MODIFIED ESCHERICHIA COLI STRAIN NISSLE AND TREATMENT OF GASTROINTESTINAL DISORDER

Abstract

The invention relates to the field of modified Escherichia coli strain Nissle 1917 (EcN) and its use for treating gastro-in-testinal disorders. The invention is based on the study of the mechanisms implicated in the probiotic properties of the Escherichia coli strain Nissle 1917 (EcN). This study has allowed the inventors to decouple the probiotic activity of EcN from its genotoxic activity by demonstrating that EcN ClbP protein, the enzyme that activates the genotoxin colibactin, is also required for the siderophore-microcins activity of probiotic EcN, but interestingly, not its enzymatic domain that cleaves precolibactin to form active colibactin. Furthermore, inventors demonstrate in an in vivo animal model infected by a bacterial pathogen that administration of an EcN modified strain with clbP gene encoding ClbP protein inactive for the peptidase domain, is non-genotoxic (do not produce colibactin) but keeps the bacterial antagonist activity, and reduces colonization and virulence of the pathogen by maintaining the siderophore-microcin production. Thus this study opens the way to safe use of EcN and accordingly the present invention provides an Escherichia coli strain Nissle 1917 (EcN) bacterium carrying a gene encoding ClbP protein which is inactive for the peptidase domain, and its use as a drug and more particularly for use in the treatment of gastro-intestinal disease.

Claims

1. An Escherichia coli strain Nissle 1917 (EcN) bacterium carrying a gene encoding a ClbP protein comprising an inactive peptidase domain, wherein said EcN bacterium (i) has antibacterial activity; and ii) is devoid of the capacity to activate the genotoxin colibactin.

2. The EcN bacterium according to claim 1 wherein the ClbP protein comprising an inactive peptidase domain, is selected from the group consisting of: ClbP protein mutated at position S95, ClbP protein mutated at position K98, ClbP protein mutated at position Y186, and ClbP protein without peptidase domain.

3. The EcN bacterium according to claim 2, wherein the ClbP protein is mutated at position S95.

4. The EcN bacterium according to claim 2, wherein the ClbP protein is mutated at position K98.

5. The EcN bacterium according to claim 2, wherein the ClbP protein is mutated at position Y186.

6. The EcN bacterium according to claims 2 wherein the ClbP protein comprising an inactive peptidase domain, is selected from the group consisting of ClbP S95A mutant (SEQ ID N° 4), ClbP K98T mutant (SEQ ID N° 6), ClbP S95R mutant (SEQ ID N° 8), ClbP Y186G mutant (SEQ ID N° 10), and ClbP protein with a peptidase domain substituted by an alkaline phosphatase enzymatic domain of PhoA (SEQ ID N° 11).

7. (canceled)

8. A method of providing a probiotic to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an EcN bacterium carrying a gene encoding a ClbP protein having an inactive peptidase domain, wherein said EcN bacterium (i) has antibacterial activity; and ii) is devoid of the capacity to activate the genotoxin colibactin.

9. A method of treating a gastro-intestinal disease in a subject in need thereof, comprising, administering to the subject a therapeutically effective amount of an Escherichia coli strain Nissle 1917 (EcN) bacterium carrying a gene encoding a ClbP protein having an inactive peptidase domain, wherein said EcN bacterium (i) has antibacterial activity; and ii) is devoid of the capacity to activate the genotoxin colibactin.

10. The method according to claim 9 wherein the gastro-intestinal disease is selected from the group consisting of bacterial gastrointestinal infections, gut inflammatory disease and visceral pain.

11. The method according to claim 10, wherein the bacterial gastrointestinal infections is Salmonella enterica serovar Enteritidis, a Typhimurium infection or an enterohemorrhagic E. coli infections.

12. The method according to claim 10, wherein the gut inflammatory disease is Inflammatory Bowel Diseases (IBD) or Irritable Bowel Syndrome (IBS).

13. The method according to claim 12, wherein the Inflammatory Bowel Diseases (IBD) is selected from the group consisting of Crohn's Disease, Ulcerative Colitis, Celiac disease, Gluten hypersensitivity and Pouchitis.

14. The method according to claim 10, wherein the visceral pain is pain associated with inflammatory bowel disease (IBD) or with Irritable Bowel Syndrome (IBS).

