM (Fig. 13B and C).

[0274] To determine the minimum inhibitory concentrations where PIRBE-CPP, PIEISR-CPP, P1RI3E,EI5R-CPP were active, the abovementioned secretion assay was used to test different concentrations of each peptide (Fig. 14). A best-fit sigmoidal curve was then plotted onto these data points to reveal the IC50 of peptides PIRBE-CPP, PIEISR-CPP, and P1RI3E,EI5R-CPP as 1.29 pM, 0.59 pM, and 0.77 pM, respectively (Fig. 14B, C, and D). Notably, PIEISR-CPP has improved activity as compared to Pl-CPP (IC50 0.59 vs. 0.69, respectively).

[0275] Testing peptides in B. bronchiseptica

[0276] Next, the efficacy of the peptides to inhibit the FhaB secretion in B. bronchiseptica was investigated. Treatment of B. bronchiseptica with 10 pM of Pl-CPP peptides reduced the release of ACT and FhaB into the culture supernatant fraction, demonstrating that these peptides were effective in reducing release of exoproteins in B. bronchiseptica (Fig. 15). Probing of B. bronchiseptica treated with 10-20 pM of Pl-CPP or PIEISR-CPP also showed that there was significantly reduced expression levels of FhaB in the bacterial cells indicating complete inhibition of FhaC, similiarly to the phenotype of AfhaC knockout strain (Fig. 16)

[0277] A Chinese Hamster Ovary (CHO) cell adhesion assay was then used to determine whether Pl-CPP treatment prevents B. bronchiseptica from adhering to CHO cells. B. bronchiseptica treated for 15 or 30 minutes with Pl-CPP exhibited reduced specific binding to the CHO cells and also formed bacterial aggregates that are not otherwise exhibited during the characterized infection process (Fig. 17).

[0278] Pl: FhaC binding simulations.

[0279] The binding of Pl to FhaC POTRA1 was simulated in molecular dynamics simulations (Fig. 18). In these experiments the structure of Pl remained highly constrained and stably associated with the TpsA binding groove of P0TRA1 throughout the period of the simulation.

[0280] EXAMPLE 2

[0281] Materials and Methods

[0282] Bacteria and Growth Conditions

[0283] E. coli B strain BL21(DE3), B. bronchiseptica strain RB50 and B. pertussis strain LI 423, LI 728, and L2228 were used.

[0284] For A. coli, experiments were conducted on subcultures of a 10-mL overnight culture of the respective strains (from a single colony). E. coli transformed with appropriate plasmids were grown in lysogeny broth (LB, Lennox) containing selection agents at their appropriate concentrations (50 pg mL'1trimethoprim, 50 pg mL'1kanamycin, or 100 pg mL'1ampicillin). For constructs controlled by a PRIM promoter, expression was induced with 0.2% L-rhamnose (w / v) and repressed with 0.2% glucose (w / v). For constructs controlled by a PTK promoter, expression was induced with 0.4 mM of IPTG and repressed with 0.2% glucose (w / v).

[0285] For B. bronchiseptica, experiments were conducted from a 10 mL overnight culture in Stainer-Scholte (SS) media from a single colony (unless otherwise indicated). B. bronchiseptica were grown overnight (in BvgAS+ conditions), washed, resuspended in media and then used for experiments.

[0286] For B. pertussis, experiments were conducted from a 10 mL overnight culture in Stainer-Scholte (SS) media containing TJIS supplement and from a single colony. Overnight cultures are washed twice (to remove heptakis), resuspended in SS media with added TJIS supplement (without heptakis) and then used for experiments.

[0287] Plasmid and Strain Construction

[0288] Plasmids and strains used are listed in Tables 7 and 8, and the oligonucleotides and dsDNA gene blocks used to construct plasmids are listed in Tables 2-4 and 9. To construct pMTDS263, pRJ72 was digested with restriction enzymes Hindlll and Xbal. mtdl85 was similarly digested with Hindlll and Xbal. The parent template and theEP1 -containing (from mtdl85) encoding fragments were then purified by agarose gel electrophoresis and T4 ligated. To construct pMTDS630, pRJ64 were digested with restriction enzymes Ncol and Xbal. pMTDS33 was similarly digested with Ncol and Xbal. The parent template and theHlsFhaC-containing (from pRJ64) fragments were then purified by agarose gel electrophoresis, and T4 ligated. Plasmids expressing deletion mutants of pMTDS263 (pMTDS608) were constructed via Gibson Assembly (with primers mtd363 / 372 and mtd364 / 371). For deletion mutants of pMTDS323, pMTDS497 (AHL) was constructed by Q5 site-directed mutagenesis (with primers mtd227 / 228) while pMTDS607 (AH) was constructed via Gibson Assembly (with primers mtd363 / 370 and mtd364 / 369). To make pMTDS323, site-directed mutagenesis was conducted using Q5 to insert a His-tag (with primers mtdl91 / 192) into pRJ72.

