Methods for producing vesicles and applications thereof

Abstract

We have shown that cprA expression leads to the production of outer membrane vesicles (OMVs) that block autophagic flux and have a greater capacity to activate the non-canonical inflammasome pathway. In a murine model of sepsis, a P. aeruginosa strain deleted for cprA was less virulent than the wild-type strain. These results demonstrate the important role of CprA in the pathogenicity of P. aeruginosa. It is worth noting that CprA is also a functional ortholog of HlyF, which is encoded by virulence plasmids of Escherichia coli. We have shown that other cryptic SDRs encoded by mammalian and plant pathogens, such as Yersinia pestis and Ralstonia solanacearum are functional orthologs of CprA and HlyF. These SDRs also induce the production of OMVs which block autophagic flux. This study uncovers a new family of virulence determinants in Gram-negative bacteria, offering potential for innovative therapeutic interventions and deeper insights into bacterial pathogenesis. The present invention relates to a method for producing vesicles, the method comprising a step of expressing or overexpressing hemolysin F (hlyF) gene or an ortholog of hemolysin F gene in Gram-negative bacteria, said hlyF gene or ortholog of hlyF gene encoding respectively a HlyF protein or an ortholog of HlyF protein.

Description

[0001] METHODS FOR PRODUCING VESICLES AND APPLICATIONS THEREOF

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to methods for producing vesicles.

[0004] BACKGROUND OF THE INVENTION:

[0005] Pseudomonas aeruginosa is a leading cause of chronic and healthcare-associated infections, exhibiting alarming levels of antimicrobial resistance, morbidity and mortality1. Contributing significantly to global bacterial fatalities, P. aeruginosa ranks among the top five pathogens, accounting for half of these deaths2. The escalation of this threat is propelled by the pathogen’s remarkable ability to develop resistance through chromosomal mutations and the rising prevalence of transferable resistance determinants3. Annually, over 300,000 deaths are attributed to antibiotic-resistant P. aeruginosa infections, warranting urgent attention from the WHO for novel research and development4. Polymyxins (i.e. polymyxin B and colistin), represent a crucial class of cyclic lipopeptides antibiotics, that maintain efficacy against extensively-drug resistant P. aeruginosa5. Among the proteins involved in polymyxin resistance, PA14_44311 (accession number uniport KB A0A0H2ZLT3), also known as CprA (for cationic peptide resistance), emerges as a hypothetical protein playing a role in P. aeruginosa resistance to polymyxins and antimicrobial peptides6. The expression of cprA is positively regulated by the two-component system PmrAB, a key regulator of LPS modifications involved in polymyxin resistance in / < aeruginosa1, but also indirectly by others two-component systems such as ParRS, CprRS and PhoPQ8–10. Activation of PmrB is triggered by various signals including polymyxins, antimicrobial peptides and low Mg2+7,11. The predicted CprA structure is typical of an extended short-chain dehydrogenase / reductase (SDR) family member12. Despite this insight, the function and substrate of CprA remain unknown. However, CprA exhibits homology with E. coli hemolysin F (HlyF), a protein which is encoded by a virulence plasmid found in various E. coli pathotypes and in Salmonella enterica serovar Kentucky13,14. It is important to note that HlyF itself lacks hemolytic activity but instead acts as a cytoplasmic enzyme triggering the formation of outer membrane vesicles (OMVs), capable of delivering ClyA, a bona fide hemolysin13. The initial assumption that HlyF was a hemolysin was based on this phenotype15. Recently, we demonstrated that HlyF induces the formation ofOMVs that not only transport various toxins but also have the intrinsic ability to block autophagic flux and exacerbate inflammasome activation in host cells16–19.

[0006] SUMMARY OF THE INVENTION:

[0007] The invention is defined by the claims. In particular, the present invention relates to a method for producing vesicles, the method comprising a step of expressing or overexpressing hemolysin F (hlyF) gene or an ortholog of hemolysin F gene in Gram-negative bacteria, said hlyF gene or ortholog of hlyF gene encoding respectively a HlyF protein or an ortholog of HlyF protein.

[0008] DETAILED DESCRIPTION OF THE INVENTION:

[0009] Here, the Inventors demonstrate that CprA from P. aeruginosa exhibits similar properties to HlyF. Furthermore, their study reveals that functional HlyF / CprA orthologs are also encoded by various Gram-negative bacteria. This constitutes a newly identified family of virulence factors and of bacterial enzymes with particular properties.

[0010] A first aspect of the present invention relates to the use of hemolysin F (hlyF) gene and / or an ortholog of hemolysin F gene (hlyF) for the production of vesicles from membrane lipids of microorganisms, in particular for the production of liposomes derived from bacterial membrane and / or Outer Membrane Vesicles (OMVs). In some embodiments, the present invention also relates to a method for producing vesicles, wherein the method comprises a step of expressing or overexpressing hemolysin F (hlyF) gene and / or an ortholog of hemolysin F gene in a bacteria, in particular a Gram-negative bacteria, said hlyF gene and / or said ortholog of hlyF gene encoding respectively HlyF protein and / or an ortholog of HlyF protein. In some embodiments, the gene is not hlyF gene and the protein is not HlyF protein when the vesicles are OMVs.

[0011] In some embodiments, the present invention relates to the use of hlyF gene and / or an ortholog of hlyF gene for the production of liposomes derived from bacterial membrane, preferentially from Gram-negative bacterial membrane. In some embodiments, the present invention relates to a method for producing liposomes derived from bacterial membrane wherein the method comprises a step of expressing or overexpressing hlyF gene and / or an ortholog of hlyF gene in bacteria, said hlyF gene and / or ortholog of hlyF gene encoding respectively HlyF protein and / or an ortholog of HlyF protein.In some embodiments, the present invention relates to the use of an ortholog of hlyF gene for the production of outer membrane vesicles (OMVs) in Gram -negative bacteria. In particular, the present invention relates to a method for producing outer membrane vesicles (OMVs) wherein the method comprises a step of expressing or overexpressing an ortholog of hlyF gene in Gram-negative bacteria, said ortholog of hlyF gene encoding an ortholog of HlyF protein.

[0012] In some embodiments, any of the methods for producing vesicles, in particular liposomes derived from bacterial membrane and / or OMVs of the present invention are performed ex-vivo or in-vitro. The invention also relates to vesicles, in particular liposomes derived from bacterial membrane and / or OMVs obtained by any of the methods herein described.

[0013] In one embodiment, the vesicles produced from membrane lipids of microorganisms are derived from yeast membrane.

[0014] As used herein, the term “vesicle” refers to a structure comprising liquid or cytoplasm enclosed by a lipid bilayer. The term encompasses both artificial (e.g. liposomes derived from bacterial or yeast membrane) and naturally formed vesicles (e.g. extracellular vesicles such as OMVs). Such vesicles can be used as such or as a compound carrier (e.g. carrying anticancer drugs, antibiotics, antiviral drugs, immunosuppressants, DNA, RNA, antibodies, fluorescent dyes). Accordingly, the term encompasses both liposomes derived from bacterial membrane and outer membrane vesicles. In some embodiments, the vesicles are not OMVs.

[0015] As used herein, the term “liposome derived from bacterial or yeast membrane” refers to a small spherical artificial vesicle having a lipid bilayer, said lipid bilayer being formed from lipids of the bacterial or yeast membrane (i.e. lipids extracted from plasmatic, inner-, outer- and endo-membranes; and / or from extracellular vesicles released after budding of the bacterial membrane). In some embodiments, the liposomes are derived from plasmatic, inner-, outer- and endo-membranes. In some embodiments, the method for producing vesicles comprises the steps of:

[0016] - Extracting lipids from the membrane of the bacteria or yeast expressing hlyF gene or hlyF ortholog to obtain a lipidic fraction; and

[0017] - Forming liposomes from said lipidic fraction to obtain liposomes derived from bacterial membrane.As used herein, the term “Outer Membrane Vesicle” or “OMV” refers to a vesicle released from the outer membranes of a bacterium, in particular a Gram-negative bacterium. These vesicles are often involved in pathogenic processes since they contribute to the long-distance delivery of bacterial virulence factors, promote inflammation and stimulate host immune response. Typically, OMVs formed by bacteria can also mediate intercellular exchange events including cell-cell signalling, protein, RNA and DNA exchange (see for example Berleman J et al., 2013).

[0018] As used herein, the term “yeast” relates to a microscopic fungus. The term encompasses Saccharomyces cerevisiae, Pichia pastoris or Yarrowia lipolytica.

[0019] As used herein, the term “bacteria” encompasses both Gram-positive and Gram-negative bacteria. In some embodiments, the bacteria are non-pathogenic bacteria. In some embodiments, the bacteria are from the genera Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia and Lactobacillus. In some embodiments, the bacteria are selected from the list comprising or consisting in: Lactococcus lactis, Micrococcus luteus, Bacillus subtilis, Bacillus coagulens, Lactobacillus acidophilus, Lactobacillus plantarum, Streptomyces griseus, Streptomyces coelicolor, Staphylococcus epidermitis, Pediococcus acidilactici, Leuconostoc me senter oides, Bacillus thuringiensis, Bacillus cereus, Propionibacterium freudenreichii, Lactobacillus rhamnosus, Carnobacterium piscicola, Corynebacterium glutamicum, Streptomyces avermitilis, Kocuria rhizophila, Lactococcus cremoris, Brevibacillus brevis, Bacillus megaterium, Lactobacillus casei, Lactobacillus delbrueckii, Geobacillus stearothermophilus, Saccharopolyspora erythrea, Nocardia brasiliensis, Clostridium acetobutylcum, Escherichia coli, Salmonella enterica Kentucky, Acinetobacter baumannii, Bordetella pertussis, Vibrio cholerae, Neisseria meningitis, Shigella flexneri, Cedecea neteri, Escherichia albertii, Enterobacter clocae, Klebsiella michiganensis, Klebsiella pneumoniae, Raoultella ornithinolytica, Klebsiella aerogenes, Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter sakazakii, Serratia marcescens, Serratia odorifera, Yersinia enter ocolitica, Yersinia pseudotuberculosis, Yersinia pestis, Dickeya zeae, Dickeya dadantii, Pantoea ananatis, Pantoea agglomerans, Pantoea vagans, Pseudomonas oryziphila, Pseudomonas mosselli, Pseudomonas aeruginosa, Pandoraea pnomenusa, Pandoraea apista, Pandoraeasputorum, Pandoraea commovens, Ralstonia pickettii, Ralstonia solanacearum, Collimonas fungivorans or Cupriavidus brasilensis.

[0020] As used herein, the term “Gram-negative bacteria” refers to bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. Gram -negative bacteria, having two membranes, are also known as “diderm bacteria”. Their defining characteristic is their cell envelope, which consists of a thin peptidoglycan cell wall sandwiched between an inner membrane and an outer membrane. In some embodiments, the Gram-negative bacteria are Gammaproteobacteria, in particular Pseudomonadales or Enterobacterales; Betaproteobacteria, in particular Burkholderiales; or Bacteroidia. In some embodiments, the Gram-negative bacteria are selected from the list comprising or consisting in Escherichia coli, Salmonella enterica Kentucky, Acinetobacter baumannii, Bordetella pertussis, Vibrio cholerae, Neisseria meningitis, Shigella flexneri, Cedecea neteri, Escherichia albertii, Enterobacter clocae, Klebsiella michiganensis, Klebsiella pneumoniae, Raoultella ornithinolytica, Klebsiella aerogenes, Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter sakazakii, Serratia marcescens, Serratia odorifera, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia pestis, Dickeya zeae, Dickeya dadantii, Pantoea ananatis, Pantoea agglomerans, Pantoea vagans, Pseudomonas oryziphila, Pseudomonas mosselli, Pseudomonas aeruginosa, Pandoraea pnomenusa, Pandoraea apista, Pandoraea sputorum, Pandoraea commovens, Ralstonia pickettii, Ralstonia solanacearum, Collimonas fungivorans or Cupriavidus brasilensis. In some embodiments, the Gram-negative bacteria are not Escherichia coli.

[0021] In some embodiments, the Gram-negative bacteria are Pseudomonas sp. As used herein, the term “Pseudomonas” denotes a large family of Gram-negative bacteria that includes, P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. jlavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. agarici, P. asplenii, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugate, P.fragi, P. lundensis, P. taetrolens, P. Antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. jluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridian, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P.cremoricolorata, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva, P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdale, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthi, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. Uni, P. lutea, P. moraviensis, P. otitidis, P. pachastr ellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophila, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septic, P. simiae, P. suis, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis or P. xanthomarina. In some embodiments, the Gram-negative bacteria are Pseudomonas aeruginosa.

[0022] As used herein, the term “Hemolysin F gene” or ‘HlyF gene” refers to a gene which encodes a protein (HlyF) that was attributed a putative hemolysin function and which is expressed by certain strains of the Enterobacteriaceae family. An exemplary sequence for the gene hlyF is deposited in GenBank: AF 155222.1. For example, HlyF gene is located on the pS88 plasmid from strain S88 (Accession number CAQ87216).