15. A method for treating gastro-intestinal disease in a subject thereof comprising administering to said subject a therapeutically effective amount of an EcN bacterium according claim 1.

16. The EcN bacterium according to claim 3, wherein the ClbP protein is not substituted with an amino acid that is a polar and non-charged equivalent of Serine.

17. The EcN bacterium according to claim 4, wherein the ClbP protein is not substituted with an amino acid that is a positively charged equivalent of Lysine.

18. The EcN bacterium according to claim 5, wherein the ClbP protein is not substituted with an amino acid that is a hydrophobic equivalent of Tyrosine.

Description

FIGURES

[0080] FIG. 1. Role of the pks/clb island in EcN antibacterial activity on LF82. (A) Serial dilutions of 24-hour cocultures of E. coli LF82 (rifampicin resistant) with wild-type (WT) E. coli strain Nissle 1917 (EcN) or the mutant for the colibactin maturing peptidase ClbP, spotted (10 μL) on LB plate containing rifampicin and incubated overnight at 37° C. (B) Colony forming unit (CFU) counts of E. coli LF82 following a 24-hour co-culture in M63 medium with WT

[0081] EcN, gene deletion for the phosphopantetheinyl transferase ClbA, the peptidase ClbP and the corresponding complemented mutant (pclbP), the polyketide synthases (PKS) ClbC and ClbO, the nonribosomal peptide synthases (NRPS) ClbH and ClbN, the hybrid PKS-NRPS ClbB, the putative amidase ClbL, the efflux pump ClbM, and the thioesterase ClbQ. LF82 was also cultured alone as a control (Ø). The medians and individual results of independent experiments are shown. One-way ANOVA and Bonferroni post-tests in comparison with co-culture with WT; .star-solid..star-solid..star-solid.P<0.001.

[0082] FIG. 2: Role of the microcin gene cluster in EcN antibacterial activity on LF82. Colony forming unit (CFU) counts of E. coli LF82 following a 24-hour co-culture in M63 medium with wild-type (WT) E. coli strain Nissle 1917 (EcN), EcN mutant for microcin M (MccM) precursor gene mcmA, for microcin H47 (MccH47) precursor gene mchB, for both mcmA mchB genes; EcN mutants and complemented strains for mchC mchD genes responsible for posttranslational modifications, and for mchE mchF genes that encode the MccM and MccH47 efflux pump. LF82 was also cultured alone as a control (Ø). The medians and individual results of independent experiments are shown. One-way ANOVA and Bonferroni post-tests in comparison with co-culture with WT; .star-solid..star-solid.0.001<P<0.01; .star-solid..star-solid..star-solid.P<0.001.

[0083] FIG. 3: Role of siderophores in EcN antibacterial activity on LF82. (A) Colony forming unit (CFU) counts ofE. coli LF82 following a 24-hour co-culture in M63 medium with wild-type (WT) E. coli strain Nissle 1917 (EcN), EcN mutant for entE that encodes the enterobactin synthase E, and the double mutant for the phosphopantetheinyl transferases ClbA and EntD; EcN mutant and complemented strain for the glucosyltransferase IroB, the cytoplasmic esterase IroD, the periplasmic esterase IroE, and the export protein IroC. LF82 was also cultured alone as a control (Ø). The medians and individual results of independent experiments are shown. One way ANOVA and Bonferroni post-tests in comparison with co-culture with WT; .star-solid..star-solid..star-solid.P<0.001.

[0084] FIG. 4: Role of ClbP catalytic and transmembrane domains in EcN antibacterial activity on LF82.

[0085] Colony forming unit (CFU) counts of E. coli LF82 following a 24-hour co-culture in M63 medium with wild-type (WT) E. coli strain Nissle 1917 (EcN), the clbP gene deletion and complemented mutant with a plasmid that encodes wild-type ClbP (pclbP), plasmids that encode ClbP with a mutation S95A or K98T in the catalytic site, and a plasmid that encodes a fusion between the alkaline phosphatase PhoA and the ClbP C-terminal sequence from amino-acid 390 (pclbP-3H). LF82 was also cultured alone as a control (Ø). Medians and individual results of independent experiments are shown. One way ANOVA and Bonferroni post-tests in comparison with co-culture with WT; .star-solid..star-solid..star-solid.P<0.001.