[0289] Table 7. Bacterial strains Abbreviations: TS = TwinStrepII-tag, His = His x 8 or 10 tag, BamAss= BamA signal sequence,EP1 = Codons encoding BamAss- Pl peptide (VAAMAAEASARARAEFAAR), Hl = FhaC Helix 1 (31-63), Hl-L = FhaC Helix 1 and linker (31-86), ApR= ampicillin resistance, TpR= trimethoprim resistance, SS = signal sequence, Hisio = His x 10 tag.

[0290] 1. Cotter and Miller 1994. 2. Octavia et al. 2012.

[0291] Table 8. Additional Plasmids.

[0292] Abbreviations: TS = TwinStrepII-tag, IPTG = Isopropyl P-D-l -thiogalactopyranoside, His = His x 8 or 10 tag, BamAss= BamA signal sequence,EP1 = Codons encoding BamAss - Pl peptide (VAAMAAEASARARAEFAAR), Hl = FhaC Helix 1 (31-63), Hl-L = FhaC Helix 1 and linker (31-86), ApR = ampicillin resistance, TpR = trimethoprim resistance, SS = signal sequence, HislO = His x 10 tag.

[0293] Table 9. Single-stranded Oligonucleotides

[0294] Abbreviations: Hl = FhaC Helix 1 (31-63), Hl-L = FhaC Helix 1 and linker (31-86), TpR= trimethoprim resistance, GA = Gibson Assembly method,EP1 = Codons encoding BamAss- Pl peptide (VAAMAAEASARARAEFAAR), SS = signal sequence, Hisio = His x 10 tag.

[0295] Western Immunoblotting

[0296] Western blot was conducted as above. Briefly, after proteins were separated by SDS- PAGE on 8-16% Tris-glycine gels (Invitrogen) and transferred to nitrocellulose membranes using an iBlotll (Life Technologies). Immunoblotting buffer [1 : 1 Odyssey Blocking Buffer (Li- Cor):PBS + 0.01% Tween-20] was used for all blocking and antibody incubations, PBS-T (PBS + 0.01% Tween-20) for initial washes, and PBS for final washes. Monoclonal mouse anti- StrepII and anti -His antibodies were obtained from Genscript (catalogue number A00186 and A01732, respectively). Secondary antibody goat anti -mouse 800CW IRDye® was obtained from Li-Cor (catalogue numbers 926-32210). Membranes were blocked overnight, incubated with 1° antibody (aHis, aStrep - 1 : 5,000 dilution; aDegP - 1 : 10,000 dilution) for 4 h, washed with PBS-T (3 x 1 min), incubated with 2° antibody (1 :5,000 dilution) for 2 h, before final washes (2 x PBS-T, 3 x PBS). Membranes were then air-dried at 37 °C for 20 min. Membranes were then imaged as below.

[0297] Image Analysis

[0298] All Coomassie-stained gels and western blot membranes were imaged with an Odyssey infrared imager (Li cor, model 9120) using maximum quality and resolution settings and a 700 nm laser (for Coomassie-stained gels and goat anti-rabbit. Pixel intensities were measured using Fiji software (v2.14.0 / 1.54f). FhaBsec Secretion Assay

[0299] E. coli strain MTDS110 was cultured at 37 °C in LB media overnight. Bacteria were then subcultured to an ODeoo of 0.05 and grown for 2h. Subcultures were then supplemented with appropriate concentrations of treatments and followed immediately by adding 0.2% L- rhamnose (w / v) to induce expression of FhaBsec and FhaC. Subcultures were then grown for another hour. An ODeoo reading was taken, and the remaining bacterial cultures were centrifuged (3,220 x g, 10 min, 25 °C). The supernatant was then isolated, filter-sterilised (with 0.22 pm filter), and aliquoted (5 mL). Aliquots were then TCA-precipitated (0.4 mM PMSF, 10% TCA (v / v)) and air-dried (20 min, 37 °C). Air-dried precipitates were then resuspended in 40 pL of 2x SDS Protein Loading Dye (100 mM Tris-HCl pH 8, 20% glycerol (v / v), 0.2% bromophenol blue (w / v), 4% SDS (v / v)). Gels were then Coomassie-stained. After image analysis, within-lane intensity values of a band corresponding to FhaBsec were quantified and normalised to their respective mock-treated (vehicle control) pair for each replicate. Appropriate statistical tests were then conducted in GraphPad Prism vl0.3.0 (461) to compare each treatment group’s relative FhaBsec secretion (relative to mock-treated) and their respective mock-treated control values.

[0300] The assay was conducted as above for experiments involving the endogenously expressed Pl peptide (EP1), but the respective strain and plasmid (pMTDS263) was used instead. For assays involving the truncated variants of FhaC (AH and AHL), the strains and plasmids (pMTDS607 and pMTDS497, respectively) were used instead.

