WO2019155002A1 - Procédés et compositions anti-prevotella bactériocine - Google Patents

Procédés et compositions anti-prevotella bactériocine Download PDF

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WO2019155002A1
WO2019155002A1 PCT/EP2019/053170 EP2019053170W WO2019155002A1 WO 2019155002 A1 WO2019155002 A1 WO 2019155002A1 EP 2019053170 W EP2019053170 W EP 2019053170W WO 2019155002 A1 WO2019155002 A1 WO 2019155002A1
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prevotella
bacteriocin
seq
lmo2776
infection
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Nathalie Rolhion
Pascale Cossart
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Institut Pasteur
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Institut Pasteur
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • Prevotella is classically considered as a bacterial commensal due to its presence in several locations of the healthy human body including the oral cavity, gastrointestinal tract, urogenital tract and skin (1).
  • the Prevotella genus encompasses more than 40 different culturable species of which three, P. copri, P salivae and P. stercorea, can be isolated from the gut. Only a few strains have been reported to be associated with opportunistic infections, e.g. periodontitis or bacterial vaginosis (1).
  • Prevotella has been described as the major genus of one of the three reported human enterotypes (2).
  • the microbiota plays a central role in protecting the host from pathogens (9).
  • pathogens for example, in the case of Listeria monocytogenes, the foodborne pathogen responsible for listeriosis, the intestinal microbiota provides protection, as germfree mice are more susceptible to bacterial infection than conventional mice (10, 11). Unravelling the interactions between the host, the microbiota and pathogenic bacteria is critical for the design of new therapeutic strategies via manipulation of the microbiota.
  • identifying the specific molecules and the mechanisms used by the commensals to elicit their beneficial action is challenging due to the high complexity of the microbiomes, together with technical issues such as unculturability of many commensal species.
  • enteric pathogens have developed various strategies to outcompete other species in the intestine and access nutritional and spatial niches, leading to successful infection (20, 21).
  • enteric pathogens have developed various strategies to outcompete other species in the intestine and access nutritional and spatial niches, leading to successful infection (20, 21).
  • the contribution of bacteriocins and type VI secretion systems effectors during pathogen colonisation of the gut is an emerging field of investigation.
  • Lmo2776 targets Prevotella and reduces its abundance in the microbiota of both the mouse and human. This effect is direct and specific as (i) Prevotella are killed by Listeria culture supernatant (containing Lmo2776) in vitro and (ii) despite the complexity of the microbiota and its well-controlled equilibrium, no other genus of the intestinal microbiota was found to decrease in the presence of Lmo2776.
  • the examples further demonstrate that the Lmo2776 bacteriocin targets B. subtilis, a Gram-positive bacterium found in the soil, suggesting that Lmo2776 could give an advantage to Listeria in the environment. B. subtilis, a Gram-positive bacterium found in the soil, suggesting that Lmo2776 could give an advantage to Listeria in the environment.
  • subtilis is also found in the human gastrointestinal tract (60) and could also be targeted by Lmo2776 in the intestine.
  • the antibacterial effect of the Lmo2776 bacteriocin against Prevotella was confirmed using a synthetic Lmo2776 peptide.
  • Lmo2776 peptide inhibits Prevotella copri growth in vitro and in vivo in conventional mouse microbiota.
  • Lmo2776 peptide is capable of inhibiting the growth of P. copri isolates from Rheumatoid arthritis patients.
  • this invention provides methods of treating or preventing inflammation in a subject, comprising administering an anti -Prevotella bacteriocin to the subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.
  • the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the subject has or is at risk of developing an inflammatory disease selected from rheumatoid arthritis, metabolic syndrome, inflammatory bowel disease, and HIV infection.
  • the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 80%.
  • this invention provides methods of treating or preventing an enteropathogenic bacterial infection in a subject, comprising administering an anti -Prevotella bacteriocin to the subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.
  • the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the subject has or is at risk of developing an Listeria infection.
  • the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 80%.
  • this invention provides methods of reducing the Prevotella load of a subject by at least 80%, comprising administering an anti -Prevotella bacteriocin to the subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.
  • the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 80%. In some embodiments the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 90%. In some embodiments the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 99%. In some embodiments the Prevotella load is reduced in the gut microbiota of the subject.
  • this invention provides pharmaceutical compositions comprising an anti -Prevotella bacteriocin and a pharmaceutically acceptable carrier.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.
  • the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • this invention provides pharmaceutical compositions comprising an anti -Prevotella bacteriocin and a pharmaceutically acceptable carrier for use in treating or preventing inflammation in a subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.
  • the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the subject has or is at risk of developing an inflammatory disease selected from rheumatoid arthritis, metabolic syndrome, inflammatory bowel disease, and HIV infection.
  • the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 80%.
  • this invention provides pharmaceutical compositions comprising an anti -Prevotella bacteriocin and a pharmaceutically acceptable carrier for use in treating or preventing an enteropathogenic bacterial infection in a subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.
  • the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the subject has or is at risk of developing an Listeria infection.
  • the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 80%.
  • this invention provides pharmaceutical compositions comprising an anti -Prevotella bacteriocin and a pharmaceutically acceptable carrier for use in reducing the Prevotella load of a subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1.
  • the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 1 SEQ ID NO: 2 or SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin reduces the Prevotella load by at least 80%.
  • the anti -Prevotella bacteriocin reduces the Prevotella load by at least 90%.
  • the anti -Prevotella bacteriocin reduces the Prevotella load by at least 99%.
  • said anti -Prevotella bacteriocin comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • said Prevotella is Prevotella copri.
  • Figures 1A to IF Lmo2776 limits Listeria virulence in a microbiota- dependent manner.