[0023] As used herein, the term “gene orthologs” refers to a gene common to different species. These ortholog genes encode ortholog proteins, proteins having the same function in said different species. The term encompasses both natural (e.g. naturally expressed by a microorganism) or synthetic (e.g. designed, artificially synthetised) orthologs. In some embodiments, the ortholog of hlyF gene is selected in Table 1. In some embodiments, the ortholog of hlyF gene is cprA gene, encoding CprA protein. An exemplary CprA amino acid sequence is depicted in SEQ ID NO:1

[0024] SEQ ID NO: 1 > CprA MNMHADGEQITAIDTRERILLTGATGFLGGSVSAQLIAAGHGANLSFLVRAESRQQGLERLRGNLLMHGVDETDC LALRAEQILCGDFLDTSWLARETPRLMQVERVINCAAVASFSKNPTIWPVNVDGTFAFADVLSRSKRLKRFLHVG TAMCCGPQRESPISESWEFPAAEQQLVDYTASKAEIERRMREELPGLPLWARPSIWGHRTLGCQASGSIFWVF RMGFALESFTCGLDEQIDVI PVDYCAEALIGLALKPCLGHSLYHISAGHRAACTFGEIDEAFARANGAAPVGERYRKVEVDDLKELAKSFESRIGPANPRLVLRALRLYSGFADLNYLFDNSRLLEEGISAPPRFTDYLDVCVQSSSAVS IPAQMQWDFK

[0025] In some embodiments, the ortholog of HlyF protein is CprA. In some embodiments, the ortholog of HlyF is selected in Table 1. In some embodiments, the ortholog of HlyF protein is a short-chain dehydrogenase / reductase (SDR) comprising a NAD(P)H binding site and a catalytic site. As used herein, the term “short-chain dehydrogenase / reductase” or “SDR” refers to a family of enzymes known to be NADH- or NADPH-dependant oxidoreductases. Typically, the SDR have two main specific sequences, one responsible for the specific binding of the NAD(P)H coenzyme (“NAD(P)H binding site”) and the other including amino acids directly involved in the catalysis of various SDR substrates (“catalytic site”).

[0026] In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500 residues to 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500 residues, preferentially from 360 to 385 residues. In some embodiments, the ortholog of HlyF protein exhibits at least 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid identity with HlyF protein, preferentially, 40% amino acid identity with HlyF protein, even more preferentially 35% amino acid identity with HlyF protein. In some embodiments, the ortholog of HlyF protein includes the residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site. In some embodiments, the ortholog of HlyF protein includes the residue YTXSK (SEQ ID NO:3) in said catalytic site. In some embodiments, the ortholog of HlyF protein includes the residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region. In one embodiment, the ortholog of HlyF protein includes the residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region wherein the amino acid D is conserved. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues and includes the residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and the residue YTXSK (SEQ ID NO:3) in said catalytic site. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to385 residues, exhibit at least 35% amino acid identity with HlyF protein and includes the residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and the residue YTXSK (SEQ ID NO:3) in said catalytic site. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibit at least 35% amino acid identity with HlyF protein, includes the residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and the residue YTXSK (SEQ ID NO:3) in said catalytic site and includes the residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibit at least 40% amino acid identity with HlyF protein and includes the residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and the residue YTXSK (SEQ ID NO:3) in said catalytic site. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibit at least 40% amino acid identity with HlyF protein, includes the residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and the residue YTXSK (SEQ ID NO:3) in said catalytic site and includes the residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region. In some embodiments, the ortholog of HlyF protein includes the residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region and said C-ter region of said ortholog of HlyF protein exhibits at least 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid identity with the C-ter region of said HlyF protein, preferentially, 31% amino acid identity with the C-ter region of said HlyF protein. In some embodiments, the ortholog of HlyF protein possesses a fatty acyl CoA reductase (FARs) domain. In some embodiments, the ortholog of HlyF protein lacks the FAR-C superfamily domain (i.e. C-terminal domain of FAR). In some embodiments, the ortholog of HlyF protein possesses a fatty acyl CoA reductase (FARs) domain and lacks the FAR-C superfamily domain. In some embodiments, the ortholog of HlyF protein has a thioester-reductase domain.

[0027] The term “expressing” refers to the most fundamental level at which the genotype gives rise to a phenotype (e.g. to the production of outer membrane vesicle). The term “overexpressing” refers to an increased expression of a gene (e.g. increased frequency of transcription). The terms “expressing” or “overexpressing” encompass both natural or artificial expression. According to the invention, hfyF gene orthologs may be expressed or overexpressed by different means. In some embodiments, endogenous hlyF gene orthologs expressed by the bacterium may beoverexpressed. In this case, bacterial genome may be modified to obtain an overexpression of the hlyF gene ortholog. For example, natural promoter of the hlyF gene may be modified to obtain an overexpression of this gene. In another embodiment, the medium used to cultivate the bacterium may be modified to induce an expression or an overexpression of the hlyF gene ortholog. For example, a depletion of Mg2+should be done to induce this overexpression. In still another embodiment, the bacterium may be transformed with an ortholog of hlyF gene. For example, the bacterium may be transformed with a plasmid which expresses an ortholog of hlyF gene. In some embodiments, the ortholog of hlyF gene can be also expressed directly into the bacterium chromosome or into a plasmid in the cytoplasm of the bacterium. In a particular embodiment, the transformation of the bacterium with the ortholog of hlyF gene can be made using a plasmid. The term “using a plasmid” denotes the fact that the ortholog of hlyF gene is expressed by a plasmid which is inserted into the bacterium cytoplasm. Alternatively, the ortholog of hlyF gene can be integrated into the bacterial chromosome, in a neutral or silent location, under the control of an inducible promoter. In a particular embodiment, the bacteria plasmid is pGEX-6P-1 or pSA10 (see for example Schlosser-Silverman E et al., 2000). In some embodiments, the ortholog of hlyF gene expression is controlled by a promoter which induced a strong expression of the ortholog of hlyF gene. In some embodiments, the promoter used is the IPTG inducible promoter pTAC or the arabinose inducible promoter pBAD.

[0028] Methods for producing OMVs

[0029] In a more specific aspect, the present invention also relates to a method for producing OMVs, said method comprising a step of expressing or overexpressing an ortholog of hlyF gene in bacteria, preferentially Gram-negative bacteria. An exemplary method is described in the material and method section.

[0030] In some embodiments, the method for producing OMVs further comprises the steps of:

[0031] Cultivating the Gram-negative bacteria expressing or overexpressing the ortholog of hlyF gene in a culture medium; and

[0032] Centrifugating the culture medium to remove bacteria and obtain a supernatant comprising OMVs.

[0033] In some embodiments, the method for producing OMVs further comprises the steps of:Transforming the Gram-negative bacteria with the ortholog of hlyF gene and / or overexpressing the ortholog of hlyF gene in the Gram-negative bacteria;

[0034] Cultivating the Gram-negative bacteria in a culture medium

[0035] Centrifugating the culture medium to remove bacteria and obtain a supernatant comprising OMVs;

[0036] - Filtering the supernatant;

[0037] Centrifugating the filtered supernatant to obtain a pellet of OMVs. Several steps of centrifugation or ultracentrifugation can be added. To remove the OMVs from bacteria, the medium can be centrifuged with forces like 5,000 to 20,000 r.p.m for about 5 min to 2 hours and particularly 6,500 r.p.m for 10 min. To pellet the OMVs, the medium can be centrifuged with forces like 100,000 to 200,000 r.p.m for about 1 min to 4 hours and particularly 150,000 r.p.m for 3 hours. In some embodiments, a step of suspension can be added to obtain the OMVs in an appropriate medium. For example, the OMVs can be suspended in Tris-HCL. In some embodiments, a step of detoxification of the OMVs may be added. This step may be useful for example to deplete the OMVs from LPS and others toxic particles.

[0038] Methods for producing liposomes derived from bacterial or yeast membrane In a more specific aspect, the present invention also relates to a method for producing liposomes derived from bacterial membrane, said method comprising a step of expressing or overexpressing hlyF gene and / or an ortholog of hlyF gene in bacteria, preferentially Gramnegative bacteria. An exemplary method is described in the material and method section.

[0039] In some embodiments, the present invention also relates to a method for producing liposomes derived from bacterial membrane, said method comprising the steps of:

[0040] Cultivating bacteria, preferentially Gram-negative bacteria, expressing or overexpressing hlyF gene and / or an ortholog of hlyF gene in a culture medium;

[0041] - Extracting lipids from the bacterial membrane of the bacteria expressing hlyF gene or the ortholog hlyF gene to obtain a lipidic fraction; and

[0042] - Forming liposomes from said lipid fraction to obtain liposomes derived from bacterial membrane.

[0043] As example, a conventional protocol for the formation of liposomes derived from bacterial membrane can include the steps of culturing the bacterial strain of interest, harvesting cells bycentrifugation and washing the pellet with cold phosphate-buffered saline (PBS). The method can also include the steps of resuspending the bacterial pellet in a lysis buffer (e.g. Tris-HCL) containing protease inhibitors, lysing the cells using a mechanical method (e.g. French press, cell disruptor, sonication) or enzymatic treatment (e.g. lysozyme) to disrupt the cell wall, centrifuging the lysate at low speed to remove unbroken cells and debris and collecting the supernatant to perform ultracentrifugation to pellet the membrane fraction.

[0044] In some embodiments, the present invention also relates to a lipidic fraction obtained by extracting lipids from bacterial membrane, preferentially Gram-negative bacteria bacterial membrane, said bacteria expressing or overexpressing hlyF gene and / or an ortholog of hlyF gene. Then, the protocol can comprises the steps of resuspending the membrane pellet in a lipid extraction solution (e.g. chloroform:methanol, acetic acid), vortexing and incubating the suspension at room temperature. A further step of removing any unencapsulated material by size-exclusion chromatography or ultracentrifugation can be included.

[0045] Vaccine composition and therapeutic use

[0046] In a further aspect of the invention, the vesicles (e.g. OMVs or liposomes derived from bacterial membrane) obtainable by any of the methods herein described may be used for the preparation of a vaccine composition, in particular as therapeutic agent, vehicle or adjuvant. Accordingly, the present invention also relates to a vaccine composition comprising vesicles obtained by any of the methods herein described, and its therapeutic use, in particular in a method of treating a subject suffering from a Gram -negative bacteria infection. In some embodiments, the present invention also relates to a vaccine composition comprising OMVs obtained by any of the methods herein described, and its therapeutic use, in particular in a method of treating a subject suffering from a Gram-negative bacteria infection. In some embodiments, the present invention also relates to a vaccine composition comprising liposomes derived from bacterial membrane obtained by any of the methods herein described, and its therapeutic use, in particular in a method of treating a subject suffering from a Gram-negative bacteria infection.

[0047] As used herein, the term “subject” or “patient” denotes any vertebrate, in particular any warm blood vertebrate, in particular any mammal. In some embodiments, the subject is a human, a mammal (e.g. animal, marsupial, primate, rodent, cetacean, seal), a bird, a fish, an amphibian or a reptile. In some embodiments, the subject is a poultry, a bovine, a sheep, a goat, a pig, anequine, a cam elid or a deer. In some embodiments, the subject suffers from a Gram -negative bacteria infection. In some embodiments, the subject is asymptomatic.

[0048] As used herein, the term “Gram-negative bacteria infection” refers to any infectious disease caused by Gram-negative bacteria (e.g. gastroenteritis, pneumonia, peritonitis, urinary tract infections, bloodstream infections, wound and surgical site infections, meningitis). In some embodiments, the Gram-negative bacteria are antibiotic-resistant Gram-negative bacteria. The term encompasses both symptomatic and asymptomatic colonisation.

[0049] As used herein, the terms “treating”, “treatment” or “therapy” refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. In some embodiments, the term “treatment” also refers to the preventive treatment of a disease or condition. The treatment may be administered to a subject having a medical disorder or a subject likely to suffer from the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, ortreatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]). The term encompasses human medicine and veterinary medicine.

[0050] Vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) obtained by the method of the invention may be used for vaccine purpose and may express specific antigens (e.g. tumoral, viral, bacterial, fungal, parasite antigen) to induce vaccine response against these antigens. As used herein the term “antigen” refers to a molecule which is capable of being recognized by the immune system and / or being capable of inducing a humoral immune response and / or cellular immune response leading to the activation of B- and / or T-lymphocytes. An antigen can have one or more epitopes or antigenic sites (B- and T- epitopes). In some embodiments, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) contain at their surface (e.g. at their membranes) or as luminal cargo antigens which are specific of the bacteria used to produce the vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane). In this way, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) may be directly used for a vaccine purpose and in function of the bacteria used, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) obtained by the method of the invention may induce a vaccine response against the antigen expressed at its surface. As example, the antigen used may be the protein FyuA, the receptor for the yersiniabactin siderophore.