[0086] FIG. 5: A genomic point mutation that inactivates ClbP catalytic domain abrogates EcN genotoxicity but not the antibacterial activity on LF82. (A) HeLa cells were transiently infected with wild-type E. coli Nissle (WT), a genome edited mutant with a single chromosomal nucleotide change in the clbP gene that inactivates the catalytic site (clbP-S95R), and the genome edited mutant complemented with the plasmid pclbP. Cells were then fixed, permeabilized and stained with rabbit monoclonal anti-gamma-H2AX followed by an infrared fluorescent secondary antibody. DNA was counterstained with RedDot2. (B) HeLa cells were transiently infected with wild-type E. coli Nissle (photo B), a clbP gene deletion mutant (C), and the genome edited clbP-S95R mutant (D). These cells were then washed and incubated with gentamicin for 72 hours before staining with Giemsa. The control is shown in photo A. Bars represent 50 (C) Colony forming unit (CFU) counts of E. coli LF82 following a 24-hour co-culture in M63 medium with wild-type (WT) E. coli strain Nissle 1917 (EcN), the clbP gene deletion mutant (AclbP), and the genome edited mutant with a single nucleotide change in the clbP gene that results in an S95R mutation in the catalytic site (clbP-S95R). LF82 was also cultured alone as a control (Ø). Medians and individual results of independent experiments are shown. One way ANOVA and Bonferroni post-tests in comparison with co-culture with WT; .star-solid..star-solid..star-solid.P<0.001

[0087] FIG. 6: Gene clusters involved in the production of microcins H47 and M (MccH47 and M) in E. coli strain Nissle (EcN) represented on a genomic map. The loci that encode enterobactin (ent), colibactin (pks), yersiniabactin (ybt) on EcN genomic island (GEI) IV, salmochelin (iro) and MccH47 (mch) and M (mcm) on GEI I are represented. The arrows represent the interplays between the different gene clusters involved in MccH47 and M production in EcN.

[0088] FIG. 7: Model proposed for biosynthesis of siderophore-microcins H47 and M in E. coli Nissle. An enterobactin precursor is modified by the enterobactin glucosyltransferase IroB. This siderophore moiety is transferred onto the C-terminal extremity of the precursor peptide by the MchCD complex. The active form of the siderophore-microcin is the result of the leader peptide cleavage during its export by the specific efflux pump MchEF-To1C. The C-terminal domain of ClbP allows this final step of siderophore-microcin production, while its N-terminal enzymatic domain cleaves precolibactin to produce colibactin.

[0089] FIG. 8: A clbP deletion, but not a genomic point mutation that inactivates ClbP catalytic domain, impairs EcN protection against the enteric pathogen Salmonella Typhimurium in mice.

[0090] C57BL/6 female mice were treated with 20 mg streptomycin per os, then 24 h later infected orally with 10.sup.9 S. Typhimurium (STm) in PBS or co-administered with 10.sup.9 S. Typhimurium and 10.sup.9 EcN wild-type, ΔclbP or clbPS95R strains. (A) The mice were monitored for clinical signs (weight loss, diarrhea, signs of abdominal pain) daily during 4 days. Each point corresponds to the mean clinical score +/−SEM of 10 to 15 animals per group in three independent experiment. The animals were scored blindly (without knowledge of the infecting bacteria) in the last two of the three experiments. Two way ANOVA with Bonferonni post-test compared to STm+EcN, a: p<0.05, c: p<0.001. (B) The fecal shedding of STm was examined by enumeration of the feces collected at day 2 and 4 after infection. The median and individual result are shown. One way ANOVA of log-transformed CFU counts compared to STm +PBS, a: p<0.05 (C) Fecal counts of STm and EcN were used to determine the competitive index (CFU STm/CFU EcN). One way ANOVA compared to STm+EcN clbPS95R, a: p<0.05.

[0091] FIG. 9: Detection of N-acyl-Asn-GABAOH in EcN wild-type, ΔclbN and clbPS95R strains.

[0092] EcN wild-type, ΔclbN or clbPS95R strains were grown 8 hours in DMEM Hepes then N-acyl-Asn-GABAOH was quantified in bacterial pellets by high-performance liquid chromatography (Agilent 1290 Infinity) coupled to a triple quadrupole mass spectrometer (G6460 Agilent). The concentration C is shown as picogram per 1E+8 CFU).