  • A-C BALB/c mice were inoculated orally with 5xl0 9 Listeria WT, D lmo2776 or Lmo2776 complemented ( p2776 ) bacteria.
  • CFUs in the intestinal luminal content (A), the spleen (B) and the liver (C) were assessed at 72h post-infection.
  • D-F Germ-free C57BL/6J were infected with 5x10 9 Listeria WT or D lmo2776 for 72h and CFUs in the intestinal luminal content (D), the spleen (E) and the liver (F) were assessed. Each dot represents the value for one mouse. Statistically significant differences were evaluated by the Mann- Whitney test. (*p ⁇ 0.05).
  • FIGS 2A to 2D Lmo2776 targets Prevotella in mouse microbiota.
  • FIGS 3A to 3J Lmo2776 targets P. copri in human microbiota and in vitro.
  • E-F Numbers of P. copri (E), B. subtilis or E. coli (F) after incubation with supernatant of WT ( Lm ) or Almo2776 strains. Results are expressed as mean ⁇ SEM of a least 3 independent experiments and P-values were obtained using two-tailed unpaired Student’s t-test (*p ⁇ 0.05, ***p ⁇ 0.005).
  • A Assessment of listerial CFUs in the intestinal luminal content of germ-free (GF) C57BL/6J mice colonized or not with P. copri, B. thetaiotamicron or P. salivae for 2 weeks and then infected with L. monocytogenes WT or D lmo2776 for 72h.
  • B Numbers of P. copri CFUs in the intestinal luminal content of GF C57BL/6J mice colonized with P. copri and then infected with Listeria WT or Almo2776 for 72h.
  • each dot represents one mouse.
  • Statistically significant differences were evaluated by the Mann- Whitney test (A), one way-ANOVA test (C and D) or two-tailed unpaired Student’s t-test (B and E) (*p ⁇ 0.05, ***p ⁇ 0.005).
  • Figures 5A to 5C Presence of lmo2776 in L. monocytogenes strains and homologies with members of the Lactococcin 972 family.
  • A Schematic representation of Listeria monocytogenes (WT) genome showing a locus comprising Imo 2776. Absence of Imo 2776 locus in the non-pathogenic L. innocua strain CLIP 111262. Deletion of Imo 2776 in the Almo2776 mutant.
  • B Cluster analysis based on core genome multilocus sequence typing (cgMLST) profiles of 1,096 genomes (61) and pattern of Imo2774-lmo2775-lmo2776 genes presence (dark) or absence (white). The ten most frequent sublineages (SL) are highlighted. The last column corresponds to the sample source, represented by colour codes (upper left key).
  • Figures 6A to 6F Effect of lmo2776 on Listeria infection.
  • mice were inoculated orally with 5xl0 9 Listeria WT or A lmo2776 strains. CFUs in the intestinal luminal were assessed at 24h, 48h and 72h post-infection.
  • mice were inoculated intraveinously with 5xl0 9 Listeria WT or A lmo2776 strains. CFUs in the the spleen were assessed at 72h post-infection.
  • FIGS 8A to 8D OTUs targeted by Lmo2776 are closely related to Prevotella genus.
  • FIG. 9A to 9D Quantification of Prevotella (pg/mg) in the feces of conventional mice at day 0 and orally inoculated with 5xl0 9 Listeria WT or A lmo2776 strains at day 1.
  • Figures 9A to 9D Production of butyrate, isobutyrate, acetate and iso valerate is not modified by Lmo2776. Levels of butyrate (A), isobutyrate (B), acetate (C) and isovalerate (D) in SHIME® vessels infected with WT (dashed line) or A lmo2776 (grey) strains or non-infected (black) overtime. Results are expressed as mean ⁇ SEM for 2 to 3 individual vessels. [0024] Figures 10A to 10B: Lm secretes a bona fide bacteriocin targeting B. subtilis.
  • FIG. 11 Lmo2776 targets Prevotella in mouse microbiota. Relative abundance of OTU 355746, OTU 216524, OTU 421792, OTU 258849, OTU 331772, OTU 346870, OTU 430197, OTU 447141, OTU 465433, OTU 208409, OTU 353012, OTU 364179 in gut microbiota of mice infected with WT or Almo2776 strains at day 0, day 1 and day 2. Each dot represents the value for one mouse.
  • Figures 12A to 12C P. copri controls Listeria infection. Assessment of listerial CFUs in the intestinal luminal content (A), spleen (B) and liver (C) of germ-free (GF) C57BL/6J mice colonized or not with a mixture of 12 bacteria including V errucom i crobi a (A. muciniphila YL44), Bacteroidetes (B. caecimuris 148, M. intestinale YL27), Proteobacteria CL. muris YL45), Actinobacteria (B. longum YL2), Fimicutes (E. faecalis KB1 ; A. muris KB18; C.
  • V errucom i crobi a A. muciniphila YL44
  • Bacteroidetes B. caecimuris 148, M. intestinale YL27
  • Proteobacteria CL. muris YL45 Proteobacteria
  • treatment refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of curing a disorder, or eradicating a pathogen, or improving the subject's condition, directly or indirectly. Treatment also refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or combinations thereof. [0029] The term“preventing” includes the reducing the incidence, recurrence, spread, onset or establishment of a disorder such as a bacterial infection. It is not intended that the present disclosure be limited to complete prevention or to prevention of establishment of an infection. In some embodiments, the onset is delayed, or the severity of a subsequently contracted disease is reduced, and such constitute examples of prevention.
  • percent amino acid sequence identity with respect to the anti- Prevotella bacteriocin polypeptide sequences is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific anti -Prevotella bacteriocin polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available software such as BTAST or Megalign (DNASTAR) software.
  • subject refers to a subject to be treated and includes inter alia mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human.
  • the phrase “inhibiting growth” in reference to Prevotella refers to any mechanism that reduces the accumulation of Prevotella cells in an area over time.
