Composition for enhancing polymyxin sensibility comprising TimP peptide encoded from small noncoding RNA gene

Abstract

The present invention relates to a composition for enhancing polymyxin sensitivity comprising a TimP peptide derived from a non-coding ribonucleic acid gene. It demonstrates the utility of TimP, a peptide derived from a gene expressing non-coding ribonucleic acid in Escherichia coli, and extracellular vesicles (EVs) derived from bacteria expressing TimP, by elucidating their mechanism of action and their utilization as polymyxin-sensitizing agents. In particular, it was confirmed that combining the newly discovered substance with triclosan can be utilized to overcome the limitations of treating polymyxin-resistant bacteria using polymyxin, thereby resensitizing polymyxin-resistant bacteria to polymyxin B (PMB) by activating multiple bacterial metabolic mechanisms. Accordingly, the present invention clearly demonstrates the potential application of TimP and TimP-derived extracellular vesicles as a novel Trojan horse strategic polymyxin sensitization therapy for controlling pathogens.

Description

Technology Field

[0001] The present invention relates to a composition for enhancing polymyxin sensitivity comprising a TimP peptide derived from a small noncoding RNA (sRNA) gene. Background Technology

[0002] Polymyxins, including polymyxin B (PMB) and colistin (COL), are the last line of defense against multidrug-resistant Gram-negative bacteria. However, due to the emergence of polymyxin-resistant strains and a lack of new antibiotic development, there is a need to develop novel methodologies to extend the efficacy of currently utilized polymyxins. Polymyxins control Gram-negative bacteria through a primary mechanism that destroys the lipopolysaccharide (LPS) on the outermost layer of the bacterial outer membrane bilayer, as well as through multiple non-lipopolysaccharide-based mechanisms. Combination therapy with polymyxins and non-antibiotics is widely used as an alternative to polymyxin therapy; however, the simultaneous administration of multiple drugs carries a high risk of developing drug resistance in adapting bacteria. It has been demonstrated that modulating bacterial stress response pathways associated with polymyxin activity and their regulators has a polymyxin sensitization effect on polymyxin-resistant Gram-negative bacteria. The mechanisms of action related to bacterial-derived substances discovered in this new strategy are highly valuable for verifying their applicability as sensitization substitutes for the toxic antibiotic polymyxin, or for utilizing them as therapeutic methods to control Gram-negative bacteria.

[0003] Non-coding ribonucleic acid (NCONA) is a well-known regulator of stress response in bacteria, altering bacterial metabolism across multiple pathways through the regulation of gene expression in response to external stimuli. Based on this, it is expected that specific NCONAs and their target genes exist to influence the efficacy of antibiotics. While previous studies have confirmed the partial effects of specific NCONA overexpression or knockout strains on the efficacy of polymyxin, the NCONAs and their target genes that influence polymyxin up to the minimum inhibitory concentration (MIC) level have not been clearly identified. Furthermore, the increased sensitization to major polymyxin induced by bacterial NCONAs must be evaluated in conjunction with associated biomolecules and metabolic pathways, yet no research results regarding this aspect have been published. Prior art literature

[0004] Korean Patent Publication No. 10-2022-0100491 (Published July 15, 2022) The problem to be solved

[0005] The present invention aims to provide a composition for enhancing polymyxin sensitivity comprising a TimP peptide derived from a non-coding ribonucleic acid gene or an extracellular vesicle derived from a bacterium in which the TimP peptide is overexpressed.

[0006] In addition, the present invention aims to provide an antimicrobial composition for polymyxin-resistant Escherichia coli comprising a non-coding ribonucleic acid gene-derived TimP peptide or a bacterial-derived extracellular vesicle overexpressing the TimP peptide; polymyxin; and triclosan as active ingredients. means of solving the problem

[0007] To solve the above problem, the present invention provides a composition for enhancing sensitivity to polymyxin comprising, as an active ingredient, a TimP peptide consisting of an amino acid sequence indicated by SEQ ID NO. 1 or a bacterial-derived extracellular vesicle overexpressed with said TimP peptide.

[0008] In addition, the present invention provides an antimicrobial composition for polymyxin-resistant Escherichia coli comprising a TimP peptide consisting of an amino acid sequence indicated by SEQ ID NO. 1 or a bacterial-derived extracellular vesicle overexpressing the TimP peptide; polymyxin; and triclosan as an active ingredient. Effects of the invention

[0009] The present invention relates to a composition for enhancing polymyxin sensitivity comprising a TimP peptide derived from a non-coding ribonucleic acid gene. It demonstrates the utility of TimP, a peptide derived from a gene expressing non-coding ribonucleic acid in Escherichia coli, and extracellular vesicles (EVs) derived from bacteria expressing TimP, by elucidating their mechanisms of action and their utilization as polymyxin sensitizing agents. In particular, it was confirmed that combining the newly discovered substance with triclosan can overcome the limitations of existing polymyxin treatments by activating multiple bacterial mechanisms, which leads to the re-sensitization of polymyxin-resistant bacteria with polymyxin B (PMB). Accordingly, the present invention clearly demonstrates the potential application of TimP and TimP-derived extracellular vesicles as a new Trojan-type strategic polymyxin sensitizing agent for controlling pathogens. Brief explanation of the drawing