EXAMPLE

[0093] Material & Methods

[0094] Bacterial Strains, Mutants and Plasmids

[0095] The bacterial strains and plasmids used in this study are listed in Table 3. Gene mutagenesis was performed by the lambda Red recombinase method (37). The double mutants were constructed sequentially. The mutations and deletion of FRT cassettes were verified by PCR using primers upstream and downstream of the target genes.

[0096] The fusion between ClbP N-terminal signal sequence, the alkaline phosphatase PhoA, and the three transmembrane helices of ClbP were constructed using the HiFi DNA assembly kit (New England Biolabs, Ipswich, Mass., USA) with primers overlapping between each fragment. The constructions were verified by PCR and confirmed by sequencing. The blue-stained colony-forming units on LB plates with 40 mg/L of 5-bromo-4-chloro-3-indolyl phosphate revealed the presence of the PhoA alkaline phosphatase domain in the periplasm as previously reported (38).

[0097] To construct plasmids pmchEF and pmchCD, the genes were PCR-amplified and cloned into pCR-XL-TOPO (Invitrogen, Carlsbad, Calif., USA).

[0098] To construct plasmid pIroB, the iroB gene was PCR-amplified with EcN genomic DNA as a template and primers IRSDNG7 and IRSDNG8, digested by EcoRI and BamHI and ligated into pBbA5a-RFP (obtained from Addgene) digested to remove the rfp gene.

[0099] The strain EcN clbP-S595R chromosomal isogenic mutant was constructed using a genome editing technique (39). EcN was transformed with pORTMAGE, then grown in LB at 30° C. and 300 rpm to reach OD600=0.5. An initial mutagenesis cycle was started by inducing the expression of Lambda recombinases and the dominant negative mutLE32K allele at 42° C. for 15 minutes at 250 rpm. The culture was then cooled to 0° C., washed in water and electroporated with 50 μM of oligonucleotide IRSDNG26 that includes the S95A mutation in the clbP gene sequence. In a control experiment, the lacZ gene was targeted by a specific mutagenic oligonucleotide. Following recovery in LB at 30° C. and 300 rpm for 1 hour, two other mutagenesis cycles were performed, and the bacteria were finally plated on MacConkey agar without any antibiotic. Approximately 33% of the isolates were LacZ negative in the control experiment. Sixty candidate clbP-S595R mutants were tested for loss of genotoxicity and megalocytosis phenotype in infected HeLa cells as previously described (40). Non genotoxic mutants that had lost the pORTMAGE plasmid were selected, and were finally verified for removal of a ClaI restriction site by S95A mutation in the PCR amplified clbP sequence.

[0100] Determination of the Genotoxic Effect Induced by Colibactin

[0101] The cellular senescence induced by colibactin was with the associated cell enlargement called megalocytosis and was determined for every EcN mutant constructed in this study in the Mcc gene cluster, in the iroA locus, and for the clbP-S95R mutant. As previously described (40), HeLa cells (ATCC, CCL-2) were infected for 4 hours. The cells were then washed and incubated with gentamicin for 72 hours before staining with Giemsa. The genotoxicity of EcN and the clbP-S95R chromosomal mutant was confirmed by an In-Cell Western procedure, as previously described (36). In brief, HeLa cells were infected in 96-well plates for 4 hours at a given multiplicity of infection (number of bacteria per cell at the onset of infection). Four hours after the end of infection cells were fixed, permeabilized and stained with rabbit monoclonal anti-gamma-H2AX (Cell Signaling, 20E4, 1:200) followed by an infrared fluorescent secondary antibody. DNA was counterstained with RedDot2 (Biotum). Fluorescence was recorded with an Odyssey infrared imaging system (Li-Cor).

[0102] Competitive Growth Assay

[0103] Strains were grown in lysogeny broth (LB Lennox, Invitrogen) overnight at 37° C. with shaking at 240 rpm. Rifampicin, streptomycin, kanamycin, carbenicillin or chloramphenicol was added as required to the medium.

[0104] The media used for co-culture experiments were either M63 minimal medium with final concentrations of 15 mM ammonium sulfate, 1 mM magnesium sulfate heptahydrate, 100 mM monopotassic phosphate, 2.5 g/L glucose, 1 mg/L thiamine, and 1 g/L Bacto tryptone (BD Biosciences, Le Pont de Claix, France), or Dulbecco's Modified Eagle Medium (DMEM) GlutaMAX (Invitrogen) supplemented with 25 mM Hepes, 10% (v/v) Fetal Calf Serum (FCS, Eurobio, Courtaboeuf, France), and 1% (v/v) Non Essential Amino Acids (NEAA, Invitrogen).