[0301] FhaC Expression Assays

[0302] To determine if treatment affected the abundance of FhaC protein levels in the presence of FhaBsec expression (or not) when treated with Pl, the strain MTDS327 (which contains pMTDS323) and MTDS630, respectively, were used. Assay was conducted as in the secretion assay described previously, but the whole cell sample are taken instead. Briefly, strains were cultured at 37 °C in LB media overnight and then subcultured to an ODeoo of 0.05 and grown for 2h. Subcultures were then supplemented with appropriate concentrations of treatments and followed immediately by adding 0.2% L-rhamnose (w / v) to induce expression of FhaC (or FhaC and FhaBsec). Subcultures were then grown for another hour. An ODeoo reading was taken, and 1 mL aliquots were taken into a 1.5 mL tube. Aliquots were then TCA-precipitated (0.4 mM PMSF, 10% TCA (v / v)) and air-dried (20 min, 37 °C). Airdried precipitates were then resuspended in [ODeoo x 200] pL of 2x SDS Protein Loading Dye (100 mM Tris-HCl pH 8, 20% glycerol (v / v), 0.2% bromophenol blue (w / v), 4% SDS (v / v). Samples were then probed by Western Blot. After image analysis, within-lane intensity values of a band corresponding to FhaC were quantified and normalised to their respective mock-treated (vehicle control) pair for each construct. Appropriate statistical tests were then conducted in GraphPad Prism vl0.3.0 (461) to compare each construct’s relative FhaC expression secretion (relative to mock-treated) and their respective mock-treated control values.

[0303] Helix Mobility Assay

[0304] To determine whether Pl-CPP treatment affects the localisation of the a-helix (Hl) of FhaC, the strain MTDS630 was used. Briefly, the strain was cultured at 37 °C in LB media overnight and then subcultured to an ODeoo of 0.05 and grown for 2h. Subcultures were then supplemented with appropriate concentrations of treatments and followed immediately by adding 0.2% L-rhamnose (w / v) to induce expression of FhaC. Subcultures were then grown for 30 mins. An ODeoo reading was taken, and 100 pL aliquots were taken into 1.5 mL tubes pre-filled with 1° antibody (dilution factor of 1 : 100 for anti-His) on a thermomixer set at 37 °C. Samples were incubated with 1° antibody for 30 minutes before being pelleted (2,000 x g, 2 min, 25 °C), and have their supernatants removed (to remove unbound 1° antibody) then resuspended with 100 pL of lx PBS premixed with 2° antibody (dilution factor of 1 : 100 for goat anti-Mouse). Samples were then incubated for a further 30 minutes at 37 °C before being pelleted (2,000 x g, 2 min, 25 °C), have their supernatants removed (to remove unbound 2° antibody), washed with 1 mL lx PBS, re-pelleted (2,000 x g, 2 min, 25 °C) and have their supernatants removed. Samples are finally resuspended with 100 pL lx PBS and loaded onto Corning® 96-well Flat Clear Bottom Black Polystyrene TC-treated Microplates (Catalogue number 3603). Samples are then scanned using Varioskan Lux Multimode Microplate Reader to detect endpoint ODeoo values and then scanned with maximum quality and resolution settings with an Odyssey DLx (LiCor) at 800nm. After image analysis, within- well intensity values of each sample wells were quantified and normalised to their respective ODeoo values obtained previously. Appropriate statistical tests were then conducted in GraphPad Prism vl 0.3.0 (461) to compare relative normalised fluorescence detected from Pl-CPP-treated samples (relative to mock-treated).

[0305] Human lung epithelial adherence assay

[0306] Human A549 lung epithelial cells (ATCC #CCL-185) were seeded at 5xl05cells per well in 6 well tissue culture plates for 18 hours. Wildtype Bordetella bronchiseptica RB50 (Cotter and Miller, 1994), the isogenic b haB and AfhaC strains were grown overnight in Stainer Scholte (SS) media (Stainer and Scholte, 1970; Hulbert and Cotter, 2009) containing 50 mM MgSC which inactivates the BvgAS two component regulatory system and prevents expression of fliaB,jhaC, and all other known Bordetella virulence proteins. The bacteria were washed twice with sterile DPBS to remove MgSC and resuspended to an OD of 0.02 OD600 in SS broth without MgSC (BvgAS activating conditions) to allow expression of fhaB and fhaC. Wildtype bacteria diluted into Ham’s F12 media +10% FBS containing either 20 pM of the PIEISR-CPP peptide in DMSO or the same volume of pure DMSO as a vehicle control, such that 100 colony forming units (cfus) were added for every A549 cell in the well (Multiplicity of Infection 100). The plates were spun for 5 minutes at 500 xg to synchronize bacterial interactions with the A549 cells. After 45 minutes of incubation at 37 °C 5% CO2 the inoculation media was removed and the infected cells were washed three times with 2mL of DPBS to remove non-adherent bacteria. The A549 cells were lysed with 0.05 % triton- XI 00 detergent in DPBS for 10 minutes, the lysates were serially diluted and plated onto Bordet-Gengou agar plates supplemented with 5% defibrillated sheep’s blood and 0.24% glycerol, and grown at 37 °C for 48 hours to enumerate the number of adherent B. bronchiseptica bacteria in each sample.