  • any mechanism that causes Prevotella cells in a culture to accumulate at a slower rate than Prevotella cells in a control culture or any mechanism that that causes Prevotella cells in an in vivo context (e.g., gut microbiota, lung microbiota) to accumulate at a slower rate than Prevotella cells in a control.
  • inhibitting includes without limitation mechanisms reducing the rate of cell division and induction of cell death such as by cell lysis.
  • Prevotella load refers to a measurable quantity of bacteria in an object, organism, or organism compartment. In some embodiments the Prevotella load is determined at least in part by measuring the proportion of total bacteria present that are viable Prevotella.
  • Bacteriocins are proteinaceous or peptidic toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are similar to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse.
  • an“anti -Prevotella bacteriocin” is a bacteriocin that inibits the growth of P. copri in an in vitro culture.
  • An exemplary assay is demonstrated in Example 3.
  • assay bacteriocin is obtained by culturing L. monocytogenes Lmo2776 and collecting the supernatant of the culture that comprises bacteriocin.
  • P. copri is then grown at 37°C in anaerobic conditions in the presence of the supernatant.
  • the assay may be adapted to use bacteriocin from any suitable source at a defined concentration in the culture media.
  • a candidate bacteriocin is added to culture media at a concentration of from 10 nM to 80 mM and the effect of the candidate bacteriocin on growth of P. copri colonies is measured.
  • a candidate bacteriocin that inhibits the growth of P. copri colonies when present in culture media at a concentration within this concentration range is identified as a anti -Prevotella bacteriocin.
  • a candidate bacteriocin that inhibits the growth of P. copri colonies when present in culture media at a concentration of from 10 nM to 100 nM is identified as a anti -Prevotella bacteriocin.
  • a candidate bacteriocin that inhibits the growth of P. copri colonies when present in culture media at a concentration of from 100 nM to ImM is identified as a anti- Prevotella bacteriocin.
  • a candidate bacteriocin that inhibits the growth of P. copri colonies when present in culture media at a concentration of from ImM to 10 pM is identified as a anti -Prevotella bacteriocin.
  • a candidate bacteriocin that inhibits the growth of P. copri colonies when present in culture media at a concentration of from 10 pM to 80 pM is identified as a anti -Prevotella bacteriocin.
  • the anti -Prevotella bacteriocin is an L. monocytogenes bacteriocin. In some embodiments the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776. In some embodiments the anti -Prevotella bacteriocin is L. monocytogenes EGD-e Lmo2776, which has the following sequence:
  • the anti -Prevotella bacteriocin is a fragment of L. monocytogenes EGD-e Lmo2776.
  • the anti -Prevotella bacteriocin is a fragment of L. monocytogenes EGD-e Lmo2776 from which the signal peptide has been cleaved.
  • Bacteriocin of the lactococcin 972 family are cleaved following a GG motif (positions 67-68 of SEQ ID NO: 1; underlined) to the mature form.
  • the fragment comprises or consists of the following sequence:
  • the fragment comprises or consists of the following sequence:
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 70% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 75% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 85% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 91% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 92% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 93% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 94% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 95% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 96% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 97% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 98% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 98% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments the anti -Prevotella bacteriocin consists of the amino acid sequence of SEQ ID NO: 1.
  • the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments the anti -Prevotella bacteriocin consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments the anti -Prevotella bacteriocin comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments the anti -Prevotella bacteriocin consists of the amino acid sequence of SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to SEQ ID NO: 3. In some more preferred embodiments, the anti- Prevotella bacteriocin comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the invention encompasses variants of L. monocytogenes Lmo2776 including natural and synthetic variants thereof. Natural variants are found in the various serotypes, strains and isolates of L. monocytogenes. Synthetic variants can be made from any one of SEQ ID NO: 1, 2, and 3 by routine techniques in the art and screened for activity (i.e. anti-Prevotella bacteriocin activity) using the assays described herein such as the exemplary assay disclosed in Example 3 or other similar assays.
  • Bacteriocins may be produced using any suitable reagents and methods known in the art.
  • a bacteriocin-producing strain of bacteria is cultured and the bacteriocin is recovered from the culture.
  • the bacteriocin is recovered by collecting culture media from the culture and then optionally purifying the bacteriocin from the culture media.
  • the bacteriocin-producing strain is a Listeria strain.
  • the bacteriocin-producing strain is an E coli strain.
  • lmo2776 was cloned in pGEX4Tl plasmid and the recombinant plasmid transformed into E. coli BL2l(DE3) Gold competent cells (Novagen). The bacteria were grown at 37°C up to an OD of 0.7-0.8 and expression of GST-Lmo2776 was induced with 0.6 mM IPTG at l6°C for l6h prior to collection of cell pellets by centrifugation. Cells were resuspended in 20 mM Tris pH 8.5, 150 mM NaCl, 1 mM DTT and lysed by sonication or by passage through a French press.
  • Lmo2776 peptide as defined above is synthesized using standard peptide synthesis methods that are well-known in the art such as for example solid-phase synthesis methods.
  • compositions comprising an anti -Prevotella bacteriocin and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a solution, a suspension, an emulsion, an inhalable powder, an aerosol, or a spray.
  • the pharmaceutical composition further comprises one or more antibiotics suitable for the treatment of bacterial infection.
  • the pharmaceutical composition further comprises one or more antibiotics suitable for the treatment of Gram-negative and/or Gram-positive bacterial infection.
  • the pharmaceutical composition further comprises one or more antibiotics suitable for the treatment of Gram-negative bacterial infection.
  • the pharmaceutical composition further comprises one or more anti-inflammatory agents for the treatment of inflammation.