[0105] 500 μL of each overnight culture were cultured in 9.5 mL of co-culture medium and incubated for 2h at 37° C. with shaking at 240 rpm. Both the producing and the target strains (EcN and LF82 respectively) were inoculated from these 2-hour-cultures at 106 CFU/mL in 10 mL of co-culture medium as previously described (3) and incubated for 24 h at 37° C. with shaking at 240 rpm. For CFU numeration, the culture broth was serial-diluted in PBS and plated on selective LB agar plates containing the antibiotic required (e.g. rifampicin for LF82 (41)). In the total results section, only the growth of the target strains (mainly LF82) is reported. As a control, the growth of the competitive strains (mainly EcN and EcN mutants) was systematically checked (data not shown).

[0106] Animal Infections

[0107] The animal infections were performed following the European directives for the protection of animal used for scientific purposes (2010/63/EU). The protocol was approved by a local ethic committee (number of protocol: 2019041710292271). Female C57BL/6 (Janvier) were housed in ventilated cages, 5 animals per cage, with ad libitum access to food and water.

[0108] The animals were administered by oral gavage 20 mg of streptomycin, then 24 h later, infected per os with 10.sup.9 S. Typhimurium strain IR715 (nalidixic acid resistant) or co-administered with 10.sup.9 S. Typhimurium and 10.sup.9 EcN, EcN ΔclbP or EcN clbPS95R (with the rpsLK42R allele to confer resistance to streptomycin).

[0109] Fecal shedding of S. Typhimurium and EcN was determined by homogenization of feces in PBS, serial dilution and plating on LB agar plates supplemented with nalidixic acid or streptomycin.

[0110] The severity of the salmonellosis was evaluated by daily scoring of weight loss, signs of abdominal pain, fever and diarrhea.

[0111] The experiment was terminated at 4 days after infection to avoid lethality.

[0112] The experiment was repeated three times with five animals per group, and the clinical score was scored blindly (without knowledge of the infecting bacteria) in two out of the three independent experiments.

[0113] Bioinformatic Analysis

[0114] Genes involved in MccH47 and MccM synthesis were searched using BLASTn and the CA58 Mcc gene cluster as a reference: mchB and mcmA which encode precursor proteins, the immunity genes mchl and mcml, genes mchE and mchF which encode a specific efflux pump, and genes mcmK and mcmL (and their respective homologs in the E. coli H47 Mcc gene cluster, mchS1 and mchA) responsible for posttranslational modifications. A query cover >80%, an identity >90%, and an E value <1e 40 were chosen as cutoff values for significance. The genes clbB and clbP, as respective markers for the 5′ and 3′ regions of the pks island, were identified using the same method, and so were genes iroN and iroB as markers for the 5′ and 3′ regions of the salmochelin gene cluster (iroA locus). Phylogroups were determined in silico based on the presence/absence of 4 genes: arpA, chuA, yjaA, and tspE4.C2 (and trpA to distinguish the A and C phylogroups) (42). The phylogenetic tree was constructed with the rpoC sequence. The sequences were collected using PATRIC 3.5.8 (43), aligned by multiple sequence comparison by log expectation (MUSCLE) with the MEGA7.0.26 software (44), and the phylogenetic tree was constructed according to the maximum likelihood method with MEGA7.0.26.

[0115] Statistical Analyses

[0116] Statistical analyses were carried out using GraphPad Prism 7.0a (GraphPad, San Diego, Calif., USA). P values were calculated using one-way ANOVA followed by Bonferroni post-tests. CFU/ml were log-transformed for the analyses. P values <0.05 were considered significant and are denoted by .star-solid., P<0.01 is denoted by .star-solid..star-solid., and P<0.001 by .star-solid..star-solid..star-solid..