[0051] According to the invention, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) may also contain others antigens which are not specific of the bacteria used to produce the vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane). In this way, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) is used as carrier of heterologous antigens and these antigens are express at the surface of the vesicle (e.g OMVs and / or liposomes derived from bacterial membrane) (e.g. at their membranes). In function of the heterologous antigen used, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) obtained by the method of the invention will induce a vaccine response against this antigen and not against the bacteria used to obtain the vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane). In this case, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) will not express antigens specific of the bacteria and no vaccine response against these bacteria will be induced.In some embodiments, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) may express antigens specific of the bacteria used to obtain the vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) but also heterologous antigens which are not specific of the bacteria used. In this way, all antigens are expressed at the surface of the vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) (e.g. at their membranes). In function of the bacteria used and in function of the heterologous antigen, vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) obtained by the method of the invention will induce a vaccine response against all antigens. In this case, divalent vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) are obtained. According to the invention, several specific antigens and several heterologous antigens may be used to obtain the appropriate vaccine response. In this case, multivalent vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane) are obtained.

[0052] In some embodiments, the heterologous antigen is a viral, a bacterial, a fungal and / or a parasite antigen. In some embodiments, the heterologous antigen may be a viral antigen. In some embodiments, the heterologous antigen is a viral antigen such as an adenovirus, retrovirus, picornavirus, herpesvirus, rotaviruses, hantaviruses, coronavirus, togavirus, flavivirus, rhabdovirus, paramyxovirus, orthomyxovirus, orbivirus, bunyavirus, arenavirus, reovirus, papillomavirus, parvovirus, poxvirus, hepadnavirus, or spongiform virus antigen. In some embodiments, the heterologous antigen is a HIV, CMV, hepatitis A, B, or C, influenza, measles, polio, smallpox, rubella, respiratory syncytial, herpes simplex, varicella zoster, Epstein-Barr, Japanese encephalitis, rabies, flu, or cold viruses’ antigen.

[0053] In some embodiments, the heterologous antigen may be a bacterial antigen. In some embodiments, the heterologous antigen is a bacterial antigen such as streptococcal, mycobacterium, helicobacter, pneumococcal, haemophilus, bacillus, rickettsia, chlamydia or mycoplasma antigen. In some embodiments, when a heterologous antigen is used, said antigen gene may be selected from genes from Neisseria meningitidis or Neisseria meningitidis serogroup A. In another embodiment, the antigen gene may genes encoding for the proteins PorA (Pl.9 and Pl.20), PorB (P3.4 and P3.21) and Opc from Neisseria meningitidis.

[0054] In some embodiments, the heterologous antigen may be a fungal antigen. In some embodiments, the heterologous antigen is a fungal antigen such as histoplasma, cryptococcal, coccidiodes or tinea antigen.In some embodiments, the heterologous antigen may be a parasital antigen. In some embodiments, the heterologous antigen is a parasital antigen such as plasmodial, toxoplasmal, schistosomal, leishmaniae or trypanosomal antigen.

[0055] In some embodiments, the heterologous antigen may be a tumor associated antigen. As used herein, the term "tumor associated antigen" refers to an antigen that is characteristic of a tumor tissue. An example of a tumor associated antigen expressed by a tumor tissue may be the antigen prostatic acid phosphatase (see W02004 / 026238) or MART peptide T (melanoma antigen).

[0056] An antigen (especially a heterologous antigen) can be prepared using a variety of methods well known in the art. A gene encoding any immunogenic polypeptide can be isolated and cloned, for example, in bacterial, yeast, insect, reptile or mammalian cells using recombinant methods well known in the art and described, for example in Sambrook et al., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992) and in Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1998). A number of genes encoding surface antigens from viral, bacterial and protozoan pathogens have been successfully cloned, expressed and used as antigens for vaccine development. For example, the major surface antigen of hepatitis B virus, HbsAg, the P subunit of choleratoxin, the enterotoxin of E. coli, the circumsporozoite protein of the malaria parasite, and a glycoprotein membrane antigen from Epstein-Barr virus, as well as tumor cell antigens, have been expressed in various well-known vector / host systems, purified and used in vaccines. A pathologically aberrant cell may also be used in a vaccine composition according to the invention and can be obtained from any source such as one or more individuals having a pathological condition or ex vivo or in vitro cultured cells obtained from one or more such individuals, including a specific individual to be treated with the resulting vaccine. In a particular embodiment, the transformation of the bacteria with the heterologous antigen gene can be made using a plasmid or not. Alternatively, the antigen gene can be integrated into the bacterial chromosome, in a neutral or silent location, under the control of an inducible promoter. In a particular embodiment, the bacteria plasmid is pGEX-6P-1 or pSA10 (see for example Schlosser-Silverman E et al., 2000).

[0057] Thus, in some embodiments, the invention also relates to a method for producing vesicles, the method comprising the steps of:- Expressing or overexpressing hemolysin F (hlyF) gene and / or an ortholog of hlyF gene in bacteria, said hlyF gene and / or ortholog of hlyF gene encoding respectively a HlyF protein and / or an ortholog of HlyF protein; and

[0058] - Expressing or overexpressing an antigen gene, in particular an antigen gene of said bacteria, in said bacteria.

[0059] Preferentially, the bacteria are Gram-negative bacteria.

[0060] Hence, the present invention also relates to a vaccine composition comprising the vesicles obtained according to any of the methods herein described. The vaccine composition may comprise vesicles with specific antigens of the bacteria used to produce vesicles and / or may comprise heterologous antigens.

[0061] In some embodiments, the invention also relates to a method for producing outer membrane vesicles (OMVs) in Gram-negative bacteria, the method comprising the steps of:

[0062] - Expressing or overexpressing an ortholog of hemolysin F (hlyF) gene in Gram-negative bacteria, said ortholog of hlyF gene encoding an ortholog of HlyF protein; and - Expressing or overexpressing an antigen gene, in particular an antigen gene of said Gram-negative bacteria, in said Gram-negative bacteria.

[0063] Hence, the present invention also relates to a vaccine composition comprising the OMVs obtained according to any of the methods herein described. The vaccine composition may comprise OMVs with specific antigens of the bacteria used to produce the OMVs and / or may comprise heterologous antigens.

[0064] In some embodiments, the invention also relates to a method for producing liposomes derived from bacterial membrane, the method comprising the steps of:

[0065] - Expressing or overexpressing hemolysin F (hlyF) gene and / or an ortholog of hlyF gene in bacteria, said hlyF gene and / or ortholog of hlyF gene encoding respectively a HlyF protein and / or an ortholog of HlyF protein; and

[0066] - Expressing or overexpressing an antigen gene, in particular an antigen gene of said bacteria, in said bacteria.

[0067] Preferentially, the bacteria are Gram-negative bacteria.Hence, the present invention also relates to a vaccine composition comprising the liposomes derived from bacterial membrane, in particular Gram-negative bacterial membrane, obtained according to any of the methods herein described. The vaccine composition may comprise liposomes derived from bacterial membrane, in particular Gram-negative bacterial membrane, with specific antigens of the bacteria used to produce the liposomes derived from bacterial membrane and / or may comprise heterologous antigens.

[0068] A "vaccine composition", once it has been administered to a subject, elicits a protective immune response against said one or more antigen(s) that is (are) comprised herein. Accordingly, the vaccine composition of the invention, once it has been administered to the subject, induces a protective immune response against, for example, a microorganism, or to efficaciously protect the subject against infection.

[0069] The vaccine composition may generally include one or more pharmaceutically acceptable and / or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and / or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. This pharmaceutical composition can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers or recombinant proteins (e. g. human serum albumin) suitable for in vivo administration.

[0070] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, in particular a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0071] As used herein, the term "adjuvant" refers to a substance that enhances, augments or potentiates the host's immune response to an antigen, e.g., an antigen that is part of a vaccine. Non-limiting examples of some commonly used vaccine adjuvants include insoluble aluminum compounds, calcium phosphate, liposomes, Virosomes™, ISCOMS®, microparticles (e.g.,PLG), emulsions (e.g., MF59, Montanides), virus-like particles & viral vectors. PolylCLC (a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), which is a TLR3 agonist, is used as an adjuvant in the present invention. It will be understood that other TLR agonists may also be used (e.g. TLR4 agonists, TLR5 agonists, TLR7 agonists, TLR9 agonists), or any combinations or modifications thereof. Examples of adjuvants that may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA. In addition, immune modulating substances such as lymphokines (e.g., IFN-[gamma], IL-2 and IL-12) or synthetic IFN-[gamma] inducers such as poly I: C can be used in combination with adjuvants described herein. Suitable adjuvants include any acceptable immunostimulatory compound, such as cytokines, chemokines, cofactors, toxins, plasmodia, synthetic compositions or vectors encoding such adjuvants. Adjuvants that may be used in accordance with embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, y-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion is also contemplated. MHC antigens may even be used. Exemplary adjuvants may include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and / or aluminum hydroxide adjuvant.

[0072] As used herein, the term ‘TLR4 agonist” denotes a compound or a molecule which bind the Toll-like receptor 4 and active it. According to the invention, a TLR4 agonist may be selected from the group consisting of Ethanol, Morphine-3 -glucuronide, Morphine, Oxycodone, Levorphanol, Pethidine, Glucuronoxylomannan from Cryptococcus, Fentanyl, Methadone, Buprenorphine, Lipopolysaccharides (LPS), Carbamazepine, Oxcarbazepine. In a particular embodiment, the TLR4 agonist according to the invention is selected from the group consisting of the LPS or monophosphoryl lipid A (MPL). Various TLR4 agonists are known in the art, including Monophosphoryl lipid A (MPLA), in the field also abbreviated to MPL, referring tonaturally occurring components of bacterial lipopolysaccharide (LPS); refined detoxified endotoxin. For example, MPL is a derivative of lipid A from Salmonella minnesota R595 lipopolysaccharide (LPS or endotoxin). While LPS is a complex heterogeneous molecule, its lipid A portion is relatively similar across a wide variety of pathogenic strains of bacteria. MPL, used extensively as a vaccine adjuvant, has been shown to activate TLR4 (Martin M. et al., 2003. Infect Immun. 71(5):2498-507; Ogawa T. et al., 2002. Int Immunol. 14(11):1325-32). TLR4 agonists also include natural and synthetic derivatives of MPLA, such as 3-de-O-acylated monophosphoryl lipid A (3D- MPL), and MPLA adjuvants available from Corixa Corporation (Seattle, Wash.; see US Patents 4,436,727; 4,436,728; 4,987,237; 4,877,611; 4,866,034 and 4,912,094 for structures and methods of isolation and synthesis). A structure of MPLA is disclosed in US 4,987,237. Non-toxic diphosphoryl lipid A (DPLA) may also be used, for example OM-174, a lipid A analogue of bacterial origin containing a triacyl motif linked to a diglucosamine diphosphate backbone. Another class of useful compounds are synthetic lipid A analogue pseudo-dipeptides derived from amino acids linked to three fatty acid chains (see for example EP 1242365), such as OM-197-MP-AC, a water-soluble synthetic acylated pseudodipeptide (C55H107N4O12P). Non-toxic TLR4 agonists include also those disclosed in EP1091928, PCT / FR05 / 00575 or PCT / IB2006 / 050748. PCT / US2006 / 002906 / WO 2006 / 083706; PCT / US 2006 / 003285 / WO 2006 / 083792; PCT / US 2006 / 041865; PCT / US 2006 / 042051. TLR4 agonists also include synthetic compounds which signal through TLR4 other than those based on the lipid A core structure, for example an aminoalkyl glucosaminide 4-phosphate (see Evans JT et al. Expert Rev Vaccines. 2003 Apr;2(2):219-29; or Persing et al. Trends Microbiol. 2002;10(10 Suppl): S32-7. Review). Other examples include those described in Orr MT, Duthie MS, Windish HP, Lucas EA, Guderian JA, Hudson TE, Shaverdian N, O'Donnell J, Desbien AL, Reed SG, Coler RN. MyD88 and TRIF synergistic interaction is required for THl-cell polarization with a synthetic TLR4 agonist adjuvant. Eur J Immunol.

[0073] 2013 May 29. doi: 10.1002 / eji.201243124.; Lambert SL, Yang CF, Liu Z, Sweetwood R, Zhao J, Cheng L, Jin H, Woo J. Molecular and cellular response profiles induced by the TLR4 agonist-based adjuvant Glucopyranosyl Lipid A. PLoS One. 2012;7(12):e51618. doi: 10.1371 / journal. pone.0051618. Epub 2012 Dec 28.