[0010] Figure 1 shows the changes in polymyxin sensitization effects on TimP and RyfA as polymyxin sensitizers and on known function regulators of TimP. (a) Determination of minimum growth inhibitory concentration to confirm polymyxin sensitization activity of non-coding ribonucleic acid gene overexpression. Escherichia coli Non-coding ribonucleic acid (OmrA + , RyfA + , and SdsR + The minimum growth inhibitory concentration of ) is indicated. (b) RyfA overexpression (RyfA + Confirmation of the effect of ) on colistin sensitization. RyfA +The minimum growth inhibitory concentration of colistin was determined. (c) E. coli In the K-12 genome ryfA Schematic representation of the gene. The blue and brown DNA sequences represent the predicted ORF and TimP regions, respectively, and the sequences within the boxes represent the predicted signal peptides of the TimP region. (d) ORF1 + and TimP + Evaluation of the polymyxin B sensitization activity. Codon-optimized peptides were expressed in E. coli, and the minimum growth inhibitory concentration for polymyxin B was determined. (e) TimP(-signal peptide) + of Polymyxin B Sensitization Evaluation. TimP (-signal peptide) + The minimum growth inhibitory concentration for polymyxin B was determined. (f) Known TimP + , RyfA + TimP based on regulator + Check activity. cpxR ( cpxR - ), hfq ( hfq - ), proQ ( proQ - ) and ybeY ( ybeY - TimP regarding ) + The minimum growth inhibitory concentration of polymyxin B was determined. (g) Determination of the minimum growth inhibitory concentrations of FT-Pep and T-Pep (-signal peptide) against E. coli. (h) Confirmation of the activity of co-treatment with FT-Pep or T-Pep (-signal peptide) containing polymyxin B. The red circle indicates the minimum growth inhibitory concentration values, + represents the overexpression of non-coding ribonucleic acid genes and TimP or TimP(-signal peptide). Representative data from an experiment with n=3 are shown. Figure 2 shows the effects of TimP+: lipopolysaccharide binding, lipopolysaccharide modification, or cardiolipin synthesis results. (a) TimP + Confirmation of relative lipopolysaccharide binding degree by TimP + Relative lipopolysaccharide binding was measured for the and control groups using fluorescent-based dansyl-labeled polymyxin B. It is expressed as mean ± standard deviation (AVG ± SD) with n=3 (*P<0.001). Relative lipopolysaccharide binding was TimP + It was expressed as the value obtained by dividing the fluorescence intensity of the cells by the fluorescence intensity of the control group. (b) TimP + Zeta potential measurement. AVG±SD for n=3 is indicated (*P=0.693). (c) Effect of lipopolysaccharide modified gene knockout on TimP-mediated polymyxin sensitization. In the corresponding gene knockout, the control and TimP + The minimum growth inhibitory concentration of polymyxin B was determined. (d) outer membrane permeability and (e) TimP + Depolarization effect caused by TimP + For the and control samples, respectively NPN (λ ex 355 / λ em 460) or DiSC3(5)(λ ext 584 / λ em Measure the fluorescence intensity of 620) and compare it with the control group to TimP + The relative proportion of was measured (n=9, *P<0.001). (f) TimP for cardiolipin synthesis-coding gene knockout + Confirmation of mediated polymyxin sensitization effect. Control and TimP in the corresponding gene knockout. + The minimum growth inhibitory concentration of polymyxin B was determined. TimP stained (g) with or without acrylidine orange dye (h) was examined using a microscope (100x magnification) or SEM (30.0 kV), respectively. + The morphology of the bacteria was identified. Representative images from the n>3 experimental results are displayed. (i) minC knockout( minC- ) TimP + Confirmation of mediated polymyxin sensitization effect. Control and TimP in the corresponding gene knockout. + The minimum growth inhibitory concentration of polymyxin B was determined. (j) By SEM minC - My TimP + Morphological analysis of. SEM images at 30.0 kV are displayed, and scale bars (5 μm) are indicated. Representative data from experiments with n=3 are shown. (k) Cardiolipin-related gene knockout TimP + Confirmation of mediated polymyxin sensitization effect. Control and TimP in the corresponding gene knockout. + The minimum growth inhibitory concentration of polymyxin B was determined. (l) Confirmation of the activity of the TimP mutant at the minimum growth inhibitory concentration of polymyxin B. Numbers (2, 4, 24, and 41) indicate the amino acid positions of TimP at the N-terminus, and red circles indicate the minimum growth inhibitory concentration values. All representative data shown represent n=3. Figure 3 shows the TimP for reactive oxygen species generation and efflux activity. + Illuminates the role of (a) E. coli and (b) TimP in reactive oxygen species-related gene knockout. + Confirmation of reactive oxygen species production. Relative reactive oxygen species production is TimP + The value was determined by dividing the fluorescence intensity of the cells by the fluorescence intensity of the control group (*P<0.001, **P=0.014) . (c) oxyR knockout( oxyR- ) TimP + Determination of the minimum growth inhibitory concentration of polymyxin B. (d) TimP for ABC transporter-related gene knockout. + Confirmation of the efflux activity effect. Relative efflux activity is TimP + of Fluorescence intensity from Hoechest was expressed as the value obtained by dividing the fluorescence intensity of the control group (P<0.001). (e) TimP of ABC transporter-related gene knockout + Determination of the minimum growth inhibitory concentration of Polymyxin B. Red circles indicate the minimum growth inhibitory concentration values, and AVG±SD for n=9 experiments is shown. Figure 4 shows the results confirming the polymyxin B sensitization activity of FT-Pep or TimP-derived extracellular vesicles and the corresponding mechanism. (a) Quantification and characterization of TimP-derived extracellular vesicles. Nanoparticle trace analysis (top; *P<0.001) and transmission electron microscopy analysis (bottom) are shown. TimP + The relative extracellular endoplasmic reticulum production of TimP + or the OD of the control group 600 It was determined by the number of extracellular vesicle particles standardized to. The white arrows indicated in the transmission electron microscope images represent the size of the extracellular vesicles, expressed as AVG ± SD for experiments with n=3. (b) Effect of TimP-derived extracellular vesicles on polymyxin B sensitization efficacy. TimP-derived extracellular vesicles (0-10 mg ml -1 ) The minimum growth inhibitory concentration of polymyxin B in E. coli under treatment is indicated. (c) Determination of molecular factors for TimP-derived extracellular vesicle-mediated polymyxin B sensitization activity. TimP-derived extracellular vesicles (1 mg mL) treated with DNA degrading enzyme I or protease K, respectively. - 1 The minimum growth inhibitory concentration of polymyxin B was evaluated using ). (d) FT-Pep (25 μg ml) utilizing lipid A modified gene knockout. -1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1 Investigation of polymyxin B sensitization efficacy when ) and polymyxin B are administered in combination. arnT - , eptA -and lpxM - The minimum growth inhibitory concentration value of the polymyxin B / FT-Pep combination for this was not determined under these conditions. (e) Confirmation of relative lipopolysaccharide binding by FT-Pep or TimP-derived extracellular vesicles. The analysis was performed using single-cell labeled polymyxin B. Fluorescence intensity (λ) of E. coli treated with FT-Pep or TimP-derived extracellular vesicles ext 355 / λ em (590-10) was measured (*P<0.001), and relative lipopolysaccharide binding was determined by dividing the treated value by the untreated value of extracellular vesicles derived from FT-Pep or TimP. AVG±SD for n=9 experiments are shown. (f) oxyR Confirmation of reactive oxygen species generation by FT-Pep or TimP-derived extracellular vesicles according to. Relative reactive oxygen species generation was determined by dividing the fluorescence intensity of the values ​​treated with FT-Pep or TimP-derived extracellular vesicles by the fluorescence intensity of the control sample. Expressed as AVG ± SD for n=9 experiments (P<0.001). (g) Confirmation of the effect of efflux activity on polymyxin sensitization. λ ext 355 / λ em 460 The fluorescence intensity of efflux activity by Hoechst assay for E. coli treated with FT-Pep or TimP-derived extracellular vesicles and related gene knockouts is shown (P<0.001). Quantification of outer membrane permeability and membrane depolarization influenced by FT-Pep or TimP-derived extracellular vesicles. (h) NPN(λ ex 355 / λ em 460) or (i) DiSC3(5)(λ ext 584 / λ emFluorescence intensity of 620) (* P<0.001). Relative activity was determined by dividing the fluorescence intensity of TimP-treated bacteria by the fluorescence intensity of the control group. Expressed as AVG±SD with n=9. Cyan, purple, or pink bars represent the control group treated with FT-Pep or TimP-derived extracellular vesicles, respectively. (j) TimP against FT-Pep or TimP-derived extracellular vesicles + Effect of active regulatory factor knockout. FT-Pep (25 μg ml -1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1 The minimum growth inhibitory concentration of polymyxin B in E. coli treated with ) was determined, and representative data for n=3 are shown. The red circle indicates the minimum growth inhibitory concentration value. Figure 5 shows the results confirming the activity of polymyxin against polymyxin-resistant bacteria. (a) Confirmation of the effects of polymyxin B and FT-Pep or TimP-derived extracellular vesicles on polymyxin-resistant bacteria. (b) Analysis of the fractional inhibitory concentration index of polymyxin B / triclosan against FT-Pep or TimP-derived extracellular vesicles against Mcr-1-related polymyxin-resistant bacteria (NCCP 16283 and 16284), (c) Mcr-1 overexpressing E. coli, and (d) non-Mcr-1-related polymyxin-resistant (polymyxin-R) strains. Red or white circles indicate the minimum growth inhibitory concentration values ​​for polymyxin B (or triclosan) or the universal treatment range (FICI ≤ 0.5), respectively. Representative data are shown for n=3. (e) FT-Pep (25 μg ml) upon treatment with polymyxin B / triclosan individually or in combination against NCCP 16283. -1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1Confirmation of the mechanism of action of polymyxin. Individual polymyxin-related activities were measured and analyzed, and the ratio of individual activities to the activities of control samples was expressed as various types of TimP. AVG±SD for n=9 experiments is indicated (P<0.001). Figure 6 shows the results confirming the in vivo efficacy of combination treatment of TimP-derived extracellular vesicles / polymyxin B / triclosan in a mouse infection model. (a) Schematic representation of the experiment using an animal model. (b) Evaluation of the cytotoxicity of TimP-derived extracellular vesicles. Various concentrations of TimP-derived extracellular vesicles (0.005, 0.05, or 0.5 mg kg⁻¹, respectively). -1 After treatment, the survival rate and body weight (%) of mice (n=2 each) were determined. mcr -1 E. coli expression Confirmation of the effects of (c) polymyxin B / triclosan or (d) combination therapy of polymyxin B / triclosan / TimP-derived extracellular vesicles against (NCCP 16283). Mice (n=4 each) were treated with polymyxin B (1 mg kg -1 ), Polymyxin B / Triclosan (1 mg kg -1 / 0.5 mg kg -1 ) or extracellular vesicles derived from polymyxin B / triclosan / TimP (1 mg kg -1 / 0.5 mg kg -1 / 0.5 mg kg - 1 ) was treated 1 hour after NCCP 16283 infection. 7 hours after bacterial infection, suspensions obtained from mouse blood, liver, and kidney were plated onto LB agar plates, respectively, and bacterial colonies were grown overnight. The average bacterial count (CFU / ml) obtained by repeating the process three times was calculated. - 1 ) was obtained. Each point represents an individual mouse and the line represents the median of each group (blood: P=0.0005, kidney: P=0.0223, liver: P=0.0776). Figure 7 shows the results confirming the preservation of TimP activity in TimP-expressing Gram-negative bacteria. (a) Escherichia coli, Salmonella and confirmation of TimP conservation in Shigella. Multiple sequence analysis was performed by Clustal Omega and visualized using Jalview. Amino acid residues were color-coded according to the similarity of physicochemical properties based on the Jalview manual. (b) Confirmation of the conservation of polymyxin B sensitization activity in TimP-expressing Gram-negative bacteria. The minimum growth inhibitory concentration of polymyxin B was determined for seven TimP-expressing strains, and (c) investigation of polymyxin B sensitization activity of FT-Pep or TimP-derived extracellular vesicles on ATCC 14028S, ATCC 12022, and NCCP 16202 strains. (d) TimP-ST on ATCC 14028S, SL1344, and BW25113 strains. + Confirmation of activity. (e) Confirmation of sensitization activity of TimP-ST-derived extracellular vesicles and polymyxin B against ATCC 14028S, SL1344, and BW25113. For (d) and (e), TimP-ST+ extracellular vesicles (0.1 mg ml -1 ) was used. (f) Strains in which the TimP coding gene is absent ( Acinetobacter baumannii ATCC 17978, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus Sensitization effects of FT-Pep or TimP-derived extracellular vesicles and polymyxin B on ATCC 25923. Red circles indicate MIC values, and representative data are displayed among n=3. (g) TimP on ATCC 14028S, NCCP 16202, and ATCC 12022 +Evaluation of 5 bacterial mechanisms of action or FT-Pep (25 μg ml) against ATCC 14028S -1 Evaluation of the mechanism of action of ) and polymyxin or TimP-derived extracellular vesicles (0.1 mg ml -1 Evaluation of the mechanism of action of polymyxin. AVG±SD for n=9 is shown (P<0.05). Specific details for implementing the invention