[0307] In further experiments (Fig. 31, in particular), A549 cells were cultured in DMEM media supplemented with 10% FBS, 1% penicillin-streptomycin (1000 U / mL) and 4 mM L- glutamine. THP-1 were cultured in RPMI 1640 media supplemented with 10% FBS, 1% penicillin-streptomycin (1000 U / mL), 1% sodium pyruvate (100 mM), and 0.3% sodium bicarbonate (7.5%). All cells were incubated at 37 °C with 5% CO2. B. pertussis strain L1423 was transformed with a pBBRlMCS-2 plasmid to express free-use green fluorescent protein (fuGFP) constitutively. L1423 were then grown on Bordet-Gengou agar (BD Scientific) for 3 days at 37 °C. A loopful of pure Bvg+ LI 423 -fuGFP colonies was then resuspended in Stainer-Scholte (SS) media supplemented with 1% heptakis ((2,6-O-dimethyl) 0- cyclodextrin) and 1% SS supplement and grown for 24 hours (37 °C, 180 rpm). 24 hours prior to infection, 1 mL of A549 cells (2 x 105) cells and THP-1 cells (5 x 105) were seeded onto 24-well plates containing rounded coverslips (13 mm). THP-1 cells were induced overnight into macrophage-like cells with 5 pL of 10 pM phorbol 12-myri state- 13 -acetate (PMA). After overnight incubation, each well was washed with 1 mL lx PBS and replaced with 1 mL antibiotic-free media. Each cell line was then infected with LI 423 -fuGFP at an MOI of 100. 10 pL of PIEISR-CPP (2 mM) or DMSO vehicle control was added to the wells. Plates were then centrifuged at 500 x g for 5 minutes to allow B. pertussis cells to come into contact with host cells. Plates were then incubated (37 °C, 5%) for 2 hours. After incubation, media was removed, and each well was washed three times with lx PBS to remove nonadherent cells. Cells were then fixed with 1 mL of 4% paraformaldehyde (PF A) for 20 minutes. Three biological replicates per cell line were performed. For immunofluorescence, cells were permeabilised with 0.5% Triton X-100 and then blocked with 1% BSA. Host cells were then stained with DAPI (1 :40,000) and anti-a-tubulin (1 :3,000) with goat anti-mouse IgG (H+L) - Alexa Fluor 568 secondary antibody (1 :600). Coverslips were then mounted with ProLong diamond mounting solution. Images were then acquired on the Leica Stellaris 5 at the Katerina Gaus Light Microscopy Facility (KGLMF) at the University of New South Wales (UNSW). Five field-of-views were randomly selected per sample (3888 x 3888 pixels). All images were taken at 40x objective (water immersion, NA1.1). The resulting images were imported into ImageJ (vl.54) and the percentage ratio of attached pertussis cells to host cells were calculated.

[0308] Results

[0309] Analysing the cell pellet in an assay for FhaBsec secretion in an E. coli model revealed that treatment of cells with the Pl-CPP inhibitor does not result in a change in expression of the target FhaC transporter (Fig. 20).

[0310] Using a novel helix mobility assay (Fig. 21 A) data was collected suggesting that Pl inhibits FhaC by stabilizing the a -Helix (Hl) of the FhaC within the barrel of the transporter (Fig. 21B). Further to this finding, deletion of Hl and the Linker from FhaC reduced the effectiveness of Pl-CPP in inhibiting FhaBsec secretion (Fig. 21C and D).

[0311] Quantifying FhaBsec secretion when treated with Pl (Pl-CPP) as compared to N- terminal and C-terminal truncated Pl (PIANI-CPP and Pl Act-CPP, respectively) showed that Pl Act-CPP and Pl-CPP have equivalent activity (Fig. 22). In contrast, PIANI-CPP had reduced activity as compared to Pl-CPP (Fig. 12 and 22). Without wishing to be bound by theory, the Inventors consider that this may suggest that the N-terminal region of Pl (VAAMAA) binds and blocks the conserved, hydrophobic pocket of FhaC POTRA1.

[0312] Quantifying FhaBsec secretion in samples treated with Pl-CPP having combinations of the different mutations (i.e., PIRUE, PIRBE, and PIEBR) revealed IC50 values for PIRUE- CPP (13.0 pM), PIRBE-CPP (1.29 pM), PIEBR-CPP (0.59 pM), and PIRI 1E,R13E,E15R- CPP (5.35 pM) (Fig. 23). The effect of these mutations on FhaBsec sections was also determined via Coomassie staining (Fig. 24). In an assay using B. bronchiseplica, the PIEISR-CPP derivative prevented adherence to immortalized human A549 lung epithelial cells (Fig. 25). In a further experiment, Pl also inhibited secretion of FHA (FhaB exoprotein) into the culture media and caused FHA accumulation in cells from clinical isolates of B. pertussis (Fig. 26). In a further experiment, using B. pertussis, the PIEISR-CPP derivative prevented adherence to both immortalized human A549 lung epithelial cells and THP-1 macrophage-like cells (Fig. 31).

[0313] Structure predictions were done on the 3 macrocyclic peptides disclosed herein (RD- 4, RD-6, and RD-7) suggesting these peptides may bind and lodge within the P-barrel domain of FhaC (Fig. 27).