[0074] As used herein, the term ‘TLR9 agonist” denotes a compound or a molecule that binds the Toll-like receptor 9 and actives it. According to the invention, a TLR9 agonist may be selected from the group consisting of CpG oligonucleotides (ODN) and its derivatives. In particularembodiment, the TLR9 agonist is the CpG (ODN). Examples of TLR9 agonists (include nucleic acids comprising the sequence 5'-CG-3' (a " CpG nucleic acid") in certain aspects C is unmethylated. The terms "polynucleotide," and "nucleic acid," as used interchangeably herein in the context of TLR9 agonist molecules, refer to a polynucleotide of any length, and encompasses, inter alia, single- and double-stranded oligonucleotides (including deoxyribonucleotides, ribonucleotides, or both), modified oligonucleotides, and oligonucleosides, alone or as part of a larger nucleic acid construct, or as part of a conjugate with a non-nucleic acid molecule such as a polypeptide. Thus, a TLR9 agonist may be, for example, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA). TLR9 agonists also encompass crude, detoxified bacterial (e.g., mycobacterial) RNA or DNA, as well as enriched plasmids enriched for a TLR9 agonist. In some embodiments, a " TLR9 agonist-enriched plasmid" refers to a linear or circular plasmid that comprises or is engineered to comprise a greater number of CpG motifs than normally found in mammalian DNA. Examples of non- limiting TLR9 agonist-enriched plasmids are described in Roman et al. (1997). In general, a TLR9 agonist used in a subject composition comprises at least one unmethylated CpG motif. In some embodiments, a TLR9 agonist comprises a central palindromic core sequence comprising at least one CpG sequence, where the central palindromic core sequence contains a phosphodiester backbone, and where the central palindromic core sequence is flanked on one or both sides by phosphorothioate backbone-containing polyguanosine sequences. In other embodiments, a TLR9 agonist comprises one or more TCG sequences at or near the 5' end of the nucleic acid; and at least two additional CG dinucleotides. In some of these embodiments, the at least two additional CG dinucleotides are spaced three nucleotides, two nucleotides, or one nucleotide apart. In some of these embodiments, the at least two additional CG dinucleotides are contiguous with one another. In some of these embodiments, the TLR9 agonist comprises (TCG)n, where n = 1 to 3, at the 5' end of the nucleic acid. In other embodiments, the TLR9 agonist comprises (TCG)n, where n = 1 to 3, and where the (TCG)n sequence is flanked by one nucleotide, two nucleotides, three nucleotides, four nucleotides, or five nucleotides, on the 5' end of the (TCG)n sequence. A TLR9 agonist of the present invention includes, but is not limited to, any of those described in U. S. Patent Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705, 6,426,334 and 6,476,000, and published US Patent Applications US 2002 / 0086295, US 2003 / 0212028, and US 2004 / 0248837. Examples of others TLR4 or TLR9 agonists are described in WO 2012 / 021834, the contents of which are incorporated herein by reference.A variety of substances, used as supplemental antigens, can be added to the vaccine composition. For example, attenuated and inactivated viral and bacterial pathogens, purified macromolecules, toxoids, recombinant antigens, organisms containing a foreign gene from a pathogen, synthetic peptides, polynucleic acids, antibodies and tumor cells can be used to prepare (i) an immunogenic composition useful to induce an immune response in a subject or (ii) a vaccine useful for treating a pathological condition. According to the invention the supplemental antigen gene may contain a signal sequence which allows the expression of the antigen in the membrane of the vesicles (e.g. liposomes derived from bacterial membrane or OMVs).

[0075] Therefore, the vaccine composition of the invention can be combined with a wide variety of antigens to produce a vaccine composition useful for inducing an immune response in an individual or in an animal. Those skilled in the art will be able to select an antigen appropriate for treating a particular pathological condition and will know how to determine whether an isolated antigen is favored in a particular vaccine formulation.

[0076] In another particular embodiment, the vaccine composition according to the invention, further comprises one or more components selected from the group consisting of surfactants, absorption promoters, water absorbing polymers, substances which inhibit enzymatic degradation, alcohols, organic solvents, oils, pH controlling agents, preservatives, osmotic pressure controlling agents, propellants, water and mixture thereof.

[0077] The vaccine composition according to the invention can further comprise a pharmaceutically acceptable carrier. The amount of the carrier will depend upon the amounts selected for the other ingredients, the desired concentration of the antigen, the selection of the administration route, oral or parenteral, etc. The carrier can be added to the vaccine at any convenient time. In the case of a lyophilised vaccine, the carrier can, for example, be added immediately prior to administration. Alternatively, the final product can be manufactured with the carrier. Examples of appropriate carriers include, but are not limited to, sterile water, saline, buffers, phosphate-buffered saline, buffered sodium chloride, vegetable oils, Minimum Essential Medium (MEM), MEM with HEPES buffer, etc.Examples of suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such asNZ-Amine orNZ-Amine AS. Examples of emulsifiers include, but are not limited to, mineral oil, vegetable oil, peanut oil and other standard, metabolizable, nontoxic oils useful for injectables or intranasal vaccines compositions.

[0078] Conventional preservatives can be added to the vaccine composition in effective amounts ranging from about 0.0001% to about 0.1% by weight. Depending on the preservative employed in the formulation, amounts below or above this range may be useful. Typical preservatives include, for example, potassium sorbate, sodium metabisulfite, phenol, methyl paraben, propyl paraben, thimerosal, etc.

[0079] The vaccine composition of the invention can be formulated as a solution or suspension together with a pharmaceutically acceptable medium. Such a pharmaceutically acceptable medium can be, for example, water, phosphate buffered saline, normal saline or other physiologically buffered saline, or other solvent or vehicle such as glycol, glycerol, and oil such as olive oil or an injectable organic ester. A pharmaceutically acceptable medium can also contain liposomes or micelles, and can contain immunostimulating complexes prepared by mixing polypeptide or peptide antigens with detergent and a glycoside, such as Quil A. Liquid dosage forms for oral administration of the vaccine composition of the invention include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient(s), the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0080] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0081] Suspensions, in addition to the active ingredient(s), may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0082] Formulations of the vaccine compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing the active ingredient(s) with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or salicylate and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active ingredient(s). Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0083] Vaccine compositions of this invention suitable for parenteral administration comprise the active ingredient(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that may be employed in the vaccine compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0084] These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0085] Injectable depot forms are made by forming microencapsule matrices of the active ingredient(s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of theactive ingredient(s) to polymer, and the nature of the particular polymer employed, the rate of release of the active ingredient(s) can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable formulations are also prepared by entrapping the active ingredient(s) in liposomes or microemulsions that are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0086] The amount of vesicles (e.g. OMVs and / or liposomes derived from bacterial membrane), eventually supplemental antigen and adjuvant composition in the vaccine composition according to the invention are determined by techniques well known to those skilled in the pharmaceutical art, taking into consideration such factors as the particular antigen, the age, sex, weight, species, and condition of the particular subject, and the route of administration. While the dosage of the vaccine composition depends notably upon the antigen, species of the host vaccinated or to be vaccinated, etc., the dosage of a pharmacologically effective amount of the vaccine composition will usually range from about 0.01 pg to about 500 pg (and in particular 50 pg to about 500 pg) of the adjuvant compound of the invention per dose. Although the amount of the particular antigenic substance in the combination will influence the amount of the adjuvant compound according to the invention, necessary to improve the immune response, it is contemplated that the practitioner can easily adjust the effective dosage amount of the adjuvant compound through routine tests to meet the particular circumstances. The vaccine composition according to the invention can be tested in a variety of preclinical toxicological and safety studies well known in the art. For example, such a vaccine composition can be evaluated in an animal model in which the antigen has been found to be immunogenic and that can be reproducibly immunized by the same route proposed for human clinical testing. For example, the vaccine composition according to the invention can be tested, for example, by an approach set forth by the Center for Biologies Evaluation and Research / Food and Drug Administration and National Institute of Allergy and Infectious Diseases. Those skilled in the art will know how to determine for a particular vaccine composition, the appropriate antigen payload, route of immunization, volume of dose, purity of antigen, and vaccination regimenuseful to treat a particular pathological condition in a particular animal species. In a vaccination protocol, the vaccine may be advantageously administered as a unique dose or preferably, several times e.g., twice, three or four times per week or month intervals, according to a prime / boost mode. The appropriate dosage depends upon various parameters. As a general rule, the vaccine composition of the present invention is conveniently administered orally, parenterally (subcutaneously, intramuscularly, intravenously, intradermally or intraperitoneally), intrabuccally, intranasally, or transdermally, intralymphatically, intratumorally, intravesically, intraperitoneally and intracerebrally. The route of administration contemplated by the present invention will depend upon the antigen.

[0087] According to the invention, the vaccinal composition of the present invention may be used in human medicine or veterinary medicine. In other word, the vaccinal composition may be useful to prevent or cure a disease in human and / or animal.

[0088] Screening method

[0089] Another object of the invention relates to a method for screening a substance that may be useful for the prevention and treatment of a Gram-negative bacteria infection comprising a step of determining the ability of a candidate substance to inhibit the expression of an ortholog of hlyF gene or the activity of the protein encoded by said gene. In some embodiments, the Gramnegative bacteria are Pseudomonas sp, in particular Pseudomonas aeruginosa.

[0090] In some embodiments, the present invention also relates to a method for screening a substance that may be useful for the prevention and treatment of a Gram-negative bacteria infection, the method comprising the steps of:

[0091] a) providing a composition comprising the Gram-negative bacteria;

[0092] b) adding the candidate substance to be tested to the composition provided at step a), thereby providing a test composition;

[0093] c) comparing the activity of the ortholog of hlyF gene or the activity of the protein encoded by said gene (e.g. OMV production) in said test composition with the activity of the same ortholog of hlyF gene or protein in the absence of said candidate substance; and

[0094] d) selecting positively the candidate substance that inhibits the expression of the ortholog of hlyF gene or the activity of the protein encoded by said gene.In some embodiments, the inhibition of the gene expression according to the invention may be monitored by the production of the protein encoded by this gene. The invention also encompasses methods for the screening of candidate substances that are based on the ability of said candidate substances to inhibit the activity of the protein encoded by the ortholog of hlyF gene. Accordingly, the invention also refers to a method for screening a substance that may be useful for the prevention and treatment of a Gram-negative bacteria infection, the method comprising the steps of:

[0095] a) providing a composition comprising the proteins encoded by the ortholog of hlyF gene;

[0096] b) adding the candidate substance to be tested to the composition provided at step a), thereby providing a test composition;

[0097] c) comparing the activity of said protein in said test composition with the activity of the protein in the absence of said candidate substance; and

[0098] d) selecting positively the candidate substance that inhibits the activity of said protein.

[0099] Candidate substances that have been positively selected with one of the methods herein described may then be tested in various in vitro assays. In another embodiment, the candidate substances may be tested. As example, the anti -autophagic activity can reflect the production of specific OMVs, whereas the absence of anti -autophagic activity can reflect the absence of production of specific OMVs. Thus, activity of the candidate substance can be tested by monitoring the production or not of OMVs with anti -autophagic activity.

[0100] This invention also encompasses methods for the screening of candidate substances, that are based on the ability of said candidate substances to bind to a protein encoded by the ortholog of hlyF gene as defined herein, thus methods for the screening of potentially substances that may be useful for the prevention and treatment of a Gram-negative bacteria infection. The binding assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art. All binding assays for the screening of candidate substances that may be useful for the prevention and treatment of a Gram-negative bacteria infection are common in that they comprise a step of contacting the candidate substance with a protein as defined herein, under conditions and for a time sufficient to allow these two components to interact. These screening methods also comprise a step of detecting the formation of complexes betweensaid protein encoded by the ortholog of hlyF gene and said candidate substances. Thus, screening for substances that may be useful for the prevention and treatment of Gram-negative bacteria infection includes the use of two partners, through measuring the binding between two partners, respectively a protein as defined herein and the candidate substance. In binding assays, the interaction is binding and the complex formed between a protein encoded by the ortholog of hlyF gene as defined above and the candidate substance that is tested can be isolated or detected in the reaction mixture. In a particular embodiment, the protein as defined above or alternatively the anti-Gram-negative bacteria candidate substance is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the protein of the invention and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the protein of the invention to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.

[0101] The binding of the anti-Gram-negative bacteria candidate to a protein of the invention may be performed through various assays, including traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns.

[0102] In addition, protein-protein interactions can be monitored by using surface plasmon resonance, double hybrid in yeast, co-immunolocalization, FRET, proximity ligation assay, coimmunoprecipitation, molecular bridging or capture by “GST-pulldown. As example, a yeastbased genetic system is described by Fields and co-workers (Fields and Song, 1989; Chien et al., 1991) as disclosed by Chevray and Nathans, 1991. Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for beta-galactosidase. A complete kit (MATCHMAKER. TM.) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.

[0103] In some embodiments, the method for the screening of substances that may be useful for the prevention and treatment of a Gram-negative bacteria infection comprises the steps of:

[0104] (i) providing a candidate substance;

[0105] (ii) assaying said candidate substance for its ability to bind to the protein of the invention.

[0106] In some embodiments, the method for the screening of substances that may be useful for the prevention and treatment of a Gram-negative bacteria infection comprises the steps of:

[0107] (i) contacting a candidate substance with a protein of the invention;

[0108] (ii) detecting the complexes eventually formed between said protein and said candidate substance.