[0011] The inventors evaluated the efficacy of TimP peptide, a small protein encoded by RyfA among E. coli non-coding ribonucleic acids, and extracellular vesicles as polymyxin sensitizing agents, and demonstrated that TimP exhibits a "Trojan horse" effect as a universal polymyxin B synergistic agent against E. coli and Gram-negative bacteria containing the TimP gene. Furthermore, to control polymyxin-resistant bacteria, the inventors verified the polymyxin sensitization recovery effect in vitro and in a mouse model from the combined treatment of triclosan, a fatty acid synthase (FAS) inhibitor, with 5'-carboxyfluorescein / FAM-TimP peptide (FT-Pep) or TimP-derived extracellular vesicles, and completed the present invention.

[0012] The present invention provides a composition for enhancing sensitivity to polymyxin comprising, as an active ingredient, a TimP peptide consisting of the amino acid sequence indicated by SEQ ID NO. 1 or a bacterial-derived extracellular vesicle overexpressed with said TimP peptide.

[0013] Preferably, the composition can enhance polymyxin sensitization efficacy by inducing the generation of reactive oxygen species, but is not limited thereto.

[0014] Preferably, the TimP peptide is Escherichia coli BW25113, Shigella ATCC 12022, Shigella NCCP 16202 and SalmonellaPolymyxin sensitivity to any one or more strains selected from the group consisting of ATCC 14028S can be enhanced, and the extracellular vesicles derived from cells overexpressing the TimP peptide are Escherichia coli BW25113 or Salmonella Polymyxin sensitivity to ATCC 14028S may be enhanced, but is not limited thereto.

[0015] In the present invention, the amino acid sequence indicated by SEQ ID NO. 1 is “MKIRCFCIVLIVSGALLTEVNNNRSLSGDNLLVVNNLQSSK”, which is a TimP peptide containing a signal peptide, and is also abbreviated as “FT-Pep” in this specification.

[0016] The peptide of the present invention may be produced by genetic engineering methods. First, a DNA sequence encoding said peptide is synthesized according to conventional methods. The DNA sequence may be synthesized by PCR amplification using appropriate primers. Alternatively, the DNA sequence may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (e.g., one sold by Biosearch or Applied Biosystems). The synthesized DNA sequence is introduced into a vector containing one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate expression to perform the specific function of said DNA sequence, and the recombinant expression vector formed therefrom is transformed into a host cell. The resulting transformant is cultured in an appropriate medium and under appropriate conditions so that said DNA sequence is expressed, and a substantially pure peptide encoded by said DNA sequence is recovered from the culture. said recovery may be performed using methods known in the art (e.g., chromatography). In the above, the term 'substantially pure peptide' means that the peptide according to the present invention does not substantially contain any other protein derived from the host.

[0017] In addition, the peptide of the present invention can be easily prepared by chemical synthesis known in the art. Representative methods include, but are not limited to, liquid or solid-phase synthesis, fragment condensation, F-MOC or t-BOC chemistry.

[0018] The peptide of the present invention is a concept that includes functional variants thereof. “Functional variant” means all similar sequences in which substitution of some amino acids occurs at amino acid positions that do not affect the properties of the peptide of the present invention.

[0019] In addition, the present invention provides an antimicrobial composition for polymyxin-resistant Escherichia coli comprising a TimP peptide consisting of an amino acid sequence indicated by SEQ ID NO. 1 or a bacterial-derived extracellular vesicle overexpressing the TimP peptide; polymyxin; and triclosan as an active ingredient.