[0314] To determine the minimum concentrations where the macrocycles RD-4 and RD7 were active, the abovementioned secretion assay was used to test different concentrations of each macrocycle (Fig. 28). RD-4 was able to significantly inhibit FhaBsec secretion down to a final concentration of 1 pM (p < 0.01) (Fig. 28 Bottom, Left) and RD-7 was able to significantly inhibit secretion down to a concentration of 2 pM (p < 0.001) (Fig. 28 Bottom, Right). These data points were well fit by a sigmoidal function, allowing us to calculate the IC50 of peptides RD-4 and RD-7 as 2.71 pM and 4.09 pM, respectively.

[0315] As a further investigation of Pl binding to FhaC, distances between residues on each protein were measured over time to see if there was evidence of specific interactions. By measuring the distance between atoms in residues R11 in Pl, E7 in Pl, and N121 in FhaC it was found that residues E7 and R11 in Pl may interchangeably bind to residue N121 in FhaC (Fig. 29)

[0316] An analysis of the hydrophobic pocket of FhaC POTRA1 in different diderm species revealed that Pl binds a conserved portion, suggesting that Pl can bind POTRA1 in different species (Fig. 30).

[0317] Table 10. Further molecular properties of Pl and related peptides.

[0318]

[0319] Abbreviations: M.W = molecular weight, Da = Daltons, pl = isoelectric point, GRAVY = grand average of hydropathy, ACt = Truncation of last four C-terminal residues of Pl.

[0320] Table 11. Peptide names and sequences as used herein

[0321] EMBODIMENTS OF THE INVENTION

[0322] Other embodiments of the invention as described herein are defined in the following paragraphs.

[0323] 1. A compound for inhibiting a two-partner secretion system, wherein the compound comprises an amino acid sequence selected from the group consisting of:

[0324] VAAMAA (SEQ ID NO: 1);

[0325] VAAMAAEASARARAEFAAR (SEQ ID NO: 2);

[0326] VAAMAAEASARAEAEFAAR (SEQ ID NO: 3);

[0327] V A AM A AE AS AR AR ARFA AR (SEQ ID NO: 4);

[0328] V A AM A AE AS ARAE ARFA AR (SEQ ID NO: 5);

[0329] YVLCTLRAKRFYALFLSG (SEQ ID NO: 6);

[0330] YRKHWPDWYRTHYVVCG (SEQ ID NO: 7);

[0331] YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8);

[0332] VAAMAAEASARARAE (SEQ ID NO: 64); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences; and an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 1-8 and 64, and wherein the compound binds to a translocator pore protein (TpsB) thereby reducing translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane.

[0333] 2. The compound according to paragraph 1, wherein the amino acid sequence further comprises the amino acid sequence: KFFKFFKFFK (SEQ ID NO: 9).

[0334] 3. The compound according to paragraph 2, wherein the amino acid sequence comprises the amino acid sequence: KFFKFFKFFKGVAAMAAEASARARAEFAAR (SEQ ID NO: 10).

[0335] 4. The compound according to paragraph 1, wherein the amino acid sequence is selected from the group consisting of:

[0336] YVLCTLRAKRFYALFLSG (SEQ ID NO: 6);

[0337] YRKHWPDWYRTHYVVCG (SEQ ID NO: 7);

[0338] YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences; and an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 6-8, and wherein the amino acid sequence is cyclized between a tyrosine (Y) residue and a cysteine (C) residue.

[0339] 5. The compound according to paragraph 1, wherein the compound comprises an amino acid sequence with a two-dimensional structure selected from the group consisting of:

[0340]

[0341] 6. The compound according to any one of the preceding paragraphs, wherein the TpsB is

[0342] FhaC.

[0343] 7. The compound according to any one of the preceding paragraphs, wherein the TpsA is

[0344] FhaB. 8. The compound according to any one of the preceding paragraphs, wherein the two- partner secretion system is from Bordetella spp.

[0345] 9. The compound according to any one of the preceding paragraphs, wherein the compound also inhibits release of adenylate cyclase toxin (ACT).

[0346] 10. A pharmaceutical composition comprising the compound according to any one of the preceding paragraphs and one or more pharmaceutically acceptable diluents, excipients, or carriers. 11. A method of treating or preventing a bacterial infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound according to any one of paragraphs 1-9 or the pharmaceutical composition according to paragraph 10.

[0347] 12. Use of the compound according to any one of paragraphs 1-9 in the manufacture of a medicament for treating or preventing a bacterial infection in a subject.

[0348] 13. A compound according to any one of paragraphs 1-9 or a pharmaceutical composition according to paragraph 10 for use in the treatment or prevention of a bacterial infection in a subject.

[0349] 14. The method according to paragraph 11, the use according to paragraph 12, or the compound for use according to paragraph 13, wherein the bacterial infection is caused by Bordetella spp.

[0350] 15. The method according to paragraph 11 or 14, the use according to paragraph 12 or 14, or the compound for use according to paragraph 13 or 14, wherein the subject is a human.

[0351] 16. The method according to paragraph 11 or 14, the use according to paragraph 12 or 14, or the compound for use according to paragraph 13 or 14, wherein the subject is a non-human animal.