[0109] Thus, any substance that has been shown to behave like an inhibitor of the genes or proteins according to the invention, after positive selection at the end of any one of the screening methods that are disclosed previously in the present specification, may be further assayed for his in vivo activity. Consequently, any one of the screening methods that are described above may comprise a further step of assaying the positively selected inhibitor substance for its in vitro or in vivo activity.

[0110] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.FIGURES:

[0111] Figure 1. Amino acid homology and structural prediction of CprA and HlyF. a, Multiple sequence alignment of HlyF and CprA orthologous proteins from E. coli strain SP15 and P. aeruginosa strains PAK and PAO1 was performed using Clustal Omega. The mutant of HlyF was constructed by site-directed mutagenesis of the predicted catalytic site leading to the two substitutions Y163F and K167A. The CprA protein from P. aeruginosa strain PAO1 is truncated due to a deletion of the cytosine at the position 670 of the gene. This results in a frame shift and a premature stop codon at position 245. The residues' identity and similarity are highlighted in dark gray and light gray, respectively, b, Alphafold 2 protein structure prediction was used to show the three-dimensional structure of the HlyF and CprA proteins from E. coli and P. aeruginosa. In both panels, the NAD(P)H binding sites and catalytic sites are depicted in green and blue, respectively, c, the iPBA web server was used to compare the secondary structure of CprA (in blue) and HlyF (in pink). (https: / / www.dsimb.inserm.fr / dsimb_tools / ipba / index.php).

[0112] Figure 2. OMVs derived from CprA or HlyF-producing E. coli and OMVs from P. aeruginosa that produce a full-length CprA trigger autophagosomes in HeLa cells and inhibit the autophagy flux of HeLa cells, a, this basal autophagy flux model describes the following five stages: (1) elongation and formation of a double-membrane LC3-II-dependent vesicle containing cytoplasmic components; (2) newly formed autophagosome; (3) fusion of the autophagosome with the lysosome; (4) degradation and (5) recycling of autophagolysosome contents. (PE, phosphoethanolamine). Created with BioRender.com. b, c, Fluorescent microscopy images of GFP-LC3 (green) in HeLa cells expressing green fluorescent protein-fused with LC3. For panel b, cells were treated for 3 hours with 5 pg / mL of OMVs purified from E. coli BL21 DE3 strain expressing the wild-type hlyF (hlyF WT) from E. coli strain SP15 (pAGO-15), the mutant in the catalytic site (hlyF Y163F, K167A) (pAGO-16), or cprA from P. aeruginosa strain PAK (pAGO-32) or strain PAO1 (pAGO-30). For panel c, cells treated for 5 hours with 50 pg / mL of OMVs purified from P. aeruginosa strains PAK, PAO1 and PAO1 + pJN cprAPAK(pAGO-38), or PAO1 + pJN cprAPAO1(pAGO-37). In b and c, the scale bar in the images represents 25 pm. These images are representative of three independent experiments. d,e Western blot analysis of LC3 (forms I and II, as autophagy marker) and actin (as loading control) in HeLa cells. For panel d, cells were treated as in panel b. For panel e, cells were treated for 5 hours with 50 pg / mL of OMVs purified from P. aeruginosa strains PAK, PAKcprA, PAO1 and PAO1 + pJN cprAPAO1(pAGO-37), or for 1.5 hours with 50 pg / mL of OMVs purified from P. aeruginosa strains PAK ΔcprA attB::PRha-cprA and PAO1 + pJN cprAp AK(pAGO-38) (in bold). Each western-blot is representative of three independent experiments. In all panels, non-treated cells (NT) were used as a control, f, Schema for visualizing basal or blocked autophagy flux in the HeLa-Difluo hLC3 cell line. The cells express a LC3 protein fused with red fluorescent protein (RFP), which is resistant to acidic conditions, and green fluorescent protein (GFP), which is sensitive to acidity. In the case of basal autophagic flux, the GFP-LC3 signal decreases due to the fusion of autophagosomes with acidic lysosomes. Subsequently, the autophagolysosome undergoes degradation and recycling, preventing the accumulation of autophagosomes. Basal autophagy is visualized through fluorescence microscopy as diffuse GFP and RFP labeling. When the autophagic flux is blocked at the lysosomal fusion step, autophagosomes accumulate with a basic pH, which is visualized as the co-localization of GFP and / or RFP signals in foci. If the autophagic flux is blocked after the lysosomal fusion step, the autophagolysosome accumulates with an acidic pH, which is observed by a low GFP-LC3 signal and intact RFP-LC3 foci37,38. Created with BioRender.com g, Confocal images of DiFluo HeLa cells were captured under different conditions: untreated (NT), deprived by incubation for 5 hours in HBSS, treated for 5 hours with 50 pM chloroquine, or treated with 50 pg / mL of OMVs from P. aeruginosa strains PAK, PAO1, and PAO1 + pJN cprAPAK(pAGO-38), and PAO1 + pJN cprAPAO1(pAGO-37). Non-treated cells (NT) were used as a control. The scale bar in the images represents 10 pm. These images are representative of three independent experiments.

[0113] Figure 3. OMVs from P. aeruginosa producing the full-length of CprA enhance IL-ip secretion and cell death in human monocytes, a, Schema of simplified canonical pathway of NLRP3 inflammasome and of non-canonical inflammasome after activation with pathogen or danger-associated patterns (PAMPs or DAMPs), Gram-negative bacteria or OMVs. The assembly of the NLRP3 canonical inflammasome relies on the sensing of cytoplasmic stress signals such as K+ efflux, in response to prior detection of extracellular PAMP and DAMPs, and lead to the activation of caspase-1 (CASP1). Assembly of the non-canonical inflammasome occurs after sensing of cytoplasmic LPS (from bacteria or OMVs) by the CARD domain of caspase-4 (CASP4), leading to the activation of its own autocatalytic subunit. Pro-IL-ip was cleaved to its mature form (IL-ip) by the activated CASP1. Activated CASP1 and CASP4 cleave gadermin-D (GSDMD), leading to pore formation at the plasma membrane, that triggerscell death by pyroptosis and release of the cytosolic contents such as of LDH, IL-ip and K+, acting as stress signal to the surrounding cells. K+efflux induces the assembly of the NLRP3 canonical inflammasome, and the maturation of pro-IL-ip via CASP139 41. Here, we used human monocytic THP-1 wild-type (WT) cells, or THP-1 cells genetically invalidated for NRLP3, CASP4 or GSDMD. In NLRP3 KO, there is no more secretion of mature IL-ip, but LDH secretion, through CASP4 mediated pyroptosis, still occurs. In CASP4 KO and in GSDMD KO, the cell death is abrogated and the mature IL-ip is severely impaired when the non-canonical inflammasome is activated by OMVs. Created with BioRender.com. b, Release of LDH and c, IL1-P from primed WT, NLRP3 KO, CASP4 KO THP-1 and GSDMD KO THP-1 cells treated overnight with 12,5 pg / mL of OMVs from P. aeruginosa strains PAK, PAK cprA and PAK ΔcprA attB::PRha-cprA. For b and c, the graphs show the mean and the standard deviation of 3 independent experiments for each condition, each point represents the value obtained in one experiment. Significance was determined by a 2-way ANOVA, with Tuckey’s multiple comparisons test. ****p < 0.0001, ** p < 0.005, * p < 0.05.

[0114] Figure 4. CprA activity is dependent on the two-component system PmrA / B. Western blot analysis of LC3 and actin in Hela cells treated for 1 hour with 50 pg / mL of OMVs from P. aeruginosa strain PAI 4, the pmrAB mutant carrying the empty vector (pME6012), the mutant strain complemented with a native pmrAB allele (pABWT) or complemented with a gain-of-function pmrAB allele (pAB16.2). Bacteria were cultivated in M63 supplemented with two different concentrations of MgCh: one (0.1 mM) described to activate the PmrAB two-component regulatory system7, and one (2 mM) described to inactivate the PmrAB two-component regulatory system. Non-treated cells (NT) were used as a control. Blots are representatives of three independent experiments.

[0115] Figure 5. CprA increases pathogenicity during sepsis. C57BL / 6 mice were infected intraperitoneally with wild-type P. aeruginosa PA14 strain, cprA isogenic mutant PAM l^cprA) or with PAM cprA strain complemented with cprA (PAM cprA a B. PRha-cprA'). a, Clinical score according to Table not shown at 4 and 8-hours post-injection. Results shown are pooled from three independent experiments (n=20-25). Differences between the experimental groups were evaluated by two-way ANOVA followed by Tuckey’s results: * p < 0.05; ** p < 0.01; *** p < 0.001. Mean values ± SEM are shown, b, The time to humane euthanasia (when the clinical score reached a predefined threshold) was monitored to build the survival curve.Results are pooled from two independent experiments and the total number of animals is shown (n=10-15). The difference between the experimental groups was evaluated by the log-rank test (Mantel-Cox): ** p < 0.01. c, Protein levels of the cytokines IL1-P from mouse spleens 8 hours post-infection were determined in proteins extracted from tissues using ELISA. The graphs show the mean and the standard deviation from an independent experiment (n=10). Significance was determined by a 1-way ANOVA, ** p < 0.005, * p < 0.05. ANOVA, analysis of variance; SEM, standard error.

[0116] Figure 6. OMVs from E. coli expressing various CprA orthologs trigger the accumulation of autophagosomes in HeLa cells, a, Maximum likelihood phylogenetic tree of HlyF / CprA orthologs present in various bacterial species, rooted on the most distant and non-functional protein found in Porphyromonas gingivalis, here the only representative of the Bacteroidia class. This protein shares the amino acids present in the NAD(P)H binding site and in the catalytic site with CprA, but lacks the highly conserved C-terminus part of the protein with D-F / Y-K residues. Other bacterial species belong to two main classes: the Gammaproteobacteria and the Betaproteobacteria. The colors indicate the taxonomic order: black for the Enter obacter ales, blue for the Pseudomonadales, and red for the Burkholderales. Orthologs activity from the strains marked with an asterisk (*) are tested in panel b. The scale bar represents the number of substitutions per site, b, Western blot analysis of LC3 and actin in HeLa cells treated for three hours with 5 pg of OMVs purified from E. coll BL21 (DE3) strain expressing hlyF from E. coli SP15 (pAGO-15), or the orthologs from E. cloacae ATCC13047 (pAGO-17), K. pneumoniae SB4496 (pAGO-19), K. aerogenes ATCC13048 (pAGO-21), S. marcescens SM39 (pAGO-23), P. aeruginosa PAK (pAGO-32), R. solanacearum GMI1000 (pAGO-29), P. gingivalis ATCC33277 (pAGO-42), or with 20 pg of OMVs purified from E. coli BL21 DE3 strain expressing Y. pestis KIM6+ (pAGO-53), Y. pseudotuberculosis IP32953 (pAGO-55). Non-treated cells (NT) were used as a control. Blot shown is representative of 3 independent experiments.

[0117] Figure 7. Amino acid homology between HlyF, CprA and their orthologous proteins: focus on two most distant functional orthologs in E. coli SP15 and R. solanacearum GMI1000. a, Plot of HlyF orthologs GC% on corresponding genomes GC%. b, Structural comparison of the secondary structure of the R. solanacearum GMI100 ortholog (in orange) with CprA (in blue) or HlyF (in pink), using the iPBA web server(https: / / www.dsimb.inserm.fr / dsimb_tools / ipba / index.php). c, Three-dimensional structure of the HlyF and its ortholog protein from R. solanacaerum using Alphafold 2 protein structure prediction, d, Multiple sequence alignment of HlyF and its ortholog proteins from R. solanacaerum, using Multialign Viewer in Chimera X. Matched residues highlighted in gray were obtained using the Matchmaker tool to overlay the two 3D structures with default settings, and corresponds to structures that are fit iteratively with a cutoff of 2.0 A. In b and c, the NAD(P)H binding sites, catalytic sites and residues that are 100% conserved in all orthologs of are shown in green, blue and orange, respectively.

[0118] Figure 8. Lipids from CprA producing P. aeruginosa trigger autophagosomes in HeLa cells. Western blot analysis of LC3 and actin in Hela cells treated a, for 1 hour with 50 pg / mL of OMVs, b, for 6 hours with an equivalent amount of lipids extracted from 3.33 pg of OMVs from P. aeruginosa PAK or PAK IscprA OMVs’. In both panels, bacteria were cultivated in M63 supplemented with two different concentrations of MgCh: one (0.1 mM) described to activate the PmrAB two-component regulatory system, and one (2 mM) described to inactivate the PmrAB two-component regulatory system. Non-treated cells (NT) were used as a control.