[0020] Preferably, the polymyxin-resistant E. coli may be a Mcr-1 expressing polymyxin-resistant E. coli, but is not limited thereto.

[0021] The term "antimicrobial composition" used in the present invention collectively refers to cosmetic or pharmaceutical preservatives, disinfectants, and antimicrobial agents.

[0022] The present invention is described in detail below according to embodiments that do not limit the invention. It should be understood that the following embodiments of the present invention are merely for the purpose of embodying the invention and do not limit or restrict the scope of the rights of the present invention. Accordingly, anything that can be easily inferred by a person skilled in the art to which the present invention pertains from the detailed description and embodiments of the present invention is interpreted as falling within the scope of the rights of the present invention.

[0023] < Experimental Example >

[0024] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0025] 1. Strains, oligonucleotides, and antibiotics used

[0026] The bacterial strains used in this invention are shown in Table 1. Bacteria were cultured using Luria-Bertani liquid medium or its agar plates. Prior to the start of the experiment, the gene knockout (Keio library) and overexpression (ASKA clone) samples used were verified by PCR using universal or gene-specific primers. The antibiotics used were ampicillin (Ap), chloramphenicol (Cm), and kanamycin (Kan) at 50, 34, and 25 μg / ml, respectively. -1Target gene and non-coding ribonucleic acid gene expression were induced using isopropyl β-D-1-thiogalactopyranoside (IPTG). All antibiotics and chemical reagents were purchased (Millipore Sigma, Saint Louis, MO, USA), prepared at high concentrations, and then diluted to the desired concentrations. Non-mcr-1 mediated polymyxin-resistant (polymyxin-R) E. coli were produced using polymyxin B at minimum inhibitory concentrations (0.4–3.2 μg / ml). -1 It was prepared by culturing E. coli after administering ) 15 consecutive times. The final minimum growth inhibitory concentration of polymyxin B for polymyxin-R was 6.4 μg ml -1 It was confirmed as (Fig. 5).

[0027] [Table 1]

[0028]

[0029] KCCM: Korean Culture Collection Center (http: / / www.kccm.or.kr); NCCP: Korea Disease Control and Prevention Agency National Pathogen Resource Bank (https: / / nccp.kdca.go.kr / main.do).

[0030] 2. Polymyxin B Sensitization and for determining the minimum growth inhibitory concentration non-coding Ribonucleic acid screening

[0031] pHMB1-based non-coding ribonucleic acid gene overexpression libraries were transformed into E. coli, and the resulting colonies were induced with 0.1 mM IPTG; subsequently, the minimum inhibitory concentration of polymyxin B was determined using 96-well plates. Ap or Kan resistance cassettes (Ap, respectively) R and Kan R) did not affect the minimum growth inhibitory concentration value of polymyxin B. The 96-well plates were imaged with a digital camera (Samsung NX200, Suwon, Korea), and representative data are shown among n=3.

[0032] 3. Growth Curve Analysis

[0033] Bacteria were cultured using a SPECTROstar (BMG Labtech, Ortenberg, Germany) under conditions of 37℃, 16 hours, and 500 rpm, and at regular intervals at 600 nm (OD 600 Optical density was measured and analyzed using Nano and MARS data analysis software V3.00R2 (BMG Labtech, Ortenberg, Germany). A portion of the bacterial culture obtained after 16 hours (5 μL) was aliquoted onto LB agar plates and incubated at 37°C for an additional 16 hours. Agar plate images are from ChemiDoc ™ Images were generated using MP System and Image Lab (V5.02; Bio-Rad, Hercules, CA, USA). Representative data among n=3 are indicated.

[0034] 4. Gene synthesis, codon optimized non-coding Ribonucleic acid gene origin peptide Manufacturing and Cloning

[0035] The codons of ORF1, TimP, and non-coding ribonucleic acid gene-derived peptides were optimized and synthesized by ordering them to GenScript (Piscataway, NJ, USA). Subsequently, the optimized gene was used for pHMB1's Eco RI Department Hin of dIII or pQE60 Nco I Bgl Subcloned at position II. pSerranoRFP- mcr-1 In production Xba I Sma Codon optimized between I positions mcr-1It was prepared by subcloning the gene, and pHMB1-TimP-ST is Salmonella It was constructed by subcloning the derived TimP coding region. The clones were verified using primers designed for DNA sequencing.

[0036] 5. lipopolysaccharides Combined analysis

[0037] TimP + The lipopolysaccharide binding ability of bacteria treated with FT-Pep or TimP-derived extracellular vesicles was evaluated using monocell-labeled polymyxin B. TimP induced with 0.1 mM IPTG + Bacteria or FT-Pep (25 μg ml) -1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1 ) treated E. coli to obtain a bacterial dilution with a McFarland turbidity of 0.5, and in LB liquid medium at 37°C for 3 hours OD 600 The culture was stirred and incubated until ~0.2. The final bacterial culture was centrifuged, and the bacterial precipitate was harvested and resuspended in PBS buffer. The resuspended precipitate was then incubated with single-cell labeled polymyxin B at 37°C for 4 hours without stirring. 200 μL of the bacterial suspension was dispensed into 96-well plates (SPL, Daejeon, Korea), and FLUOStar 　 Using Omega and MARS data analysis software V3.00R2 (BMG Labtech, Ortenberg, Germany) λ ex 485-12 / λ em Fluorescence intensity was quantified at 520. The relative lipopolysaccharide binding affinity was TimP + The fluorescence intensity of bacteria treated with FT-Pep or TimP-derived extracellular vesicles was evaluated by dividing it by the fluorescence intensity of control samples. AVG±SD obtained from n=9 samples is shown.

[0038] 6. Zeta potential measurement

[0039] FT-Pep (25μg ml -1 ) treated or untreated E. coli (OD 600 The zeta potential (millivolts, mV) of ~0.5 was quantified using the Nanopartica SZ-100 (Horiba Scientific, Tokyo, Japan) according to the manufacturer's instructions. AVG ± SD is indicated for a sample size of n=3.

[0040] 7. Ribonucleic acid extraction and quantitative RT- qPCR

[0041] pHMB1-TimP(TimP + Escherichia coli harboring ) or pHMB1 (control) at OD at 37℃ 600 Incubated to ~0.5. Subsequently, 0.1 mM IPTG was added to the culture to [calculate] OD 600 Cultured up to ~1.0. Monarch according to manufacturer's instructions. Using the Total RNA Miniprep Kit (New England Biolabs, Ipswich, MA, USA), the control group and TimP + Total RNA was extracted from ReverTraAce TM qPCR RT MasterMix with gDNA remover (Toyobo, Osaka, Japan) and Turbo DNA-free according to the supplier protocol TM cDNA from whole RNA using the Kit (Thermo Fischer Scientific, Lenexa, KS, USA) Synthesized. qRT-PCR analysis was performed on CFX96 TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and IQ TM SYBR ®The study was performed using Green Supermix (Bio-Rad, Hercules, CA, USA). Statistical analysis was conducted using CFX Manager™ software (Ver. 3.1, Bio-Rad, Hercules, CA, USA). AVG ± SD is displayed for a sample size of n=3.