[0117] Results

[0118] EcN Antibacterial Activity Requires ClbP but not the Other Components of the Colibactin Synthesis Pathway

[0119] In order to specifically decouple the genotoxic activity from the probiotic activity, we tested the antibacterial activity of the EcN mutant deleted for ClbP that allows the maturation of precolibactin in genotoxic colibactin (24,25). We compared it to the pleiotropic ClbA mutant coding for a PPTase (27,35,36). We performed co-culture experiments with the wild-type EcN, the EcN ΔclbA and ΔclbP mutants, and the Crohn's disease-associated E. coli strain LF82 which have been previously shown to be susceptible to EcN (45,46). CFU showed that the EcN strain strongly inhibited LF82 growth. EcN antibacterial activity on LF82 was not altered in a ΔclbA mutant but was completely lost in a ΔclbP mutant (FIG. 1). A kinetic experiment indicated that EcN inhibitory activity on LF82 started 6 hours post-inoculation and reached its maximum 8 hours post-inoculation, at the beginning of the stationary phase (data not shown). LF82 growth was not altered at any time by the ΔclbP mutant, further proving the ClbP-dependence of the EcN antibacterial effect. This EcN ClbP-dependent inhibitory activity was also observed with other pathogenic strains of E. coli (JJ186 and NRG857c) and closely related bacteria species, such as Salmonella enterica subsp. enterica Typhimurium, and Enterobacter aerogenes (data not shown).

[0120] To further determine whether other components of the colibactin synthesis pathway besides ClbP are required for EcN antibacterial activity, the inhibitory effect of mutants for the PKS ClbC and C1bO, the NRPS ClbH and ClbN, the hybrid PKS-NRPS ClbB, the putative amidase ClbL, the efflux pump ClbM, and the thioesterase ClbQ were assessed against LF82. EcN antibacterial activity against LF82 was not altered in any of these mutants (FIG. 1). These results confirm that colibactin itself or the cleavage product N-myristoyl-D-asparagine is not essential for EcN antibacterial activity against LF82 (and other Gram-negative bacteria, data not shown). Therefore, the probiotic activities of EcN are clearly associated with the presence of the pks/clb island and ClbP but not colibactin is involved in EcN inhibitory activity.

[0121] EcN ClbP-Dependent Antibacterial Activity Requires MccH47 and MccM

[0122] Previous studies have associated EcN antibacterial activity with MccH47 and MccM (4,11,14,15). Therefore, we performed co-culture experiments with LF82, EcN and mutants in MccH47 and MccM production systems. EcN antibacterial activity against LF82 was not affected by the deletion of the MccM precursor gene mcmA alone or the MccH47 precursor gene mchB alone (FIG. 2). In contrast, deletions of both mcmA and mchB almost completely abrogated the inhibitory effect of EcN on LF82. Similarly, deletion of the MccM and MccH47 efflux pump encoding genes mchE and mchF resulted in a loss of antibacterial activity (FIG. 2). The trans-complementation of mchE and mchF increased EcN inhibitory activity compared to the wild-type EcN strain (FIG. 2), probably because of an increase in Mcc export following overexpression of the MchE-MchF efflux pump. None of these mutations in the Mcc production system affected the ability of EcN to produce active colibactin (data not shown).

[0123] To further confirm the role of MccH47 and MccM in EcN antibacterial activity, plasmids that encode MccH47 or MccM immunity genes were transformed in LF82, and the resulting resistance of the strains was assessed against EcN (data not shown). EcN AmchB mutant antibacterial activity was almost completely abrogated on LF82 that carries the MccM immunity gene mcmI (data not shown). A similar result was obtained with the AmcmA mutant and LF82 that carries MccH47 immunity gene mchl (data not shown). Overall, these results confirmed that the EcN ClbP-dependent inhibitory activity against LF82 is due to MccH47 and MccM.

[0124] EcN ClbP-Dependent Antibacterial Activity is Due to the Production of Siderophore-Mcc

[0125] MccH47 and MccM can be modified posttranslationally by the linkage of a catechol siderophore to form a “siderophore-Mcc” (13). Therefore, we hypothesized that the ClbP-dependent antibacterial activity might be dependent on these modified forms of microcins. In fact, EcN antibacterial activity against LF82 was strongly reduced in a AentE mutant deprived of the enzyme 2,3-dihydroxybenzoate-AMP ligase essential for siderophore enterobactin production (47). Similar results were obtained with the EcN ΔclbA AentD double mutant which was unable to produce enterobactin (36) (FIG. 3).