[0119] Figure 9. (a-c) Liposomes made with bacterial lipids extracted from bacterial membranes of CprA-producing bacteria inhibits autophagy. Western blot analysis of LC3 and actin in Hela cells treated a, for 1 hour with 50 pg / mL of OMVs, b, for 6 hours with an equivalent amount of lipids extracted from 3.33 pg of OMVs from P. aeruginosa PAK or PAK IscprA OMVs’, c) for 6 hours with 50 pg of lipids extracted from P. aeruginosa PAK or PAK IscprA. Non-treated cells (NT) were used as a control. In the three panels, bacteria were cultivated in M63 supplemented with 0.1 mM MgCh.

[0120] Figure 10. a, Western blot analysis for the detection of the N-terminal tagged proteins, b, Western blot analysis for the detection of LC3 and actin.

[0121] EXAMPLE:

[0122] Material and Methods

[0123] Bacterial strains and growth conditions. Bacteria were routinely grown in lysogeny broth (LB, Lennox). For OMVs production, bacteria were grown in Terrific broth (TB) (Gibco) or in M63 minimal medium (Bio Basic) supplemented with 0.5% Bacto™ casamino acids(ThermoFisher), 0.2% D+glucose solution and MgCh (0.1 or 2 mM) (Sigma Aldrich). For plasmid maintenance in E. coli, antibiotics were used at the following concentrations: 50 mg / L kanamycin sulfate, 20 mg / L gentamicin, 10 mg / L tetracycline and 30 mg / L streptomycin. For plasmid maintenance in P. aeruginosa, antibiotics were used at 500 mg / L streptomycin, 50 mg / L tetracycline, 20 mg / L gentamicin for plasmid. 10 mM L-arabinose (L-Ara) (Sigma Aldrich) was added to the bacterial culture for induction of cprA under the arabinose inducible promoter and 6 mM L-rhamnose (Sigma Aldrich) was added to the bacterial culture for the induction of cprA under the rhamnose inducible promoter.

[0124] Plasmid construction. Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega Corporation, Charbonnieres-les-Bains, France) before amplifications by PCR. Expression plasmids used in E. coli were built from plasmid pK184 using the NEB HiFi cloning Kit (NEB), with oligonucleotides. The plasmid pAGO-15 was first constructed by the insertion of a PCR product within the EcoRI and Sa A restriction sites of pK184. The insert of pAGO-15 contains the intergenic region (432 pb) upstream the start codon of the hfyF ORF and a translational fusion of a 6-His and S-tag at the N-terminus of HlyF. The upstream intergenic region and the hlyF ORF were PCR-amplified with the genome of E. coli SP15 as a matrix, and the tags were amplified from the plasmid pET-30-Ek / LIC (Novagen) with primers. The three PCR products were assembled by NEB Hifi cloning and then digested by EcoRI and SaA for cloning in pK184. The other plasmids were built from pAGO-15, after its linearization and the exchange of the hlyF ORF with the different alleles of cprA from P. aeruginosa or the different CprA orthologs. The plasmid pAGO-15 was modified with the QuikChange site-directed mutagenesis method (Agilent Technologies) to obtain pAGO-16 harboring hlyF mutated at Y163F and K167A. The expression plasmids were built from pJN105 to obtain the genes under control of the ParaBAD promoter. The N-terminally tagged hlyF and cprA were amplified by PCR from pAGO-15 and pAGO-32 with the primers, digested with Sad and PsA and ligated in pJN105 digested with the same enzymes.

[0125] Mutants and complemented P. aeruginosa strains construction. The deletion mutants of P. aeruginosa PA AcprA, PMAAcprA and PAI SpmrAB were constructed by allelic exchange20as described by Bolard etal. (2019)21. Briefly, the region upstream and downstream of the target gene were PCR amplified using primers cprAupFor / cprAupRev and cprAdownFor / cprAdownRev respectively, for PAKAc / vd and PA14AcprA and primers PCR-ipmrABCl / C2 and PCR-ipmrABC3 / C4 respectively, for PA14A / wzr 8 (data not shown). For both mutants, the PCR products were either cloned into the pKNGlOl suicide vector22by one-step sequence and ligation-independent cloning (SLIC)23for PAKAc / vA, or cloned into the pCR2.1-TOPO vector (ThermoFisher) and subcloned in pKNGlOl by restriction ligation with BamAMApaX for PAAA pmrAB. The resulting plasmids pKNGAc / vd and pKNGA / wz maintained in E. coli CCI I8X / W' and E. coli HB101, respectively, were then mobilized into PAK or PA14 by triparental mating as previously described22. Transconjugants in which the double recombination events occurred, were analyzed by PCR analysis to confirm the gene deletion24. The resulting mutant PA14A / ?mrA8 was further transcomplemented with plasmid pME6012 and its derivatives21,25. The cis-complementation of PAKAc / vd, PA14AcprA and the construction of the overproducing strain PAO1 attB: '. PRha-cprA was performed by the insertion of cprA under a rhamnose inducible promoter in the attB chromosomic site. The cprA gene and His- and S-tags were amplified by PCR from the matrix pAGO-32 using cprARhaup and cprARhadown and cloned by SLIC into the miniCTXl-rhaSR- rAaBAD26. Transfer of MiniCTX- A / za-c / z in P. aeruginosa PAV cprA and PA\4AcprA strains was carried out by triparental mating, and then was selected for the insertion of PRha-cprA in the attB site.

[0126] Purification of bacterial OMVs. Briefly, after a 8 hours culture, the supernatant was recovered after centrifugation, sterilized by filtration, concentrated through a 100-kDa MWCO tangential flow filtration unit and then ultracentrifuged. Residual and surface proteins from the sample were digested with the Pronase, and OMVs were washed out by ultracentrifugation before undergoing an iodixanol density gradient ultracentrifugation, allowing the selection of fractions containing pure OMVs. The concentration of OMVs in the suspension was correlated with the protein concentration measured by BCA protein assay.

[0127] Preparation of liposomes from OMVs lipids. Lipids from P. aeruginosa OMVs were extracted using a method adapted from Bligh and Dyer27,with the solvent ratio 2: 1 MeOH: CH2C12. This protocol has been described to recover only lipids, without protein or LPS contaminants, the latter being retained in the aqueous phase28. Following the drying the organic phase under N2, the lipid pellets were resuspended in DMEM IX without phenol red (ref 21063, Gibco). Well-calibrated - liposomes were then formed by lipid extrusion through 200 nm, 100 nm and 50 nm membranes, according to the manufacturer instruction about the Avanti Mini Extruder Extrusion Technique (Avanti Polar Lipids Inc). Lipid concentrations in the liposome solutionwere quantified by the sulfo-phospho-vanillin (SPV) lipid assay, following the method of Izard and Limberger29with Triolein as reference (ASTM® Triolein Solution, ref 44896-U, SigmaAldrich).

[0128] Eukaryotic cell culture. HeLa cells (ATCC CCL-2), GFP-LC3 HeLa cells and HeLa-Difluo hLC3 cells (Invivogen) were cultured as previously described19. THP-1 cells and associated genetically invalidated cells (Invivogen, THP1-Null2, thp-kocasp4z, thp-konlrp3, thp-kogsdmdz) were cultured and maintained in RPMI supplemented with 10% heat-inactivated FCS at 37°C, 5% CO2. THP-1 cells were first pre-stimulated with 10 ng / mL of IFNy (Invivogen, rcyec-hifng) overnight and subsequently primed with 1 pg / mL of LPS (Invivogen, tlrl-eklps) for 3 hours. Cells were washed 3 times in PBS and then seeded at the density of 106cells / mL in 24 well plates. Different amounts of specified OMVs were then incubated with cells for 18 hours before analysis.

[0129] Cell lysis and IL-ip release determination. Cell culture supernatants were harvested and centrifuged at 2000 x gfor 10 minutes to remove cellular debris. LDH release, a marker of cell lysis, was assayed in 50 pL of the supernatant by using the Pierce kit (ThermoFisher, 88953) according to the manufacturer instructions. IL-ip release was assayed by ELISA on lOOpL of the supernatant, beforehand diluted by 2, by using the Invitrogen kit (88-7261).

[0130] Transmission Electron Microscopy. Negative staining of OMVs for TEM was performed according to standard procedures. Briefly, 5 pL of OMV samples, with equivalent volume / bacteria ODeoonm, were added to carbon coated copper mesh grids and stained with 1% uranyl acetate for 1 minute. The grids were examined with a Jeol JEM- 1400 (JEOL Inc, Peabody, MA, USA) at 80 kV. Images were acquired using a digital camera (Gatan Orius, Gatan Inc, Pleasanton, CA, USA).

[0131] Cryoelectron microscopy (Cryo-EM). Isolated OMVs were visualized by cryoelectron microscopy. Brieefly, 3 pL of sample were deposited onto glow-discharged lacey carbon grids and placed in the thermostatic chamber of a Leica EM-GP automatic plunge freezer, set at 20°C and 95% humidity. Excess solution was removed by blotting with Whatman n°l filter paper for 2.5 seconds, and the grids were immediately flash frozen in liquid ethane at -185°C. Images were acquired on a Talos Arctica (Thermo Fisher Scientific) operated at 200kV in parallel beamcondition with a K3 Summit direct electron detector and a BioQuantum energy filter (Gatan Inc.). Energy-filtered (20 eV slit width) image series were acquired with Digital Micrograph software at a pixel size of 0.85A between -1 and -1.5pm defocus.

[0132] Dynamic Light Scattering (DLS). OMVs were analysed by DLS using a Zetasizer Nano ZS (Malvern Instruments Ltd.) operating at a temperature of 25°C. The OMVs were diluted 10-fold in PBS for measurements in triplicates and analysed using a spectrophotometer MACRO cuvette in crystal PS (ref BSA001, Biosigma S.p. A).

[0133] Mouse model. Male, 8-week-old, C57B16J mice (ENVIGO, France) were infected intraperitonally with 2.107CFU. Bacteria were cultivated in LB overnight and then 8 hours in M63 minimal medium (see above) with 0.1 mM MgCh for the wild type strain and the mutant strain and with 0.1 mM MgCh and 6 mM L-rhamnose (Sigma Aldrich) for the complemented strain. The severity of the clinical signs was evaluated blindly by scoring (data not shown).

[0134] Any animal presenting at least one clinical sign with a score of 3 led to humanely sacrifice the animal in accordance with decree N°2013-18 of February 1, 2013. All the experimental procedures were carried out in accordance with the European directives for the care and use of animals for research purposes and were validated by the local and national ethics committee. Protocol number 2023040715164643. 8 hours post-infection, tissue proteins were extracted from spleen with RIPA (0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 1% Igepal in Tris-buffered saline pH = 7.4) as previously described14. Clear lysates were processed for ELISA using commercial kits (Duoset R& D Systems, Lille, France) for Interleukin- ip (IL-ip). Data are expressed as picograms of cytokines per milligram of tissue protein.

[0135] Bioinformatics analysis. The tridimensional structure predictions of HlyF and CprA proteins from E. coli and P. aeruginosa were obtained using Alphafold 2 (Galaxy Version 2.3.1+galaxy2). The proteins were visualized using ChimeraX 1.3. The protein structure comparison was performed using the iPBA web-server30. For the phylogenetic tree of P. aeruginosa we constructed a Randomized Axelerated Maximum Likelihood tree (RAxML version 8.2.11) from a Mafft alignment of 1000 CDS extracted from each genome (www.bv-brc.org). The tree has been displayed and annotated with the online tool iTol (itol.embl.de / ). To analyze the allele distribution among P. aeruginosa isolates, the nucleotide sequences of cprA from the strains present in the phylogenetic tree were retrieved from the National Center forBiotechnology Information (NCBI). Then, the amino acid sequence corresponding to each allele was blasted against the P. aeruginosa database on the PubMLST.org website31and from the National Reference Center for Antibiotic Resistance of aeruginosa (NRC-RA Besangon, France). To analyze the presence of CprA orthologs in others bacterial core genome a pangenome analysis of each species was performed using reliable genomes from the NCBI genome database. Genome sequences of each species were obtained from the GenBank database and selected for analysis according to the following criteria: (i) keeping full strain genome representation instead of partial, (ii) excluding strains with assembly anomaly such as chimeric, contaminated, misassembled and (iii) filtering out strains with abnormal genome length, low-quality sequences, untrustworthy as types, unverified source organisms, many frameshifted proteins, abnormal gene to sequence ratio. The 3371 genomes selected, were annotated using prokka32and are submitted to a pangenome analysis using roary (with options: -cd 100 -i 80) for each bacterial species33. In this analysis, "core genes" are defined as those conserved in all isolates. The presence of a putative PhoP binding site upstream of the ORF of CprA orthologs was assessed using the virtual footprint tool on PRODORIC34, with PhoP matrix from S. enterica, E. coli and Y. pestis.