[0042] 8. Analysis of outer membrane permeability

[0043] TimP + The outer membrane permeability of extracellular vesicles derived from , FT-Pep, or TimP was performed as follows. TimP + , FT-Pep (25 μg ml -1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1 Bacterial suspension treated with ) (10 8 CFU ml - 1 ) was mixed with buffer (5 mM HEPES, 5 mM glucose buffer, pH 7.4), and NPN (5 μM) was added to the suspension to λ ext 355 / λ em Measure the fluorescence intensity of the sample at 460 nm and FLUOStar ® Omega and MARS data were analyzed using the analysis software V3.00R2 (BMG Labtech, Ortenberg, Germany). The normalized fluorescence intensity (fluorescence intensity / OD) of TimP-treated bacteria 600 Relative outer membrane permeability was determined by dividing by the fluorescence intensity of the control sample. AVG±SD is indicated for a sample size of n=9.

[0044] 9. Act depolarization analyze

[0045] TimP + , FT-Pep (25 μg ml -1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1The active membrane potential of the bacterial membrane depolarization was measured using the dye DiSC3 (5). The fluorescence intensity of the sample was measured using FLUOStar® Omega and MARS data analysis software V3.00R2 (BMG Labtech, Ortenberg, Germany) λ ext 584 / λ em It was measured at 620-10. Relative membrane depolarization was TimP + The fluorescence intensity of cells treated with FT-Pep or TimP-derived extracellular vesicles was measured by dividing it by the fluorescence intensity of the control sample. The results were expressed as AVG ± SD for n=9 samples.

[0046] 10. Bacterial Morphological Analysis

[0047] TimP with or without Acridin Orange + Microscopic image analysis of bacteria was performed at 100x magnification using the SELENA S cell imaging system (Logos biosystems, Annandale, VA, USA). E. coli cultures containing pHMB1 or pHMB1-TimP with a McFarland turbidity of 0.5 were diluted 1,000-fold in LB liquid medium, and 0.1 mM IPTG was added to [calculate] the OD 600 After incubating at ~0.2, the cells were further stirred and incubated at 37°C for 2 hours. Bacterial cells were harvested, and the resulting suspension was used directly after washing with PBS buffer. High-resolution morphological analysis of E. coli by scanning electron microscopy at 30 kV was performed using the VEGA3, a multi-purpose tungsten thermionic emission SEM system (TESCAN, Fuveau, France), according to the supplier's protocol. All experiments were repeated three times, and the results of the statistical quantitative analysis of the data are presented.

[0048] 11. Quantification of Reactive Oxygen Species

[0049] Quantification of reactive oxygen species production is TimP + , FT-Pep (12.5 μg ml-1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1 E. coli treated with ) was analyzed using FLUOstar Omega and MARS data analysis software V3.00R2 (BMG Labtech, Ortenberg, Germany) according to the manufacturer's instructions. Relative reactive oxygen species generation was TimP + It was calculated by dividing the fluorescence intensity of the control cell by the fluorescence intensity of the control cell and expressed as AVG±SD with n=9.

[0050] 12. Fluorescence Activity Assessment

[0051] To measure efflux activity, Hoechst analysis was performed as follows. TimP + (0.1 mM IPTG), FT-Pep (12.5 μg ml -1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1 ) treated bacteria and / or polymyxin B (0.2 μg ml -1 Bacteria treated with ) were cultured. FLUOStar 　 Fluorescence intensity was monitored using Omega and MARS data analysis software V3.00R2 (BMG Labtech, Ortenberg, Germany), and these values ​​were OD 600 Fluorescence intensity / OD, the value divided by 600 was determined. Relative efflux activity is TimP + The fluorescence intensity of bacteria treated with FT-Pep or TimP-derived extracellular vesicles was measured by dividing it by the fluorescence intensity of the control sample, and expressed as AVG±SD with n=9.

[0052] 13. Bacterial origin Extracellular Purification and characterization of endoplasmic reticulum

[0053] Bacterial-derived extracellular vesicles are ExoBacteria according to the supplier's protocol TMExtraction was performed using the OMV Isolation Kit (Systems Biosciences, CA, USA). The extracted extracellular vesicles were analyzed for size using a NanoSight NS300 (Malvern Panalytical, Malvern, UK) and a Talos L120C TEM (Thermo Fischer Scientific, Lenexa, KS, USA) according to the vendor's protocol. To identify determinants for bacterial extracellular vesicle-mediated polymyxin B sensitization, DNA degradase I (1 U μL -1 , Zymo Research, Irvine, CA, USA) or protease K (20 mg ml -1 , Thermo Fischer Scientific, Lenexa, KS, USA) TimP-derived extracellular vesicles (0.1 mg ml -1 The samples were analyzed after treatment at 37°C for 2 hours according to the manufacturer's instructions. The minimum growth inhibitory concentration of polymyxin B was determined when DNA or protease-treated bacterial extracellular vesicles were co-administered with polymyxin B, and the plates were imaged using a digital camera (Samsung NX200, Suwon, Korea). Representative data for n=3 are presented.

[0054] 14. peptide Prediction of characteristics

[0055] Predictions were made using PepCalc (www.pepcalc.com, Innovagen AB, Lund, Sweden) and the antimicrobial activity and peptide structure database (DBAASP, www.dbaasp.org).

[0056] 15. of peptides Synthesis and Characterization

[0057] Purified 5'-FAM modified TimP peptide (5'-FAM-MKIRCFCIVLIVSGALLTEVNNNRSLSGDNLLVVNNLQSSK; FT-Pep) or TimP peptide without signal peptide [TEVNNNRSLSGDNLLVVNNLQSSK; T-Pep(-signal peptide)] were synthesized at Pepmic Co. Ltd (Suzhou, Jiangsu, China) or GenScript (Picataway, NJ, USA), respectively. The peptides were purified using preliminary high-performance liquid chromatography (HPLC) with a stationary phase of C18-bonded silica and a mobile phase gradient of 0.065–0.1% (v / v) trifluoroacetic acid (TFA) in water and 0.05–0.1% (v / v) TFA in acetonitrile. Peptide sequences were analyzed under the same mobile and stationary phases using an Inertsil ODS-SP analysis column (250 x 4.6 mm, 5 μm, Shimadzu Corp., Japan), and the sequence composition was verified by electrospray mass spectrometry using the LCMS-2020 mass spectrometer (Shimadzu Corporation, Kyoto, Japan).

[0058] 16. Comparative Analysis of Amino Acid Sequences

[0059] E. coli The TimP amino acid sequence of K-12 MG1655 (WP_001380605.1) is Salmonella 14028S (WP_242617172.1), dysentery bacteria boydii (WP_252965562.1), dysentery bacteria flexneri 2a str. 2457T (EFS15410.1) and Shigella sonnei For the homology analysis of (WP_252990267.1), the National Center for Biotechnology Information database was consulted, and Jalview (https: / / www.jalview.org / ) was used for sequence alignment and visualization.

[0060] 17. Fractional Inhibition Concentration Index Analysis ( FICI )

[0061] FT-Pep (25 μg ml -1 ) or TimP-derived extracellular vesicles (0.1 mg ml -1 The antimicrobial activity of polymyxin B / triclosan was evaluated, and the sum of the Fractional Inhibition Index (FICI) analysis for polymyxin B / triclosan was presented as described. 96-well plates were imaged using a digital camera (Samsung NX200, Suwon, Korea), and aliquots (5 μl) of mature bacteria were placed on LB agar plates and incubated at 37°C for 16 hours. Plate images were taken from ChemiDoc ™ Analysis was performed using the MP System and Image Lab (V5.02) (Bio-Rad, Hercules, CA, USA), and representative data from n=3 analysis experiments are presented.