  • the term“pharmaceutically acceptable carrier” includes any and all solvents, additives, excipients, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption delaying agents, surfactants, propellants and the like that are physiologically compatible.
  • the carrier(s) must be“acceptable” in the sense of not being deleterious to the subject to be treated in amounts typically used in medicaments.
  • Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose.
  • pharmaceutically acceptable carriers are suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are“undue” when their risk outweighs the benefit provided by the composition.
  • Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, and emulsions such as oil/water emulsions and microemulsions.
  • excipients such as urea or mesna can be included to improve stability.
  • Other excipients include bulking agents, buffering agents, tonicity modifiers, surfactants, preservatives and co-solvents.
  • suitable pharmaceutically acceptable excipients include, but are not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
  • suitable pharmaceutically acceptable excipients include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and the like.
  • suitable excipients include, but are not limited to a cream, a cellulosic or oily base, emulsifying agents, stiffening agents, rheology modifiers or thickeners, surfactants, emollients, preservatives, humectants, alkalizing or buffering agents, and solvents.
  • Suitable excipients for the formulation of the foam base include, but are not limited to, propylene glycol, emulsifying wax, cetyl alcohol, and glyceryl stearate.
  • Potential preservatives include methylparaben and propylparaben.
  • compositions of the present disclosure can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, tampon applications emulsions, aerosols, sprays, suspensions, lozenges, troches, candies, injectants, chewing gums, ointments, smears, a time- release patches, a liquid absorbed wipes, and combinations thereof.
  • compositions of the present disclosure or pharmaceutically acceptable forms thereof may be topical, i.e., the pharmaceutical composition is applied directly where its action is desired, or systemic.
  • systemic administration can be enteral or oral, i.e., substance is given via the digestive tract, parenteral, i.e., substance is given by other routes than the digestive tract such as by injection or inhalation.
  • the bacteriocin polypeptides of the present disclosure can be administered to a subject orally, parenterally, by inhalation, topically, rectally, nasally, buccally or via an implanted reservoir or by any other known method.
  • the bacteriocin polypeptides of the present disclosure can also be administered by means of sustained release dosage forms.
  • the bacteriocin polypeptides of the present disclosure can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions and dispersions.
  • the compound can be formulated with excipients such as, e.g., lactose, sucrose, corn starch, gelatin, potato starch, alginic acid and/or magnesium stearate.
  • a bacteriocin polypeptide of the present disclosure is mixed with a pharmaceutical excipient to form a solid preformulation composition.
  • tablets may be sugar coated or enteric coated by standard techniques.
  • the tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • the bacteriocin of the present disclosure may also be administered by injection of a therapeutic agent comprising the appropriate amount of bacteriocin polypeptide and a carrier.
  • a therapeutic agent comprising the appropriate amount of bacteriocin polypeptide and a carrier.
  • the bacteriocin polypeptides can be administered intramuscularly, intrathecally, subdermally, subcutaneously, or intravenously.
  • the carrier may be comprised of distilled water, a saline solution, albumin, a serum, or any combinations thereof.
  • compositions of parenteral injections can comprise pharmaceutically acceptable aqueous or nonaqueous solutions of bacteriocin polypeptides in addition to one or more of the following: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • adjuvants e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents
  • liposomal formulations e.g., nanoparticles, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • an isotonic formulation is preferably used.
  • additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose.
  • isotonic solutions such as phosphate buffered saline are preferred.
  • Stabilizers can include gelatin and albumin.
  • a vasoconstriction agent can be added to the formulation.
  • the pharmaceutical preparations according to this type of application are provided sterile and pyrogen free.
  • the diluent may further comprise one or more other excipient such as, e.g., ethanol, propylene glycol, an oil or a pharmaceutically acceptable emulsifier or surfactant.
  • excipient such as, e.g., ethanol, propylene glycol, an oil or a pharmaceutically acceptable emulsifier or surfactant.
  • compositions of the present disclosure are inhalable compositions.
  • the inhalable compositions of the present disclosure can further comprise a pharmaceutically acceptable carrier.
  • bacteriocin polypeptide(s) of the present disclosure are advantageously formulated as a dry, inhalable powder.
  • bacteriocin polypeptides inhalation solution may further be formulated with a propellant for aerosol delivery.
  • solutions may be nebulized.
  • a surfactant can be added to an inhalable pharmaceutical composition of the present disclosure in order to lower the surface and interfacial tension between the medicaments and the propellant.
  • a surfactant may or may not be required.
  • a surfactant may or may not be necessary, depending in part, on the solubility of the particular medicament and excipient.
  • the surfactant may be any suitable, non-toxic compound which is non-reactive with the medicament and which substantially reduces the surface tension between the medicament, the excipient and the propellant and/or acts as a valve lubricant.
  • Suitable surfactants include, but are not limited to: oleic acid; sorbitan trioleate; cetyl pyridinium chloride; soya lecithin; polyoxyethylene(20) sorbitan monolaurate; polyoxy ethylene (10) stearyl ether; poly oxy ethylene (2) oleyl ether; poly oxypropylene-polyoxy ethylene ethylene diamine block copolymers; polyox yethylene(20) sorbitan monostearate; polyoxyethylene(20) sorbitan monooleate; polyoxypropylene- polyoxyethylene block copolymers; castor oil ethoxylate; and combinations thereof.
  • Suitable propellants include, but are not limited to: dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane and carbon dioxide.
  • excipients for use in inhalable compositions include, but are not limited to: lactose, starch, propylene glycol diesters of medium chain fatty acids; triglyceride esters of medium chain fatty acids, short chains, or long chains, or any combination thereof; perfluorodimethylcyclobutane; perfluorocyclobutane; polyethylene glycol; menthol; lauro glycol; diethylene glycol monoethylether; polyglycolized glycerides of medium chain fatty acids; alcohols; eucalyptus oil; short chain fatty acids; and combinations thereof.