[0136] Modified plasmids for the production of variant forms of HlyF and CprA. Modified plasmids for the production of variant forms of HlyF and CprA were synthesized by Genecust (France). Mono variants correspond to the WT amino acid replaced by an alanine. E. coli bearing the expression system for the production of HlyF WT, CprA WT and their variants were cultured 8h in 50 mL M63 supplemented with 0, ImM MgCh. A fraction of the cell pellets were analysed for the production of N-terminal His-tagged proteins by SDS-PAGE and western blot. The remaining cell pellets were used to extract lipids (1:2:1 MeOH: CHC13: H2O). Following the drying of the organic phase under N2, lipid pellets were resuspended in 200 pL DMEM and 30 pL were loaded in 24-wells microplate containing HeLa cells (5.104cells / mL). After a 5h incubation, cells were assessed for LC3 and actin by SDS-PAGE and western blots. Lipids extracted from a WT HlyF- or WT CprA-producing E. coli were used as positive control. Non treated cells and the catalytically inactive double variant HlyF Y163F / K167A were used as negative control.

[0137] Statistical analysis. All statistical analyses were performed with Prism 10.1.0 software. Data are represented as the mean ± SD. Significance was determined by a one-way ANOVA(analysis of variance) for IL-ip measurement in mice spleen, by a two-way ANOVA followed with Tuckeys’ multiple comparisons testing for IL-ip secretion, cell death in human monocytes and for mice clinical score, and by a Mantel-Cox test for mice survival. ****p < 0.0001, *** p < 0.001, ** p < 0.005, * p < 0.05, ns: not significant, p > 0.05.

[0138] Results

[0139] CprA exhibits structural homology with HlyF. CprA has been described as two forms: a full-length allele found in the PAK strain, and a truncated allele found in PAO1 due to an indel mutation that creates a premature stop codon at position 2456. CprA from PAK shares 50.13% amino acid residues identity with HlyF and is predicted to belong to the short-chain dehydrogenase / reductase (SDR) superfamily. The protein features a highly conserved catalytic site and an NAD(P)H binding site as shown in Figure la. In PAO1, the truncated form of CprA retains both the catalytic and NAD(P)H binding sites but has lost one alpha helice and eleven beta sheet, which constitute the lower portion of the protein, resulting in a change in its conformation. Notably, HlyF and CprA exhibit similar predicted three-dimensional structures and share a comparable catalytic pocket with the NAD(P)H cofactor binding site located next to the catalytic site (Figure lb and 1c). The predicted structural similarity between HlyF and full-length CprA prompted us to investigate whether CprA shares similar properties with HlyF, such as the ability to produce OMVs that modulate autophagy in eukaryotic cells.

[0140] When produced in E. coli, the function of CprA is similar to that of HlyF. To determine whether structural homology leads to conserved function, we have cloned both the truncated (from PAO1) and full-length (from PAK) variants of cprA and expressed them in E. coli. We then purified OMVs from E. coli expressing these variants and tested their ability to modulate autophagy in HeLa cells, by assessing the accumulation of autophagosomes (Figure 2)19. The marker of autophagosome LC3 was assessed by immunofluorescence using a fusion LC3-GFP or analyzed by western blotting to distinguish between the LC3-I (free) and the LC3-II (associated with autophagosomes) forms (Figure 2a). Treatment with OMVs derived from E. coli harboring the full-length CprA from the PAK strain resulted in a presence of LC3-GFP foci (Figure 2b), along with an accumulation of LC3-II protein, as evidenced by western blotting (Figure 2d). This phenotype closely resembles that observed in cells treated with OMVs produced by E. coli producing HlyF (i.e. the accumulation of autophagosomes, as reported in David et al. (2022)19). In contrast, OMVs produced by E. coli that produce the truncated formof CprA from PAO1 failed to induce the accumulation of LC3-II or GFP-LC3 foci. The observed outcome was comparable to the results seen in untreated cells or cells treated with OMVs produced by E. coli that produce an inactive form of HlyF with the two mutations in the catalytic domain Y167F and K164A (Figure 1, Figure 2b and 2d). Therefore, the activity of the full-length form of CprA is similar to that of HlyF when produced in E. coli.

[0141] OMVs from CprA-producing P. aeruginosa trigger the accumulation of autophagosomes. To evaluate the ability of P. aeruginosa expressing cprA to induce autophagosomes accumulation through the production of specific OMVs, we used the wild-type PAK strain, its cprA gene-deleted mutant, the complemented mutant strain PAK EcprA atB. PPha-cprA, and the PAO1 strain transformed with a plasmid expressing either the full-length or the truncated form of cprA, referred to as cprAPAKor cprAPAO1respectively. OMVs were purified from culture supernatants (data not shown) and visualized using both transmission electron microscopy (TEM) and transmission electron cryomicroscopy (Cryo-TEM) and further characterized by dynamic light scattering (DLS). The PAK strain, with or without full-length CprA, yielded heterogeneous populations of spherical OMVs with a diameter of 140-150 nm ± 50 nm (data not shown), which exhibited a single lipid bilayer, confirming their classification as bona fide OMVs, as previously reported in the literature35,36. Upon treatment with OMVs isolated from P. aeruginosa producing full-length CprA, cells showed an accumulation of GFP-LC3 foci (Figure 2c), which is consistent with the increase in LC3-II compared to the untreated sample (Figure 2e). In contrast, cells treated with OMVs derived from P. aeruginosa producing the truncated form of CprA did not show GFP-LC3 foci or elevated levels of LC3-II. Thus, full-length CprA but not its truncated variant, allows E. coli and P. aeruginosa to produce of OMVs that induce accumulation of autophagosomes in eukaryotic cells.

[0142] Pseudomonas aeruginosa producing CprA produce OMVs that impede the autophagic flux.

[0143] Autophagy is a dynamic process that involves the formation and degradation of autophagosomes. Previous research has shown that OMVs produced by E. coli producing HlyF impede the autophagic flux at the lysosome-autophagosome fusion stage, leading to the accumulation of autophagosomes19. To determine whether OMVs derived from P. aeruginosa producing a full-length CprA can impede the autophagic flux at the lysosome-autophagosome fusion stage, we used HeLa-Difluo hLC3 cells to monitor the acidification of autophagosomes (Figure 2f). In cells treated with OMVs from P. aeruginosa producing a full-length form ofCprA (PAK or PAO1 pJN c / ?PAK), we observed a pronounced colocalization of GFP and RFP-positive puncta, similar to the positive control where cells were treated with chloroquine inhibiting the lysosomal fusion (Figure 2g). In contrast, HeLa-Difluo hLC3 cells treated with OMVs from P. aeruginosa producing the truncated CprA (PAO1 or PAO1 pJN c / ? / HPAO1), showed a diffuse GFP and RFP labeling with no colocalization, similar to that observed in cells deprived by incubation in HBSS, known to induce a fully functional autophagic flux (Figure 2g). Previous work has demonstrated that the absence of acidification of autophagosomes in cells treated with OMVs from E. coli producing HlyF, is due to a fusion defect with lysosome19. Therefore, our results strongly suggest that OMVs from P. aeruginosa producing full-length CprA, but not the truncated variant, impede the autophagic flux by preventing the fusion between the autophagosomes and the lysosomes.

[0144] The production of CprA by P. aeruginosa results in OMVs with a greater capacity to activate the non-canonical inflammasome pathway. The non-canonical inflammasome pathway is induced by the activation of the NLRP3 inflammasome by cytosolic lipopolysaccharide (LPS). Previous research has shown that OMVs from bacteria producing HlyF were more prone to activate the non-canonical inflammasome pathway than OMVs purified from bacteria producing an inactive form of HlyF19. It was hypothesized that the expression of cprA could affect the ability of OMVs to activate the non-canonical pathway of NLRP3 inflammasome. Human monocytic THP-1 wild-type (WT) cells were used along with THP-1 cells knocked out for NRLP3 (THP1NRLP3KO), an intracellular sensor that is part of the caspase-1 activating complex, and for Caspase-4 (THP1CASP4KO) or Gasdermin-D (THP1GSDMDKO), two essential components of the non-canonical inflammasome pathway (Figure 3a). The cells were exposed to OMVs from P. aeruginosa PAK WT, the mutant PAK ΔcprA and the complemented strain (PAK ΔcprA attB::PRha-cprA). Althought the effect was smaller with the OMVs from the mutant PAK ΔcprA, OMVs from the 3 strains induced THP-1 cell lysis (measured by LDH release) and IL-1β release (measured by ELISA) in a Caspase-4 and GSDMD-dependent manner. However, no cell death was observed in a NLRP3 -dependant manner. These results indicate that / < aeruginosa OMVs activate the non-canonical inflammasome pathway (Figure 3b and 3c). Exposure of cells to PAK WT and PAK ΔcprA attB::PRha-cprA OMVs resulted in a significant increase in cell lysis and IL-ip release compared to PAK ΔcprA. This process was found to be dependent on Caspase-4 and GSDMD, as shown in Figure 3. Our findings indicate that that OMVs from P. aeruginosa, which produce CprA, are much more prone toactivate the non-canonical inflammasome pathway. This effect could be explained by the blockage of the autophagic flux, which inhibits the primary negative feedback mechanism of non-canonical inflammasome activation, as observed with OMVs from HlyF -producing E. coli19.

[0145] Role of the PmrAB two-component system in CprA-mediated OMVs production and autophagic disruption. Previous studies have shown that the PmrAB two-component system plays a regulatory role in the transcriptional activation of cprA, but the role and enzymatic activity of CprA in the bacteria was unclear at the time7,21. Given the functional similarities between the CprA allele of PA14 and PAK (data not shown), we chose to evaluate the role of PmrAB in CprA-mediated OMVs production in PA14, a more virulent strain that causes disease in a wide range of organisms. To test whether PmrAB boosts the toxicity of OMVs, we performed a comparative analysis of the effects of OMVs obtained from the wild-type P. aeruginosa PAM strain, its pmrA / ^-deficient isogenic mutant harboring either the empty vector (PA14 ΔpmrAB + pME6012) or the vector encoding the wild-type pmrAB allele (PA14 ΔpmrAB + pABWT)21. As PmrAB is known to be activated by a low concentration of Mg2+, we cultivated the strains in a medium with a low concentration of MgCl2(0.1 mM)7Western blot analysis showed an accumulation of LC3-II protein levels in cells exposed to OMVs from the wild-type PAM. In contrast, the strain that lacked the PmrAB did not show any accumulation of LC3-II, similarly to the untreated cells. Complementation with the wild-type pmrAB allele partially restored the ability of the mutant strain to produce OMVs that induce LC3-II accumulation (Figure 4). These findings highlight the crucial role of a functional PmrAB two- component system in producing OMVs that impede the autophagic flux when environnemental conditions activate the two-component system.

[0146] PmrB gain-of-function mutations increase the production of OMVs that block autophagic flux. Exposure to colistin may select for pmrB mutants that confer a gain of polymyxin resistance, as observed in clinical isolates11’21,42. This phenomenon is attributed to the elevated mutation rate that occurs in this locus upon colistin selection43. These mutations are referred to as “gain-of-function” mutations because they activate the PmrAB regulon in the absence of environmental signals11. To investigate whether a pmrAB allele described as gain-of-function allele could also induce the production of OMVs that block the autophagic flux, we treated Hela cells with OMVs from PAM EpmrAB complemented with either the wild-type pmrAB allele(pABWT) or with the a pmrAB harbouring the deletion ΔL172 in PmrB (pAB16.2), identified as a pmrAB gain-of-function allele21. OMVs were produced by cultivating bacteria in the minimum medium M63 supplemented with either an activating (0.1 mM) or an inhibiting (2 mM) concentration of MgCl2. We observed a clear accumulation of the LC3-II protein in HeLa cells treated with OMVs obtained from the strain complemented with the pmrAB gain-of-function allele (i.e. magnesium non-responsive allele), regardless of the concentration of MgCl2(Figure 4). In contrast, OMVs from the strain complemented with the wild-type pmrAB allele (z.e. magnesium -responsive allele) blocked the autophagy flux only when the bacteria are cultivated in a MgCh concentration activating the PmrAB two-component system. The toxicity of OMVs on cells was abolished when the bacteria were cultivated with a high concentration of MgCl2, inactivating PmrAB. This shows that pmrAB gain-of-function allele enable the constitutive production of toxic OMVs, regardless of environmental factors.

[0147] CprA is a virulence factor of P. aeruginosa in a mouse sepsis model. Subsequently, the role of CprA in the virulence of P. aeruginosa was evaluated in a mouse model of infection. Mice were intraperitoneally infected with 2.107CFU of PA14 WT, PA14 ΔcprA mutant, or the complemented PA14 cprA attB:: PRha-cprA. The clinical scores for mice infected with the mutant strain were significantly lower than those infected with the WT or complemented strain at both 4 and 8 hours post-infection (Figure 5a, data not shown). Between 20 and 24 hours post-infection, the WT and complemented strains caused mortality rates of 94% and 100%, respectively. In contrast, infection with the PA14 ΔcprA mutant strain resulted in a statistically significant delay in survival kinetics, with a median of 32 hours post-infection (Figure 5b).