[0062] 18. Analysis of in vivo efficacy through animal experiments

[0063] Animal experiments were conducted with the approval of the Animal Experimentation Management Committee of Pusan ​​National University (Approval No.: PNU-2023-0393). Female C57BL / 6 mice (6 weeks old) were injected intraperitoneally with 1.1 x 10⁶ E. coli bacteria suspended in sterile phosphate-buffered saline (PBS). 10 Infection was administered in amounts equivalent to colony-forming units (CFU). One hour later, polymyxin B (1 mg kg) was administered via the intraperitoneal route. -1 ), triclosan (0.5 mg kg -1 ), TimP-derived extracellular endoplasmic reticulum (0.005, 0.05, or 0.5 mg kg -1Polymyxin B / triclosan combinations, treated with or untreated with TimP-derived extracellular vesicles, were administered to each individual. Six hours after antibiotic administration, samples were taken from blood, kidney, and liver fractions, plated onto LB agar plates, and incubated at 37°C for 16 hours. Colony counts were determined on the incubated LB agar plates. Statistical significance was determined using an unpaired two-sided t-test in GraphPad Prism (v10.1.2) (GraphPad Software, Boston, MA, USA, www.graphpad.com).

[0064] 19. Statistical Analysis

[0065] Statistical analysis was performed using one-way analysis of variance with the Holm-Sidak method integrated into SigmaPlot (v12.5) (Inpixon HQ, Palo Alto, CA, USA).

[0066] < Examples 1> Polymyxin as an increase / decrease agent TimP

[0067] Overexpression of non-coding ribonucleic acid in E. coli (gene + The minimum growth inhibitory concentration for polymyxin B was determined by (indicated as ). Among the 92 determined non-coding ribonucleic acid genes, OmrA + , RyfA + , SdsR + showed increased sensitization to polymyxin B (Fig. 1a). Among them, RyfA + Only showed an effect regardless of general growth conditions, and increased sensitization to colistin was also confirmed (Fig. 1b).

[0068] E. coli ryfA The gene is also present in the Gram-negative bacterium Salmonella; previous results showed a sequence similar to the toxic protein TimP, and it was predicted that this gene encodes the protein ORF1 (Fig. 1c). TimP from the determination of the minimum growth inhibitory concentration of polymyxin B + RyfA + It was shown to possess polymyxin sensitization activity as shown (Fig. 1d). However, among known peptides derived from non-coding ribonucleic acid genes in E. coli, no peptide exhibiting sensitization activity to polymyxin B was identified, confirming that TimP is a unique polymyxin-sensitizing peptide derived from non-coding ribonucleic acid genes. E. coli's Sequence analysis predicted that TimP possesses a localization signal peptide (Fig. 1c). Analysis of the effect of the signal peptide on the sensitization efficacy of polymyxin B revealed that TimP without the signal peptide (-signal peptide) + Since no polymyxin sensitization characteristics were observed (Fig. 1e), it was confirmed that the signaling peptide of TimP is essential for increased polymyxin B sensitization. Additionally, the sensitization efficacy of polymyxin was investigated by overexpressing TimP in the knockout of genes encoding regulatory factors of TimP and RyfA-related factors, namely CpxR, Hfq, ProQ, and YbeY. As a result, it was confirmed that TimP-mediated polymyxin sensitization efficacy was inhibited only in the knockout of ProQ (Fig. 1f).

[0069] As a result of investigating the polymyxin sensitization of hydrophobic proteins T-Pep (-signal peptide) and FT-Pep, neither T-Pep (-signal peptide) nor FT-Pep exhibited any antimicrobial activity on their own (Fig. 1g), and it was confirmed that only FT-Pep affected polymyxin sensitization activity when treated in combination with polymyxin B (Fig. 1h). These results establish TimP as a polymyxin sensitization peptide, confirm the role of signal peptides in TimP-mediated polymyxin sensitization, and support TimP action within bacteria (Fig. 1h).

[0070] < Examples 2> Polymyxin Sensitization having an impact lipopolysaccharides deformation and Cardiolipin synthesis

[0071] Polymyxin bound to lipopolysaccharide molecules on the outer membrane of Gram-negative bacteria through hydrophobic interactions with cationic side chains. Therefore, it was hypothesized that TimP can increase polymyxin sensitization by increasing the binding of polymyxin to lipopolysaccharides, and TimP + This was evaluated by treating lipopolysaccharides purified from the control group with fluorescent monocrystalline polymyxin B, a polymyxin B analog, and measuring the fluorescence intensity. TimP + The fluorescence intensity was 3.1 times higher than the control group (Fig. 2a), which is TimP + It demonstrated that the polymyxin B-lipopolysaccharide binding of derived lipopolysaccharides is superior. This phenomenon is TimP + It can occur due to an increase in negative charge caused by TimP + No change in charge was detected in the measurement of the zeta potential of the bacteria (Fig. 2b).

[0072] TimP + Major lipid A modification gene expression affected by TimP + Total RNA was extracted from bacteria and analyzed using RT-qPCR. For this purpose, genes related to polymyxin activity and resistance were selected: 1) PhoPQ and PmrAB; 2) ArnT and EptA; 3) LpxM. Among these, the increase in mRNA was eptA , lpxM , phoP and pmrA , decrease is lpxL , phoQ ; No change arnT , pmrB It was confirmed as TimP+ Through additional analysis of gene knockout for activity arnT , eptA , lpxM , phoP or pmrA Is TimP + It was confirmed that it is a factor affecting activity (Fig. 2c). All of the above data are TimP + It is involved in modifying the activity of lipid A and polymyxin through lipid A modification genes and eptA , lpxM , phoP or pmrA TimP expression + It indicated the importance of the function.

[0073] When compared to the control group in the N-phenyl-1-naohthylamine (NPN) and DiSC3(5) assays that measure cytoplasmic membrane depolarization, TimP + It was confirmed that it increases membrane permeability and depolarizes the cytoplasmic bacterial membrane (Figs. 2d and 2e).

[0074] PhoPQ increased the amount of cardiolipin, and E. coli Cardiolipin synthase gene knockout induced increased polymyxin sensitization (Fig. 2f). TimP + To identify the cardiolipin synthase required for the function, TimP from individual knockouts of the cardiolipin synthase gene + The minimum growth inhibitory concentration for polymyxin B was determined. clsB and clsC Is completely, clsA is partially TimP + It was confirmed that the activity is controlled (Fig. 2f). TimP using acridin orange dye + Through microscopic analysis of cell division inhibition, TimP +Additional data were obtained showing that bacteria expressing [the compound] were elongated and stained better with dyes than the control group (Fig. 2g). Cells treated with polymyxin exhibit an elongated shape, and it is known that cardiolipin is associated with bacterial division. TimP + It can be seen that bacterial length increase and / or modification of cardiolipin composition caused by [subject] is the underlying mechanism of action of polymyxin sensitization. However, this hypothesis was not supported because it did not demonstrate a linear relationship between increased elongation through inhibition of cell division and polymyxin sensitization. 1) DicF + ; This is a non-coding ribonucleic acid gene that elongates the length of bacteria upon overexpression, but it showed no sensitization activity to polymyxin; 2) minC is a gene knockout that produces filamentous bacteria (Fig. 2h), TimP + It inhibited the action of (Fig. 2i). These results indicate that the membrane lipid levels and distribution of cardiolipin, rather than bacterial elongation, are TimP + This suggests that it may affect the action. This was supported by microscopy (Fig. 2g) and SEM images (Fig. 2j), which indicate TimP + This shows that it is affected by cardiolipin-dependent proteins such as ProP and MscS. Therefore, the knockout of cardiolipin-dependent proteins is TimP + It can interfere with the activity of, and actually proP or mscS TimP for polymyxin sensitization in knockout +As a result of evaluating activity, it was confirmed that both strains lost activity (Fig. 2k). TimP, a hydrophobic protein, contains basic residues (2 Arg and 2 Lys) (Fig. 1c), which implied that it binds to the phosphate group of cardiolipin. Therefore, this implies that TimP function can be influenced by cardiolipin, and the elimination of binding ability and the interaction between TimP and cardiolipin could inhibit polymyxin sensitization activity. To verify this, base residues 2, 4, 24, or 41 of TimP were mutated with alanine, and polymyxin sensitization was confirmed to be completely or partially inhibited (Fig. 2l). Thus, it was confirmed that the interaction of cardiolipin through TimP base sequences 2, 4, and 24 is essential for polymyxin sensitization.