  • compositions of the present disclosure comprise nasal applications.
  • Nasal applications include for instance nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, mouthwashes or gargles, or through the use of ointments applied to the nasal nares, or the face or any combination of these and similar methods of application.
  • compositions of the present disclosure contain a complementary agent, including one or more antimicrobial agents, one or more conventional antibiotics, and/or one or more anti-inflammatory agents.
  • the therapeutic agent containing one or more bacteriocin polypeptides of the present disclosure may further include at least one complementary agent which can also potentiate the bactericidal activity of the bacteriocin polypeptide.
  • the complementary agent may be one or more antibiotics.
  • the pharmaceutical compositions of the present disclosure comprise an anti -Prevotella bacteriocin comprising an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to SEQ ID NO: 3; preferably an anti -Prevotella bacteriocin comprising or consisting of the amino acid sequence of SEQ ID NO: 3.
  • Examples 1 and 2 demonstrate that Lmo2776 reduces the amount of Prevotella in the human gut microbiota.
  • Example 3 demonstrates that Lmo2776 is a bona fide bacteriocin that targets directly P. copri in an in vitro assay. Accordingly, this disclosure provides methods of inhibiting growth of Prevotella and/or methods of reducing Prevotella load.
  • this invention also provides methods of inhibiting growth of Prevotella, comprising contacting Prevotella with an anti -Prevotella bacteriocin.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti- Prevotella bacteriocin comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to SEQ ID NO: 3; preferably the anti -Prevotella bacteriocin comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin is in an amount sufficient to reduce the growth of Prevotella by at least 80%.
  • the anti -Prevotella bacteriocin is in an amount sufficient to reduce the growth of Prevotella by at least 90%.
  • the anti -Prevotella bacteriocin is in an amount sufficient to reduce the growth of Prevotella by at least 95%. In some embodiments the anti -Prevotella bacteriocin is in an amount sufficient to reduce the growth of Prevotella by at least 99%.
  • This invention also provides methods of inhibiting growth of Prevotella in a subject, comprising administering an anti -Prevotella bacteriocin to the subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti- Prevotella bacteriocin comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to SEQ ID NO: 3; preferably the anti -Prevotella bacteriocin comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin is in an amount sufficient to reduce the growth of Prevotella by at least 80%.
  • the anti -Prevotella bacteriocin is in an amount sufficient to reduce the growth of Prevotella by at least 90%.
  • the anti -Prevotella bacteriocin is in an amount sufficient to reduce the growth of Prevotella by at least 95%. In some embodiments the anti -Prevotella bacteriocin is in an amount sufficient to reduce the growth of Prevotella by at least 99%.
  • This invention also provides methods of reducing the Prevotella load in a subject, comprising administering an anti -Prevotella bacteriocin to the subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti- Prevotella bacteriocin comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to SEQ ID NO: 3; preferably the anti -Prevotella bacteriocin comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin is in an amount sufficient to reduce the Prevotella load by at least 80%.
  • the anti -Prevotella bacteriocin is in an amount sufficient to reduce the Prevotella load by at least 90%.
  • the anti- Prevotella bacteriocin is in an amount sufficient to reduce the Prevotella load by at least 95%. In some embodiments the anti -Prevotella bacteriocin is in an amount sufficient to reduce the Prevotella load by at least 99%. In some embodiments the Prevotella load in the gut microbiota of a subject is reduced. In some embodiments the Prevotella load in the lung microbiota of a subject is reduced. In some embodiments the Prevotella load in an organ or another compartment of a subject is reduced. In some embodiments the Prevotella load in at least one first compartment of a subject is reduced while the Prevotella load in at least one first compartment of a subject is not reduced.
  • said Prevotella is Prevotella copri.
  • Prevotella bacteria are associated with inflammation. As shown in Example 5, the presence of Prevotella in the intestine is associated with a thinner mucus layer and increased levels of faecal Lipocalin-2 (LCN-2). These results indicate that Prevotella is a bacterium promoting a pro-inflammatory phenotype. Thus, the identification in this disclosure of anti- Prevotella bacteriocin provides a new and useful reagent for treating and/or preventing inflammation and diseases having an inflammation component. Thus, this invention provides methods of treating and/or preventing inflammation in a subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti- Prevotella bacteriocin comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to SEQ ID NO: 3; preferably the anti -Prevotella bacteriocin comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the subject has or is at risk of developing an inflammatory disease selected from rheumatoid arthritis, metabolic syndrome, inflammatory bowel disease, HIV infection and lung disorders, in particular cytic fibrosis.
  • administration of the anti- Prevotella bacteriocin to the subject reduces inflammation in the subject.
  • the reduction in inflammation results in treatment and/or prevention of an inflammatory disease selected from rheumatoid arthritis, metabolic syndrome, inflammatory bowel disease, HIV infection and lung disorders, in particular cystic fibrosis in the subject.
  • an inflammatory disease selected from rheumatoid arthritis, metabolic syndrome, inflammatory bowel disease, HIV infection and lung disorders, in particular cystic fibrosis in the subject.
  • the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 80% in the subject.
  • an inflammatory disease selected from rheumatoid arthritis, metabolic syndrome, inflammatory bowel disease, HIV infection and lung disorders, in particular cystic fibrosis in a subject
  • administering an anti -Prevotella bacteriocin comprising administering an anti -Prevotella bacteriocin to the subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti- Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to SEQ ID NO: 3; preferably the anti -Prevotella bacteriocin comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • administration of the anti -Prevotella bacteriocin to the subject reduces inflammation in the subject.