[0148] The lower clinical scores and the related mortality rates observed in the PA14 ΔcprA mutant compared to PA14 with cprA (WT or complemented strain), may be explained by a lower level of inflammation exerted by a strain lacking cprA, as observed in THP-1 cells (Figure 3). To verify this assessment, the levels of the pro-inflammatory cytokine IL-ip induced by the non-canonical pathway of NLRP3 inflammasome were evaluated in mice spleen 8 hours postinfection. A reduction in the inflammatory response was observed in the spleen of mice infected with the PA14 ΔcprA mutant strain in comparison to the-wild type or complemented strain. This was demonstrated by the observation of reduced levels of interleukin IL-ip (Figure 5c).

[0149] These results demonstrate that CprA contributes to the virulence of P. aeruginosa and is responsible for a stronger inflammatory response. This is likely responsible for the observed clinical scores and associated survival kinetics.The cprA gene is ubiquitously present in P. aeruginosa. Analysis of available genomes in the databases indicated that the cprA gene and its genetic environment are part of the core genome of P. aeruginosa. This is true for the PAO1, PAK, PA14 strains, as well as the more phylogenetically distant PA7 strain. The genetic organization upstream and downstream of cprA includes essential genes, such as those encoding subunits of cytochrome C oxidase which is an enzyme necessary for aerobic respiration, and the aerotaxis receptor Aer (data not shown). Out of the 5,286 genomes obtained from the PubMLST database (2,797 genomes) and the collection of the French National Reference Center for Antibiotic Resistance P. aeruginosa strains (2,489 genomes), only 17 (0.32%) displayed a truncated variant of CprA. Among these, 14 had a variant identical to the strain PAO1 (data not shown). Twelve major alleles were identified, and their distribution was not dependent on the P. aeruginosa clades, unlike the well-known type III secretion system exotoxins ExoU (Clade B) and ExoS (clade A), and the exolysin ExlA (most of the clade C) (data not shown). The most common allele is found in CprAATCC27853, found in 81 % of the isolates (data not shown). This allele is present in the most prevalent epidemic high-risk clones such as ST111, ST175, ST233, ST235, ST244, ST257, ST308, ST357, and ST65444. The CprA allele from the outlier strain PA7 is notably the most genetically divergent (data not shown) compared to the other alleles. We assessed the capacity of the most frequent allele, CprAATCC2785, the allele with the highest rate of SNPs CprAPA7, and the CprAPAKand CprAPA14alleles, which have already been shown to be functional in this study, to produce toxic OMVs. The two E. coli hosting the constructs CprAATCC27853and CprAPA7led to the production of OMVs blocking autophagy, just as does CprAPA14and CprAPAK(data not shown). We also confirmed the production of autophagy -blocking OMVs in clinical strains isolated from patients at the University Hospitals of Toulouse with cystic fibrosis, ear-nose-throat infections, and infections due to medical devices (data not shown). After sequencing the cprA gene in these isolates, we identified the CprAATCC27853allele in all of them, which is consistent with the prevalence of this allele among P. aeruginosa. These results show that all P. aeruginosa strains, with the exception of a few strains harboring a truncated CprA allele such as PAO1, are potentially capable of producing OMVs that inhibit autophagy.

[0150] CprA and HlyF represent a novel family of virulence determinants in Gram-negative bacteria. Given the relative taxonomic distance between E. coli and P. aeruginosa, it was of interest to ascertain whether CprA and HlyF orthologs were found in other bacteria. A number of cryptic SDRs, similar to CprA and HlyF, have been identified in the genome of Gram-negative bacteria. The potential orthologs exhibit a protein size ranging from 360 to 385 residues and a minimum of 35% amino acid identity, including the key residues of the NAD(P)H binding site (TG. TGF. G) and the catalytic site (YT. SK), which are typical of SDRs. CprA / HlyF orthologs have been found mainly in bacteria belonging to the class of the Gammaprote obacteria, and especially the orders of Pseudomonadales and Enterobacterales. Orthologs are also found in the Betaproteobacteria class, especially in the Burkholderiales order (Figure 6a and data not shown). The majority of these orthologs are encoded on the bacterial chromosome, with a similar GC% to the rest of the genome, suggesting that they belong to the core genome (Figure 7a). This is particularly true for 18 bacterial species for which several genomes could be used for a pangenome analysis (data not shown). Although the number of core genes varied among species, more than 93% of isolates in each species possess an HlyF ortholog, with the exception of D. dadantii and P. agglomerans, indicating that the genes are highly conserved among the genome. The absence of an HlyF ortholog in a minor portion of genomes could be explained by sequencing or assembly errors in retrieved genomes. We also observed a larger amount of P. agglomerans missing the HlyF ortholog, certainly because the orthologs is encoded by a large plasmid (110 to 500 kbp), such as those from E. coli, E. albertii, S. enterica and P. vagans (Figure 6a). To investigate the roles of these putative SDRs, we purified OMVs produced by E. coli expressing different orthologous proteins from pathogens that are of interest in terms of human, mammal and plant health such as S. marcescens, K. pneumoniae, Y. pestis, and R. solanacearum (Figure 6a). Strikingly, we consistently observed an accumulation of LC3-II protein, similar to the results obtained with OMVs from E. coli expressing hlyF or cprA (Figure 6b). All validated orthologs, including the two most distant forms (HlyF from E. coli SP 15 and HlyF / CprA ortholog from R. solanacearum GMI1000), are functional and maintain three-dimensional structural similarity and conserved residues (Figure 7b, 7c, 7d). Furthermore, all of these orthologs possess a protein domain classified as fatty acyl CoA reductases (FARs) in the National Library of Medicine's conserved domain database (CDD)45. All these orthologs, including HlyF and CprA, are characterized by the FAR-N, SDRe domain (accession: cd05236), but lack the FAR-C superfamily domain (C-terminal domain of FAR, accession: cd0971). Fatty acyl-CoA is reduced to fatty alcohols by FARs, which can catalyze both saturated and unsaturated C16 or C18 fatty acids46. The homology of CprA, HlyF, and their orthologs to FARs suggests that these SDRs may modify the bacteria's outer membrane composition by targeting lipids. This leads us to the hypothesis that these SDRs could modify the lipid composition of the bacterial outer membrane vesicles.Liposomes made with bacterial lipids extracted from OMVs of CprA-producing bacteria, inhibits autophagy. We extracted lipids from the OMVs of P. aeruginosa PAK expressing or not cprA, using a method adapted from Bligh and Dyer27. Following liposomes formation trough lipid extrusion, HeLa cells were treated with a lipid amount equivalent to the dose of 3.33μg protein in OMVs (Figure 8a and 8b). We observed an accumulation of LC3-II protein in cells treated with OMVs or liposomes from P. aeruginosa PAK, but not from its isogenic mutant in cprA, suggesting that lipids extracted from OMVs are as toxic to eukaryotic cells as whole OMVs. Although the lipids responsible for the anti -autophagic activity of OMVs have not yet been identified, we confirmed here that the component responsible for this activity is present in the lipidic fraction (see also Figure 9a, 9b, 9c wherein liposomes are made with bacterial lipids extracted from bacterial membranes of CprA-producing bacteria). This result, together with the in silico analysis of the conserved enzymatic domain of HlyF and CprA, strongly supports the hypothesis that this new family of SDRs modifies lipid(s) of the bacterial membrane leading to the production of OMVs and liposomes with anti -autophagic activities.

[0151] Variants of HlyF and CprA were produced in E. coli. Western blot analysis of the cell pellet with anti-his tag showed that variants HlyF D367A, CprA D383A, HlyF K369A and CprA K385A are well produced. Cell pellets were then extracted to recover lipids. Such lipids were assessed for their potential to induce autophagy blockade. The lipids extracted from variants HlyF D367A and CprA D383A do not trigger autophagy blockade anymore whereas lipids extracted from variant HlyF K369A and CprA K385A reproduce a WT phenotype. These results indicate that the D is essential for activity (Figures 10a and 10b).

[0152] TABLE:

[0153] Table 1. Non-exhaustive list of HlyF orthologs

[0154] E. cloacae ATCC13047 (CP001918) ECL_04112 / ADF63646.1

[0155] K pneumoniae SB4496 (NZ_CAAHGC010000001.1) SB04496_04318 / VGP43912.1

[0156] K aerogenes ATCC13048 GCA_003417445.1

[0157] Enterobacter aerogenes KCTC2199 (CP002824.1) EAE_01945 / AEG95324.1

[0158] S. marscesens SM39 (AP013063.1) SM39_2772 / BAO34761.1

[0159] Y pestis CO92 (AL590842.1) YPO1559 / CAL20204.1

[0160] Y pestis CO92 (NC_003143) YPO_RS08785 / WP_002211905.1

[0161] Y. pseudotuberculosis IP32953 (BX936398.1) YPTB1570 / CAH20809.1

[0162] Y. enterocolitica 8081 (AM286415.1) YE2757 / CAL12791.1

[0163] P. aeruginosa PAK (CP020659.1) Y880_0993 / ARI01029.1

[0164]

[0165] P. aeruginosa PAK(LR657304.1) PAKAF_03592 / VUY45701.1

[0166] P. aeruginosa PA14 (NC_008463) cprA / WP_003140184.1

[0167] UCBPP-PA14 (CP000438.1) PA14_44311 / ABJ15632.1

[0168] P. aeruginosa PA7 (CP000744.1) PSPA7_3771 / ABR86037.1

[0169] P. aeruginosa ATCC27853 (CP015117.1) A4W92_03795 / AMX86105.1

[0170] R. solanacearum GMI1000 (AL646052) RSc1462 / CAD15164.1

[0171]

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Claims

CLAIMS:

1. A method for producing vesicles, the method comprising a step of expressing or overexpressing hemolysin F (hlyF) gene or an ortholog of hemolysin F gene in Gramnegative bacteria, said hlyF gene or ortholog of hlyF gene encoding respectively a HlyF protein or an ortholog of HlyF protein.

2. The method according to claim 1, wherein the ortholog of HlyF protein is a short-chain dehydrogenase / reductase (SDR) comprising a NAD(P)H binding site and a catalytic site.

3. The method according to claim 2, wherein the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibits at least 35% amino acid identity with HlyF protein, includes the residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and a residue YTXSK (SEQ ID NO:3) in said catalytic site and includes the residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region.

4. The method according to any of claims 1 to 3, wherein the ortholog of HlyF protein possesses a fatty acyl CoA reductase (FARs) domain and lacks the FAR-C superfamily domain.

5. The method according to any of claims 1 to 4, wherein the ortholog of hlyF gene is CprA gene, encoding CprA protein.

6. The method according to any of claims 1 to 4, wherein the Gram-negative bacterium is a Gammaproteobacteria, in particular Pseudomonadales or Enterobacterales; a Betaproteobacteria, in particular Burkholderiales; or a Bacteroidia.

7. The method according to any of claims 1 to 6, wherein the Gram-negative bacterium is selected from the list consisting in Escherichia coli, Salmonella enterica Kentucky, Cedecea neteri, Escherichia albertii, Enterobacter clocae, Klebsiella michiganensis, Klebsiella pneumoniae, Raoultella ornithinolytica, Klebsiella aerogenes, Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter sakazakii, Serratia marcescens, Serratia odorifera, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersiniapestis, Dickeya zeae, Dickeya dadantii, Pantoea ananatis, Pantoea agglomerans, Pantoea vagans, Pseudomonas oryziphila, Pseudomonas mosselli, Pseudomonas aeruginosa, Pandoraea pnomenusa, Pandoraea apista, Pandoraea sputorum, Pandoraea commovens, Ralstonia pickettii, Ralstonia solanacearum, Collimonas fungivorans or Cupriavidus brasilensis.

8. The method according to any of claims 1 to 6, wherein the Gram-negative bacterium is a Pseudomonas sp.

9. The method according to any of claims 1 to 6, wherein the Gram-negative bacterium is Pseudomonas aeruginosa.

10. The method according to any of claims 1 to 9 for producing Outer Membrane Vesicles (OMVs), wherein the method comprises a step of expressing or overexpressing an ortholog of hemolysin F (hlyF) gene in Gram-negative bacteria, said ortholog of hlyF gene encoding an ortholog of HlyF protein.

11. OMVs obtained with the method according to claim 10.

12. The method according to any of claims 1 to 9 for producing liposomes derived from bacterial membrane.

13. The method according to claim 12, wherein the method comprises the steps of:- Extracting lipids from the bacterial membrane of the Gram-negative bacteria expressing hlyF gene or hlyF ortholog gene to obtain a lipidic fraction; and- Forming liposomes from said lipidic fraction to obtain liposomes derived from bacterial membrane.

14. A vaccine composition comprising vesicles obtained according to any of the methods described in any of claims 1 to 13.

15. The vaccine composition according to claim 14 for use in a method of treating a subject suffering from a Gram-negative bacterium infection.

16. A lipidic fraction obtained by extracting lipids from Gram-negative bacteria bacterial membrane, said Gram-negative bacteria expressing or overexpressing hlyF gene or an ortholog of hlyF gene.

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