[0075] < Examples 3> TimP's Reactivity Oxygen species Inhibition of generation and efflux activity

[0076] TimP + The bacteria produced 10.8 times more ROS than the control group (Fig. 3a). TimP + To confirm the roles of OxyS and SoxS in the production of reactive oxygen species oxyR or soxS TimP in knockout bacteria + Reactive oxygen species for were investigated. oxyR of TimP at the knockout + It was confirmed that the reactive oxygen species generation function caused by [this] was eliminated (Figs. 3b and 3c). Therefore, OxyR confirmed the polymyxin sensitization activation phenomenon caused by the reactive oxygen species of TimP.

[0077] TimP for effluent activity using the Hoechst assay + The activity of TimP was verified. +The bacteria exhibited stronger fluorescence intensity than the control (Fig. 3d), indicating inhibition of efflux activity. In addition, genes associated with efflux activity lptB , tolC TimP through knockout + As a result of evaluating activity, effluent activity lptB It was lost in the knockout, but tolC It was confirmed that there was no loss in the knockout (Fig. 3d). The same set of TimP + In the experiment to determine the minimum growth inhibitory concentration of polymyxin B, TimP + The polymyxin sensitization activity of lptB It was confirmed that it was lost in the knockout (Fig. 3e). Also, TimP + from bacteria lptB and tolC of Transcriptome through RT-qPCR analysis lptB is increasing tolC It was further confirmed that it decreased. This is in polymyxin-resistant bacteria tolC and lptB Regulation of the expression level of controls polymyxin sensitization, and TimP + This suggests that it regulates efflux activity when exposed to polymyxin.

[0078] < Examples 4> TimP origin Extracellular Mass production of endoplasmic reticulum and Polymyxin Sensitization Efficacy Analysis

[0079] The cardiolipin required for outer membrane assembly and surface exposure is TimP + It can be seen that it is necessary for the function (Fig. 2). Through this, TimP + enabled the mass production of extracellular vesicles through polymyxin sensitization activity. To verify this hypothesis, TimP +Extracellular vesicles were extracted from bacteria, their characteristics were analyzed using a transmission electron microscope, and the number and concentration of particles were quantified using nano-particle analysis and BCA protein quantification to determine TimP relative to the same bacterial count. + It was confirmed that a large amount of extracellular endoplasmic reticulum was produced (Fig. 4a). TimP + The sensitizing efficacy of polymyxin was evaluated using derived extracellular vesicles, and dose-dependent polymyxin sensitizing activity was also assessed (Fig. 4b). To identify the key biological components of this mechanism, TimP + Polymyxin sensitization activity was evaluated by treating derived extracellular vesicles with DNA degrading enzyme I or protease K, and it was confirmed that polymyxin sensitization activity was eliminated in extracellular vesicles treated with protease K (Fig. 4c). This is TimP + Polymyxin sensitization of derived extracellular vesicles suggests that it is mediated by proteins labeled on the surface of extracellular vesicles.

[0080] < Examples 5> Polymyxin Sensitization FT-Pep and TimP origin Extracellular Mechanism of action of the endoplasmic reticulum

[0081] The TimP from before + The same study was conducted. First, as a result of evaluating lipid A modification gene knockout: three regulatory forms were identified depending on the type of TimP. 1) Repression: minC -, oxyR - , prmr A - , tolC - (FT-Pep and TimP-derived extracellular vesicles) and arnT- , lpxM - , lpxP -(TimP-derived extracellular vesicles); 2) No effect: lptB - (FT-Pep); 3) Opposite function: eptA - (FT-Pep and TimP-derived extracellular vesicles) and arnT - , lpxM - and lpxP- (FT-Pep) (Fig. 4d). Second, evaluation of the lipid A binding capacity of the two substances revealed that FT-Pep increased single-cell polymyxin B binding to lipid A (~3-fold), whereas TimP-derived extracellular vesicles showed no effect (Fig. 4e). This difference in results suggests that lipid A modification is not general, and that the mechanism of TimP-mediated polymyxin sensitization is substance-dependent. Third, both substances increased the production of reactive oxygen species (Fig. 4f). Since all types of TimP generate reactive oxygen species, this mechanism indicates that it is key to TimP's polymyxin sensitization. Fourth, Hoechst analysis showed that TimP + Unlike, no change in effluent activity was observed upon treatment with the two substances (Fig. 4g). Fifth, FT-Pep is TimP + It was confirmed that while membrane depolarization was induced as shown, TimP-derived extracellular vesicles reduced depolarization (Figs. 4h and 4i). Finally, the activity of the two substances on bacterial elongation changes, cardiolipin biosynthesis, cardiolipin-related proteins, and transcription factors was verified using gene knockout, and the results showed proQ - While only [the other] was completely deactivated for the two substances, mscS -, phoP - and proP - Is It was confirmed that only the mechanism of TimP-derived extracellular vesicles is inactivated (Fig. 4j). All of the above results show that it operates differently depending on the type of TimP.

[0082] < Examples 6> TimP In Gram-negative bacteria expressing TimP Active preservation efficacy

[0083] As shown in Figure 7, there were almost no Gram-negative bacteria containing TimP homologues. Therefore, this suggests that TimP function may be conserved in these bacteria. To verify this hypothesis, the minimum growth inhibitory concentration of polymyxin B was determined in seven TimP homologue bacteria using the TimP plasmid from E. coli. Among them, ATCC 12022, ATCC 14028S, and NCCP 16202 exhibited polymyxin sensitization activity identical to that of E. coli. Furthermore, among the three TimP-active species mentioned above, ATCC 14028S showed polymyxin sensitization activity in FT-Pep and TimP-derived extracellular vesicles, indicating that TimP function is conserved in E. coli and Salmonella. However, TimP... all Salmonella It did not exhibit activity against the strain, indicating that the activity is strain- or sequence-dependent. To confirm this, E. coli's TimP + go Non-functional Salmonella TimP-ST overexpressing the TimP sequence of the SL1344 strain + Polymyxin sensitization activity on Escherichia coli and Salmonella (ATCC 14028S) was confirmed using [the method]. The results showed that TimP of the same species + TimP of a different species + It was confirmed to be more effective, and TimP-ST + It was confirmed that it increased the sensitization activity of polymyxin B in both strains. However, TimP-ST + The derived extracellular endoplasmic reticulum had polymyxin sensitization activity only in Salmonella species (Fig. 7).