[0352] 17. A method of screening for inhibitors of TpsA secretion, the method comprising: providing a bacterial strain comprising: a nucleotide sequence encoding a TpsB protein; and a nucleotide sequence encoding a TpsA protein; exposing the bacterial strain to a candidate inhibitor; and measuring the amount of TpsA protein or TpsB protein in a fraction isolated from the bacterial strain.

[0353] 18. A method of screening for inhibitors of TpsA secretion, the method comprising: providing a bacterial strain comprising: a nucleotide sequence encoding a TpsB protein; a nucleotide sequence encoding a TpsA protein; and a nucleotide sequence encoding a signal peptide and a candidate inhibitor; measuring the amount of TpsA protein or TpsB protein in a fraction isolated from the bacterial strain.

[0354] 19. The method according to paragraph 17 or 18, wherein the TpsA protein has an increased ability to secrete from the TpsB protein when compared to a wildtype TpsA protein.

[0355] 20. The method according to any one of paragraphs 17-19, wherein the TpsB is FhaC.

[0356] 21. The method according to any one of paragraphs 17-20, wherein the TpsA is FhaB.

[0357] 22. The method according to any one of paragraphs 17-21, wherein the fraction isolated from the bacterial strain is a supernatant fraction or a pellet fraction.

[0358] 23. A method of treating or preventing a plant disease, the method comprising applying the compound according to any one of paragraphs 1-9 to a plant in an amount effective to treat or prevent the plant disease.

[0359] 24. The method according to paragraph 23, wherein the plant disease is bacterial soft rot.

[0360] 25. A method of preventing or reducing bacterial replication, the method comprising exposing bacteria to the compound according to any one of paragraphs 1-9.

[0361] 26. The method according to paragraph 25, wherein the bacteria are in a biofilm.

[0362] 27. The method according to paragraph 25 or paragraph 26, wherein the bacteria is on a biological surface or a non-biological surface.

[0363] 28. A compound for binding a translocator pore protein (TpsB), wherein the compound comprises an amino acid sequence selected from the group consisting of: VAAMAA (SEQ ID NO: 1); VAAMAAEASARARAEFAAR (SEQ ID NO: 2);

[0364] VAAMAAEASARAEAEFAAR (SEQ ID NO: 3); VAAMAAEASARARARFAAR (SEQ ID NO: 4); V A AM A AE AS ARAE ARFA AR (SEQ ID NO: 5); YVLCTLRAKRFYALFLSG (SEQ ID NO: 6); YRKHWPDWYRTHYVVCG (SEQ ID NO: 7);

[0365] YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8); YRKHWPEWYRTHYVVCG (SEQ ID NO: 11); YRKHWPDWYRTHYVACG (SEQ ID NO: 12); YRKHWPDWYRTHYVICG (SEQ ID NO: 13); YHILYYKVLYYLYCG (SEQ ID NO: 14); YRIICWYLICG (SEQ ID NO: 15); YRYPTWYSTYYNQCG (SEQ ID NO: 16); YYWYYLFWSCG (SEQ ID NO: 17);

[0366] YAIVVSPKYLFIRVCG (SEQ ID NO: 18); YHYYLYQSKFIWECG (SEQ ID NO: 19); YGGGKDRKSTRLNSSH (SEQ ID NO: 20); YIVSTIPSNVICLICG (SEQ ID NO: 21); YEEHTSELQSLGIISYAVFC (SEQ ID NO: 22); YLKISTCTWVLSISFCG (SEQ ID NO: 23); YFILKLLLVCG (SEQ ID NO: 24); YFVLTLVYIIVIHAVCG (SEQ ID NO: 25); YYVVYFDYLFWIVVGCG (SEQ ID NO: 26); YFLIWWLLLCG (SEQ ID NO: 27);

[0367] YLILVFDSSEVWVVLICG (SEQ ID NO: 28); YAVAYWSRNFVLVYTICG (SEQ ID NO: 29); YLIVTRVNERWHFSHVCG (SEQ ID NO: 30); YVFLYEKGFLIVFTICG (SEQ ID NO: 31); YTWLYVLLVCG (SEQ ID NO: 32); YWQKRHICFEIVLCG (SEQ ID NO: 33); YRLTIVRNHIGWTLYVCG (SEQ ID NO: 34); YINVIWFWYCG (SEQ ID NO: 35); YLCLLFVFLTLCIIICG (SEQ ID NO: 36); YEEHTSELQSRTPISYAVFC (SEQ ID NO: 37); YVLCTLRAKRFYALFLCG (SEQ ID NO: 63); VAAMAAEASARARAE (SEQ ID NO: 64); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences; and an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 1-8, 11-37, 63, and 64.

[0368] 29. The compound according to paragraph 28, wherein the amino acid sequence further comprises the amino acid sequence: KFFKFFKFFK (SEQ ID NO: 9).

[0369] 30. The compound according to paragraph 28 or 29, wherein the compound further comprises a fluorophore, radio ligand, colorimetric label, histidine tag, streptavidin tag, or avidin tag.

[0370] 31. A peptide comprising an amino acid sequence as set forth in any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93; an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93; or an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93.

[0371] 32. The peptide according to paragraph 31, wherein the peptide is isolated.