  • the reduction in inflammation results in treatment and/or prevention of an inflammatory disease selected from rheumatoid arthritis, metabolic syndrome, inflammatory bowel disease, HIV infection and lung disorders, in particular cystic fibrosis in the subject.
  • an inflammatory disease selected from rheumatoid arthritis, metabolic syndrome, inflammatory bowel disease, HIV infection and lung disorders, in particular cystic fibrosis in the subject.
  • the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load by at least 80% in the subject.
  • said Prevotella is Prevotella copri.
  • Example 4 demonstrates that the ability of the D lmo2776 mutant to better grow in the intestine compared to the WT is dependent on the presence of Prevotella in the microbiota of the intestine, as it is observed either in conventional mice or mice colonized with P. copri.
  • Example 5 demonstrates that the presence of Prevotella in the intestine is associated with a thinner mucus layer and increased levels of faecal Lipocalin-2 (LCN-2). These results indicate that Prevotella is a bacterium promoting a pro-inflammatory phenotype. These results also strongly indicate that P. copri, by modifying the mucus layer thickness and changing the gut inflammatory condition, promotes infection. This suggests that people with high abundance of intestinal Prevotella are at risk for enteropathogens infection.
  • this invention provides methods of treating and/or preventing an enteropatho genic bacterial infection in a subject.
  • this invention also provides methods of treating or preventing an enteropathogenic bacterial infection in a subject, comprising administering an anti -Prevotella bacteriocin to the subject.
  • the anti -Prevotella bacteriocin is L. monocytogenes Lmo2776.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
  • the anti -Prevotella bacteriocin comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical to SEQ ID NO: 3; preferably the anti -Prevotella bacteriocin comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the subject has or is at risk of developing an Listeria infection.
  • the anti -Prevotella bacteriocin is administered in an amount sufficient to reduce the Prevotella load of the subject by at least 80%.
  • said Prevotella is Prevotella copri.
  • Bacterial strains and plasmids For standard experiments, Lm EGD-e, E. coli and B. subtilis were grown at 37°C with shaking in Brain Heart Infusion (BHI) medium (Difco) and Luria-Bertani (LB) medium (BD). If needed, Lm were selected on Oxford medium (Oxoid).
  • BHI Brain Heart Infusion
  • LB Luria-Bertani
  • P. copri DSM 18205
  • B. thetaiotamicron CIP 104206T, CRB Institut Pasteur
  • the Lmo2776 deletion mutant was constructed using the pMAD shuttle plasmid (22) as described previously (23) and confirmed by DNA sequencing.
  • pAD-based plasmid fragment obtained by PCR with EGD-e genomic DNA as template, was cloned into the Smal/Sall sites of the pAD vector (24) derived previously from the pPL2 vector (25). Plasmid was verified by sequencing with primers pPL2-Rv and pPL2-Fw and were transformed into A lmo2776 by electroporation. Integration in the chromosome was verified by PCR using primers NC16 and PL95 (24).
  • mice 9- to 12-week-old female B ALB/C conventional (Charles River), C57BL6/J conventional (Charles River), C57BL6/J germfree (CDTA or Pasteur) or Oligo- Mouse-Microbiota (Oligo MM12) C57BL6/J (Brugiroux el ah, Nature Microbiology, 2016, 16131) wild-type mice were used for experiments. Germfree mice were housed in plastic gnotobiotic isolators.
  • mice infection Lm overnight cultures were diluted in BHI and bacteria were grown to an optical density at 600 nm (OD600) of 1. Bacterial cultures were centrifuged at 3,500 x g for 15 min and washed three times in phosphate-buffered saline (PBS). Mice were infected orally with 5 x 10 9 bacteria diluted in 200 pl of PBS supplemented with 300 m ⁇ of CaC03 (50 mg/ml). Serial dilutions of the inoculum were plated to control the number of bacteria inoculated.
  • PBS phosphate-buffered saline
  • mice were killed at subsequent time points, and intestines, mesenteric lymph nodes, spleens, and livers were removed.
  • the small intestine was opened, and the luminal content was recovered in a l.5-mL tube and weighed.
  • the small intestine (duodenum, jejunum, and ileum) tissue was washed four times in DMEM (ThermoFisher), incubated for 2 h in DMEM supplemented with 40 pg/mL gentamycin, and finally washed three four in DMEM.
  • DMEM ThermoFisher
  • mice were infected with 5 x 10 bacteria diluted in lOOul of PBS. Mice were killed at subsequent time points, and spleens, and livers were removed and processed as described above.
  • mice colonisation P. copri, P. salivae and B. thetaiotamicron were grown to log phase under anaerobic conditions in M20 liquid media and 107 CFU were used to inoculate mice. Feces were collected at 2 weeks post-gavage to confirm colonization. Feces were homogenized, serially diluted and plated on M20 agar plates
  • Fecal microbiota analysis by 16S rRNA gene sequencing using Illumina technology Before infection, 8 BAFB/c conventional mice were co-housed for 5 weeks in order to stabilize and homogenize their microbiota. After oral infection, animals were single- housed. Feces were collected and frozen at -20°C. 16S rRNA gene amplification and sequencing were done using the Illumina MiSeq technology following the protocol of Earth Microbiome Project with their modifications to the MOBIO PowerSoil DNA Isolation Kit procedure for extracting DNA (www.earthmicrobiome.org/emp-standard-protocols) (27, 28).
  • 16S rRNA gene sequence analysis Analysis of the 16S rRNA gene sequence was performed exactly as previously described (29).
  • M-SHIME M-SHIME system is a dynamic in vitro model which simulates the lumen-associated and mucus-associated human intestinal microbial ecosystem (ProDigest, Ghent University, Belgium) (30-32). It consists of consecutive pH-controlled, stirred, airtight, double-jacketed glass vessels kept at 37°C and under anaerobic conditions. The system was operated as described earlier (33) with minor modifications The set-up used here consisted of 3 stomach-small intestine vessels and nine proximal colon vessels in parallel. The colon vessels were inoculated at the start with 40 mL fresh human faecal suspension in 500 mL sterile nutritional medium.