[0084] Next, additional Gram-negative bacteria without TimP homologs ( Acinetobacter baumannii and Pseudomonas aeruginosa ) and positive bacteria( Staphylococcus aureus FT-Pep and TimP regarding ) + The effects of derived extracellular vesicles were verified. The results showed that neither substance affected the polymyxin B sensitization activity for all strains. Therefore, the spectrum of TimP activity was limited to Gram-negative bacteria with TimP homologues (Fig. 7).

[0085] The mechanism of action of TimP-active bacterial species (ATCC 12022, ATCC 14028S, and NCCP 16202) treated with FT-Pep or TimP-derived extracellular vesicles was evaluated using polymyxin-mediated phenotypes. Additionally, the polymyxin sensitization activity of FT-Pep or TimP-derived extracellular vesicles was confirmed against ATCC 12022 and NCCP 16202, and no activity was observed (Fig. 7). These results reveal two distinct characteristics: 1) TimP + 1) induced binding with different lipopolysaccharides, membrane depolarization, and reactive oxygen species generation with different species specificities: ATCC 14028S (lipopolysaccharide binding and reactive oxygen species), NCCP 16202 (reactive oxygen species), and ATCC 12022 (lipopolysaccharide binding, membrane depolarization, and reactive oxygen species); 2) FT-Pep induced lipopolysaccharide binding and reactive oxygen species generation, whereas TimP-derived extracellular endoplasmic reticulum induced only reactive oxygen species generation. From all of the above results, it was found that reactive oxygen species induction is a conserved mechanism of action independent of TimP type.

[0086] < Examples 7> Polymyxin FT-Pep and for controlling resistant bacteria TimP origin Extracellular Combined treatment of endoplasmic reticulum and polymyxin B / triclosan

[0087] Based on the hypothesis that TimP can be effectively utilized against polymyxin-resistant bacteria because it affects phospholipid and outer membrane-related pathways (Fig. 4) and reduces outer membrane fluidity by altering the phospholipid composition of polymyxin-resistant bacteria, mcr -1 For strains (NCCP 16283 and 16284), the minimum growth inhibitory concentration was determined using polymyxin B and extracellular vesicles derived from FT-Pep or TimP. mcr -1 For the strain, the two forms of TimP did not have polymyxin sensitization activity (Fig. 5a), which means that TimP mcr -1 It was indicated that polymyxin sensitization activity could not be restored for the strain. However, this result is predictable from the previously shown results, as the mcr-1 strain is known to regulate polymyxin sensitization by lipid A modification, so TimP activity affected by lipid A modification may be reduced (Figs. 2 and 4). Therefore, to determine whether FT-Pep or TimP-derived extracellular vesicles could subtly sensitize the strain, activity was verified using triclosan, a fatty acid synthesis inhibitor. For NCCP 16283 and 16284, analysis of the fractional inhibition concentration index of FT-Pep or TimP-derived extracellular vesicles and triclosan / polymyxin B confirmed that the effect of the combination treatment was enhanced (Fig. 5b). Since the NCCP 16284 strain is resistant to multi-cell wall-targeting antibiotics and polymyxin B, it is unclear why the effect of the above combination treatment appears weak. Additionally, the combined effect of polymyxin B / triclosan treatment on FT-Pep and TimP-derived extracellular vesicles in E. coli expressing codon-optimized Mcr-1 protein was evaluated, and it was confirmed that the same effect was observed (Fig. 5c). Furthermore, mcr- 1 We evaluated other types of polymyxin-resistant bacteria (polymyxin-R) that do not contain [specific component] (Fig. 5d). We confirmed that the effect of both FT-Pep and TimP-derived extracellular vesicles increased upon co-treatment with polymyxin B / triclosan. This suggests that the polymyxin B / triclosan / TimP combination could be a potential therapeutic agent capable of resensitizing polymyxin-resistant bacteria to polymyxin.

[0088] On the effects of combined treatment with polymyxin B / triclosan mcr -1 including Evaluations were conducted against Salmonella and Shigella. It was confirmed that the combined treatment of polymyxin B / triclosan was more effective than the polymyxin B / triclosan / TimP combination. Therefore, it was confirmed that polymyxin B / triclosan / TimP is capable of controlling only polymyxin-resistant E. coli. To determine the mechanism of action of antimicrobial activity induced by FT-Pep or TimP-derived extracellular vesicles in the polymyxin B / triclosan combination against polymyxin-resistant E. coli, five polymyxin-mediated phenotypes were evaluated using NCCP 16283. 1) The polymyxin B / triclosan combination induced outer membrane permeability and efflux activity compared to antibiotic treatment, respectively. 2) The addition of FT-Pep induced outer membrane permeability to the polymyxin B / triclosan combination. 3) The addition of TimP-derived extracellular vesicles increased the binding of lipopolysaccharides to the polymyxin B / triclosan combination (Fig. 5e). All of the above data indicate that extracellular vesicles derived from FT-Pep and TimP can be utilized to control polymyxin-resistant E. coli through various mechanisms in combination with triclosan and polymyxin B.

[0089] < Examples 8> mcr -1 medium Polymyxin Regarding the killing of resistant E. coli TimP origin Extracellular In vivo efficacy of endoplasmic reticulum

[0090] TimP-derived extracellular vesicles are promising drug biomolecules that exhibit high production rates as bacteria grow, and Mcr-1 in vitro The action of polymyxin B / triclosan on expressing E. coli was evaluated (Fig. 5b). To confirm the in vivo efficacy of the combination treatment, A polymyxin-resistant isolate-infected mouse model was constructed using NCCP 16283, and TimP-derived extracellular vesicles and polymyxin B / triclosan were injected intraperitoneally (Fig. 6a). First, the dose-dependent cytotoxicity of TimP-derived extracellular vesicles was evaluated by measuring the body weight and survival rate of the mice for one week. At the highest dose, there was a maximum body weight loss of 15%, but it did not affect survival (Fig. 6b). Second, regarding the combined treatment effect of polymyxin B / triclosan and TimP-derived extracellular vesicles with NCCP 16283, bacterial killing in the blood, liver, and kidneys increased by approximately 100-fold compared to treatment with polymyxin B / triclosan alone (Fig. 6c). TimP-derived extracellular vesicles were confirmed to be a potential combination treatment biomolecule for resensitization with triclosan and polymyxin B in the control of Mcr-1 resistant bacteria expressed in animals.

[0091] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A TimP peptide comprising the amino acid sequence indicated by SEQ ID NO. 1 or a bacterial-derived extracellular vesicle overexpressing the TimP peptide, comprising as an active ingredient Escherichia coli BW25113 or Salmonella Composition for enhancing polymyxin sensitivity to ATCC 14028S. Claim 2 A composition according to claim 1, characterized in that the composition enhances polymyxin sensitization efficacy by inducing the generation of reactive oxygen species. Claim 3 delete Claim 4 delete Claim 5 An antimicrobial composition for Mcr-1 expressing polymyxin-resistant Escherichia coli comprising, as active ingredients: a TimP peptide consisting of the amino acid sequence indicated by SEQ ID NO. 1 or a bacterial-derived extracellular vesicle overexpressing the TimP peptide; polymyxin; and triclosan. Claim 6 delete

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