[0372] 33. The peptide according to paragraph 31 or 32, wherein the peptide binds to a translocator pore protein (TpsB).

[0373] 34. The peptide according to any one of paragraphs 31-33, wherein the peptide reduces translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane.

[0374] 35. A nucleic acid molecule comprising a nucleotide sequence encoding the peptide according to any one of paragraphs 31-34.

[0375] 36. The nucleic acid molecule according to paragraph 35, wherein the molecule is isolated.

[0376] 37. A vector comprising a nucleotide sequence encoding the peptide according to any one of paragraphs 31-34.

[0377] 38. A cell comprising: a nucleotide sequence encoding the peptide according to any one of paragraphs 31-34, the nucleic acid molecule according to paragraph 35, or the vector according to paragraph 37.

[0378] 39. The cell according to paragraph 38, wherein the cell is a bacterial cell.

[0379] 40. The cell according to paragraph 38 or 39, wherein the cell is isolated.

[0380] 41. A kit comprising the compound according to any one of paragraphs 1-9, and 28-30, or the peptide according to any one of paragraphs 31-34.

[0381] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, and in particular features of any one of the various described examples may be provided in any combination in any of the other described examples. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.

[0382] REFERENCES

[0383] Cardona & Valvano. 2005. An expression vector containing a rhamnose-inducible promoter provides tightly regulated gene expression in Burkholderia cenocepacia. Plasmid, 54, 219- 228.

[0384] Cotter et al. 1998. Filamentous Hemagglutinin of Bordetella bronchiseptica Is Required for Efficient Establishment of Tracheal Colonization. Infect Immun 66:5921-5929.

[0385] Cotter & Miller. 1994. BvgAS-mediated signal transduction: analysis of phase-locked regulatory mutants of Bordetella bronchiseptica in a rabbit model. Infect Immun 62:3381- 3390.

[0386] Hanson et al. 2023. The patatin-like protein PlpD forms novel structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane. bioRxiv, 2023.04.17.537245.

[0387] Hulbert & Cotter. 2009. Laboratory Maintenance of Bordetella pertussis. Curr Protoc Microbiol 15:4B.1.1-4B.1.9.

[0388] Johnson et al. 2021. DegP Initiates Regulated Processing of Filamentous Hemagglutinin in Bordetella bronchiseptica. Mbio 12:e01465.

[0389] Norman et al. 2021. Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein Using mRNA Display. ACS Central Science, 7, 1001-1008.

[0390] Octavia et al. 2012. Newly Emerging Clones of Bordetella pertussis Carrying pm2 and ptxP3 Alleles Implicated in Australian Pertussis Epidemic in 2008-2010. The Journal of Infectious Diseases, 205, 1220-1224.

[0391] Passioura & Suga. 2017. A RaPID way to discover nonstandard macrocyclic peptide modulators of drug targets. Chemical Communications, 53, 1931-1940.

[0392] Stainer & Scholte. 1970. A Simple Chemically Defined Medium for the Production of Phase I Bordetella pertussis. Microbiology 63:211-220.

[0393] Tsiamantas et al. 2001. Discovery of Functional Macrocyclic Peptides by Means of the RaPID System. Cyclic Peptide Design, Chapter 14, 299-315.

[0394] Yamamoto et al. 2022. Scaffold size-dependent effect on the enhanced uptake of antibiotics and other compounds by Escherichia coli and Pseudomonas aeruginosa. Scientific Reports, 12, 5609.

Claims

CLAIMS1. A compound for inhibiting a two-partner secretion system, wherein the compound comprises an amino acid sequence selected from the group consisting of:VAAMAA (SEQ ID NO: 1);VAAMAAEASARARAEFAAR (SEQ ID NO: 2);VAAMAAEASARAEAEFAAR (SEQ ID NO: 3);V A AM A AE AS AR AR ARFA AR (SEQ ID NO: 4);V A AM A AE AS ARAE ARFA AR (SEQ ID NO: 5);YVLCTLRAKRFYALFLSG (SEQ ID NO: 6);YRKHWPDWYRTHYVVCG (SEQ ID NO: 7);YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8);VAAMAAEASARARAE (SEQ ID NO: 64); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences; and an amino acid sequence with at least 70% sequence identity to any one of SEQ ID Nos: 1-8 and 64, and wherein the compound binds to a translocator pore protein (TpsB) thereby reducing translocation of a two-partner secretion exoprotein (TpsA) across a lipid membrane.

2. The compound according to claim 1, wherein the amino acid sequence further comprises the amino acid sequence: KFFKFFKFFK (SEQ ID NO: 9).

3. The compound according to claim 2, wherein the amino acid sequence comprises the amino acid sequence: KFFKFFKFFKGVAAMAAEASARARAEFAAR (SEQ ID NO: 10).

4. The compound according to claim 1, wherein the compound comprises an amino acid sequence with a two-dimensional structure selected from the group consisting of:

5. The compound according to any one of the preceding claims, wherein the TpsB is FhaC.

6. The compound according to any one of the preceding claims, wherein the TpsA is FhaB.

7. A pharmaceutical composition comprising the compound according to any one of the preceding claims and one or more pharmaceutically acceptable diluents, excipients, or carriers.