  • RNA extraction and qRT A total of 25 ml cultures of bacteria (Fm in stationary phase or Fm in exponential phase) was grown in medium (BHI, FB or M20) overnight. Cultures were subsequently pelleted for 20 min at 3000g. Pellets were resuspended in 1 ml TRI Reagent (Sigma), transferred to 2-ml Fysing Matrix tubes (MP Biomedicals) and mechanically lysed by bead beating in a FastPrep apparatus (twice 45s, speed 6.5). Tubes were then centrifuged for 5 min at 8000g at 4°C to separate beads from lysates. The lysates were drawn off and transferred to a 2-ml Eppendorf tube.
  • RNA pellets were resuspended in 50 to 100 uF water. For each sample, 10 ug of RNA was treated with Dnase (Turbo DNA-free, Ambion) following manufacturer’s instructions.
  • cDNA was synthetiszed from 1 ug of RNA using QuantiTect Reverse Transcription (Qiagen) and reactions were subsenquently diluted with 180 ul of water. qRT-PCR reactions were prepared with SYBR Green master mix. Reaction cycling and quantification was carried out in an Cl 000 touch Thermal cycler (CFX384, Biorad). Expression levels were normalized to the rpoB gene. Samples were evaluated in triplicate and results represent at least three independent experiments.
  • Co-culture assays For co-culture assays, 10 bacteria from overnight cultures were inoculated into 5 mF of fresh BHI either in single culture or in coculture with another strain as indicated in the figures and incubated at 37°C for 6 hours. Serial dilutions were plated on selective media for CFU enumeration. [0097] For culture of target in presence of Listeria supernatant, 25 ml of overnight culture of Listeria were centrifuged at l3000g and the supernatants were collected and centrifuged further to remove cells and cells debris. Supernatants were filtered through a
  • Lmo2776 (SEQ ID NO : 3) was prepared by solid-phase synthesis (Polypeptide Group) with a purity of more than 95 %. The molecular weight of the peptide was confirmed by mass spectrometry (7402.5 ES+).
  • Example 1 Lmo2776 limits Listeria intestinal colonization and virulence in a microbiota-dependent manner
  • Lmo2776 is predicted to be a secreted bacteriocin of 107 amino acids (36, 38), belonging to the bacteriocin 972 family, which contains bacteriocins secreted by Lactococcus lactis (39) and also by pathogenic Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus iniae (Figure 5C).
  • Bacteriocin of the lactococcin 972 family are cleaved following a GG motif to the mature form, indicating that Lmo2776 mature form is predicted to be a peptide of 63 amino acids (SEQ ID NO: 3).
  • Lmo2776 shares between 38 to 47% overall sequence similarity with the other members of the bacteriocin 972 family.
  • Lmo2774 and Lmo2775 have sequence similarity with ABC transporters and immunity proteins of the related bacteriocin 972 family members, respectively.
  • the Almo2776 strain displayed significantly higher bacterial loads in the small intestinal content compared to the WT strain at 24h, 48h and 72h post-infection (Figure 6B). No difference was observed between the growth curves of the Almo2776 strain and the wild-type in vitro in BHI medium, indicating that lmo2776 deletion does not affect bacterial growth (Figure 6D). Similar differences were observed in C57BL/6J mice (data not shown). Altogether, these data indicate a key role for Lmo2776 in bacterial growth in the intestine and deeper organs. The higher number of Almo2776 strain in the spleen and the liver directly correlate with the high number of bacteria in the intestinal content, crossing the intestinal barrier and consequently reaching deeper organs such as the spleen and the liver.
  • Example 2 Lmo2776 specifically targets Prevotella in mouse and human microbiota
  • the increased levels in the Firmicutes were mainly due to an increase of the Clostridia class (relative abundance before infection: 27.4% and at day 1 post-infection: 50.7%).
  • the relative abundance of Tisteria was around 0.1% and cannot therefore explain by itself the increased levels of Firmicutes observed between day 0 and day 1.
  • microbial community compositions of the intestine differed in mice orally infected with the A lmo2776 strain compared to the WT strain ( Figure 2A and 11).
  • Prevotella abundance is known to be low in the mouse feces (less than 1%) and can reach up to 50 % in the human feces (41, 42).
  • M-SHIME® a mucosal simulator of the human intestinal microbial ecosystem. This ex vivo model allows to stably maintain a human microbiota in vitro, in the absence of host cells (30-33). The microbiota of a healthy human volunteer with high levels of Prevotella was inoculated into the system, which was then infected with WT or D lmo2776 bacteria.
  • short-chain fatty acid levels serve as a general read-out for gut microbiota metabolism.
  • propionate is produced by many bacteria
  • the decrease in propionate production observed during infection of M- SHIME® with WT Listeria is in agreement with the decrease in Prevotella population, as Prevotella are known to produce this short chain fatty acid (43).
  • Example 3 Lmo2776 targets P. copri in vitro
  • Example 4 Colonization of germ-free mice by P. copri recapitulates the effect of the microbiota on Listeria intestinal growth in conventional mice
  • P. copri P. salivae
  • B. thetaiotaomicron another commensal bacterium
  • a mixture of 12 bacteria including V errucom i crobi a (A. muciniphila YL44), Bacteroidetes (B. caecimuris 148, M. intestinale YL27), Proteobacteria CL. muris YL45), Actinobacteria (B. longum YL2), Fimi cutes (E. faecalis KB1; A. muris KB 18; C.
  • Example 5 P. copri modifies the intestinal environment
  • Mucus in the intestine of conventional animals forms a physical barrier of about 30pm that is able to keep bacteria at a distance from the epithelium (45).