8. A method of treating or preventing a bacterial infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7.

9. Use of the compound according to any one of claims 1-6 in the manufacture of a medicament for treating or preventing a bacterial infection in a subject.

10. A compound according to any one of claims 1-6 or a pharmaceutical composition according to claim 7 for use in the treatment or prevention of a bacterial infection in a subject.

11. The method according to claim 8, the use according to claim 9, or the compound for use according to claim 10, wherein the bacterial infection is caused by Bordetella spp.

12. The method according to claim 8 or 11, the use according to claim 9 or 11, or the compound for use according to claim 10 or 11, wherein the subject is a human.

13. A method of screening for inhibitors of TpsA secretion, the method comprising: providing a bacterial strain comprising: a nucleotide sequence encoding a TpsB protein; and a nucleotide sequence encoding a TpsA protein; exposing the bacterial strain to a candidate inhibitor; and measuring the amount of TpsA protein or TpsB protein in a fraction isolated from the bacterial strain.

14. A method of screening for inhibitors of TpsA secretion, the method comprising: providing a bacterial strain comprising: a nucleotide sequence encoding a TpsB protein; a nucleotide sequence encoding a TpsA protein; and a nucleotide sequence encoding a signal peptide and a candidate inhibitor; measuring the amount of TpsA protein or TpsB protein in a fraction isolated from the bacterial strain.

15. A method of treating or preventing a plant disease, the method comprising applying the compound according to any one of claims 1-6 to a plant in an amount effective to treat or prevent the plant disease.

16. A method of preventing or reducing bacterial replication, the method comprising exposing bacteria to the compound according to any one of claims 1-6.

17. A compound for binding a translocator pore protein (TpsB), wherein the compound comprises an amino acid sequence selected from the group consisting of: VAAMAA (SEQ ID NO: 1); VAAMAAEASARARAEFAAR (SEQ ID NO: 2); VAAMAAEASARAEAEFAAR (SEQ ID NO: 3); VAAMAAEASARARARFAAR (SEQ ID NO: 4); VAAMAAEASARAEARFAAR (SEQ ID NO: 5); YVLCTLRAKRFYALFLSG (SEQ ID NO: 6); YRKHWPDWYRTHYVVCG (SEQ ID NO: 7);YLLLFHSRRGLLVLHLCG (SEQ ID NO: 8); YRKHWPEWYRTHYVVCG (SEQ ID NO: 11); YRKHWPDWYRTHYVACG (SEQ ID NO: 12); YRKHWPDWYRTHYVICG (SEQ ID NO: 13); YHILYYKVLYYLYCG (SEQ ID NO: 14); YRIICWYLICG (SEQ ID NO: 15); YRYPTWYSTYYNQCG (SEQ ID NO: 16); YYWYYLFWSCG (SEQ ID NO: 17);YAIVVSPKYLFIRVCG (SEQ ID NO: 18); YHYYLYQSKFIWECG (SEQ ID NO: 19); YGGGKDRKSTRLNSSH (SEQ ID NO: 20); YIVSTIPSNVICLICG (SEQ ID NO: 21); YEEHTSELQSLGIISYAVFC (SEQ ID NO: 22); YLKISTCTWVLSISFCG (SEQ ID NO: 23); YFILKLLLVCG (SEQ ID NO: 24); YFVLTLVYIIVIHAVCG (SEQ ID NO: 25); YYVVYFDYLFWIVVGCG (SEQ ID NO: 26); YFLIWWLLLCG (SEQ ID NO: 27);YLILVFDSSEVWVVLICG (SEQ ID NO: 28); YAVAYWSRNFVLVYTICG (SEQ ID NO: 29); YLIVTRVNERWHFSHVCG (SEQ ID NO: 30); YVFLYEKGFLIVFTICG (SEQ ID NO: 31); YTWLYVLLVCG (SEQ ID NO: 32); YWQKRHICFEIVLCG (SEQ ID NO: 33); YRLTIVRNHIGWTLYVCG (SEQ ID NO: 34); YINVIWFWYCG (SEQ ID NO: 35);YLCLLFVFLTLCIIICG (SEQ ID NO: 36); YEEHTSELQSRTPISYAVFC (SEQ ID NO: 37); YVLCTLRAKRFYALFLCG (SEQ ID NO: 63); VAAMAAEASARARAE (SEQ ID NO: 64); an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of the preceding amino acid sequences; and an amino acid sequence with at least 70% sequence identity to any one of SEQ ID Nos: 1-8, 11- 37, 63, and 64.

18. A peptide comprising an amino acid sequence as set forth in any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93; an amino acid sequence with between 1 and 3 amino acid deletions, additions, and / or substitutions as compared to any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93; or an amino acid sequence with at least 70% sequence identity to any one of SEQ ID Nos: 1-8, 10-37, 58-60, 63-68, and 89-93.

19. A nucleic acid molecule comprising a nucleotide sequence encoding the peptide according to claim 18.

20. A kit comprising the compound according to any one of claims 1-6 and 17, or the peptide according to claim 18.

Images