  • Prevotella through production of sulfatases that induce actively mucus degradation (46), might impair the mucosal barrier function and therefore contribute to local inflammation and better accessibility to intestinal epithelial cells for pathogens.
  • faecal LCN-2 levels of faecal LCN-2 are used as a marker of intestinal inflammation (47). Faecal LCN-2 levels were thus analysed after colonization of germ-free mice with P. copri compared to non-colonized mice or mice colonized with B. thetaiotaomicron. A significant increase of faecal LCN-2 was observed in germ-free mice monocolonized with P. copri compared to non-colonised animals or to animals monocolonized with B. thetaiotaomicron ( Figure 4E), revealing that P. copri induces intestinal inflammation. Altogether, these results showed that presence of Prevotella in the intestine is associated with a thinner mucus layer and increased levels of faecal LCN-2. They are consistent with previous reports describing Prevotella, as a bacterium promoting a pro-inflammatory phenotype (i, 4, 48).
  • mice germ-free C57BT/6J mice were inoculated orally with P. copri, P. salivae or B. thetaiotaomicron, two weeks later mice were orally infected with WT Listeria or D lmo2776 strains and mucus layer thickness was compared 72h post-infection.
  • the average distance of bacteria from colonic epithelial cells was significantly smaller in P. copri colonized mice infected with D lmo2776 compared to P. copri colonized mice infected with WT Listeria at 72h ( Figure 4D), non- colonised animals or to animal monocolonized with B. thetaiotaomicron or P. salivae, showing that Prevotella decreases specifically the mucus layer thickness.
  • Example 6 Lmo2776 peptide inhibits P. copri growth in vitro and in vivo
  • Tmo2776 peptide of SEQ ID NO: 3 was synthesized and tested in vitro and in vivo. In vitro, Tmo2776 peptide inhibits Prevotella copri growth in a dose dependent manner and has no effect on the growth of B. thetaiotaomicron ( Figure 3G). Tmo2776 peptide also inhibits B. subtilis growth but has no effect on the growth of other Prevotella strains, intestinal bacteria or Tactic acid bacteria (IPTA) ( Figure 3H). In addition, conventional mice treated with Tmo2776 peptide displayed significantly lower number of P. copri in the feces compared to untreated mice ( Figure 3J).
  • Tmo2776 peptide is also capable of inhibiting the growth of various P. copri isolates from Rheumatoid arthritis (RA) patients ( Figure 31). These results confirm that Tmo2776 is a bona fide bacteriocin that targets directly both P. copri and B. subtilis in vitro and in vivo. These results show the therapeutic potential of Tmo2776 peptide in the treatment of inflammatory diseases and enteropathogenic bacterial infections.
  • Listeriolysin S decreases Allobaculum and Alloprevotella genera known to produce butyrate or acetate, two SCFAs reported to inhibit transcription of virulence factors or growth of Listeria (50, 51).
  • LLS Listeriolysin S
  • a direct or an indirect role on these genera is still under investigation.
  • Salmonella enterica serovar Typhimurium infection killing of intestinal Klebsiella oxytoca via the Salmonella T6SS is essential for colonisation of the murine gut (52), but whether K. oxytoca and other members of the gut microbiota are targeted by the Salmonella T6SS in conventional mice is unknown.
  • Shigella sonnei uses a T6SS to outcompete E. coli in vivo but the effectors responsible for this effect are unknown (53).
  • B. thetaiotaomicron enhances Clostridium rodentium colonization by producing succinate (54, 55) and Akkermansia muciniphila exacerbates S. 1 y phi mu rium - i nduccd intestinal inflammation by disturbing host mucus homeostasis (5b).
  • P. copri decreases the mucus layer thickness and increases propionate concentration and levels of fecal LCN-2 ( Figure 4F), in agreement with previous studies reporting that Prevotella exacerbates inflammation (4, 48).
  • Prevotella enrichment within the lung microbiome of HIV-infected patients has been observed and is associated with Thl7 inflammation (57).
  • Lmo2776 bacteriocin targets B. subtilis, a Gram positive bacterium found in the soil, suggesting that Lmo2776 could give an advantage to Listeria in the environment.
  • B. subtilis is also found in the human gastrointestinal tract (60) and could also be targeted by Lmo2776 in the intestine.
  • B. subtilis is also targeted by Sil, another member of the bacteriocin 972 family (44).
  • the role of the homologs of bacteriocin 972 in other human pathogenic bacteria such as S. pneumoniae and S. aureus remains to be determined.
  • the conservation of the bacteriocin in different pathogenic bacteria strongly suggests an important role.
  • Lmo2776 bacteriocin against Prevotella was confirmed using a synthetic Lmo2776 peptide.
  • Lmo2776 peptide inhibits Prevotella copri growth in vitro and in vivo in conventional mouse microbiota.
  • Lmo2776 peptide is capable of inhibiting the growth of P. copri isolates from Rheumatoid arthritis patients.
  • Faecalibacterium prausnitzii a commensal bacterium deficient in Crohn's disease. Gut 65, 415- 425 (2016).

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Abstract

L'invention concerne des procédés de réduction de la charge de Prevotella, chez un sujet, de traitement ou de prévention d'une inflammation chez un sujet et le traitement ou la prévention d'une infection bactérienne entéropathogène chez un sujet, comprenant l'administration d'une bactériocine anti-Prevotella au sujet. L'invention concerne également des compositions pharmaceutiques comprenant une bactériocine Anti-Prevotella et un support pharmaceutiquement acceptable.
PCT/EP2019/053170 2018-02-08 2019-02-08 Procédés et compositions anti-prevotella bactériocine Ceased WO2019155002A1 (fr)

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