[0126] The two genes responsible for enterobactin glycosylation and esterification (mcmL and mcmK) are missing from the EcN Mcc gene cluster (18,48). As a result, whether MccH47 and MccM are siderophore-Mcc or unmodified Mcc is still being debated (13). Considering that EcN carries the McmL and McmK homologs, glucosyltransferase IroB and esterase IroD respectively (13), we investigated the interplay between the Mcc and the salmochelin production systems. The antibacterial activity of EcN mutants for genes that encode the glucosyltransferase IroB, the cytoplasmic esterase IroD, the periplasmic esterase IroE, and the export protein IroC (49,50) was compared to the activity of the wild-type EcN strain. Only iroB deletion led to a significant decrease in EcN antibacterial activity (FIG. 3). Complementation of the AiroB mutant fully restored the antibacterial activity. None of these mutations in the iroA locus affected EcN ability to cause megalocytosis linked with the colibactin genotoxic effect (data not shown). These results suggest that IroB could be responsible for enterobactin glycosylation, which enables the linkage of Mcc precursor proteins to the siderophore-derived moiety in the absence of McmL.

[0127] MchC and MchD are respective homologous to MceJ and MceI of K. pneumoniae strain E492 (13). These proteins form a complex responsible for the linkage of glycosylated enterobactin derivatives to MccE492 the precursor peptide MceA (51). The EcN mutant for mchC and mchD lost the antibacterial effect against LF82, whereas complementation restored the initial phenotype (FIG. 2). These results indicate that the posttranslational modification of MccH47 and MccM with an enterobactin-derived moiety is required for EcN antibacterial activity. In short, EcN ClbP-dependent antibacterial activity is due to siderophore-Mcc.

[0128] The ClbP Transmembrane Domain, Rather Than the Periplasmic Peptidase Catalytic Site, is Required for the Antibacterial Activity of EcN

[0129] To further elucidate the role of ClbP in siderophore-Mcc production, we examined whether ClbP catalytic activity is required for EcN antibacterial activity. S95 and K98 are key residues for ClbP peptidase activity, and mutants for these residues fail to cleave precolibactin to release mature active genotoxin (24,25). Co-culture experiments were performed with LF82 and the EcN ΔclbP mutant complemented with plasmids that encode the wild-type ClbP protein, or the ClbP protein that harbors the substitutions S95A or K98T. EcN ΔclbP mutants complemented with ClbP S95A or K98T demonstrated antibacterial activities similar to those of the wild-type ClbP protein (FIG. 4), whereas they lost their ability to cause megalocytosis linked with the colibactin genotoxic effect (data not shown).

[0130] To exclude the role of another putative catalytic site of ClbP enzymatic domain, this enzymatic domain was replaced by alkaline phosphatase enzymatic domain of PhoA, as previously reported (38). The PhoA domain was fused with the ClbP N-terminal signal sequence which allows the translocation to periplasm, and the ClbP C-terminal sequence from amino-acid 390; the residues forming the three transmembrane helices being 390-412, 433-455, and 465-485 (24). An EcN ΔclbP mutant transformed with a plasmid bearing this fusion demonstrated a similar inhibitory activity against LF82 as the EcN WT strain (FIG. 4), whereas it did not cause megalocytosis (data not shown). Therefore, the C-terminal domain of ClbP that comprises the three transmembrane helices is essential for EcN antibacterial activity, as opposed to the ClbP periplasmic peptidase domain which is crucial only for genotoxic activity.

[0131] To confirm this observation, and as a proof of the concept that a non-genotoxic EcN probiotic strain could be engineered, we used genome editing to construct an EcN mutant strain that exhibits a single nucleotide mutation in the chromosomic clbP gene, which leads to an S95R mutation in the ClbP catalytic site at the amino-acid level. This mutant did not produce colibactin and is not genotoxic but still exhibited an antibacterial activity towards LF82 that is similar to that of the wild-type genotoxic EcN strain (FIG. 5).

[0132] An EcN Strain with a Point Mutation in the clbP Gene is Non-Genotoxic but Keeps the Antagonist Activity, and Reduces S. Typhimurium Intestinal Colonization and Virulence

[0133] The EcN probiotic is well known to offer protection against enteric pathogens such as Salmonella, by competing for iron and producing the siderophore-microcins (3,4). Thus, we examined whether the EcN wild-type, ΔclbP and clbP-S95R mutants reduces S. Typhimurium intestinal colonization and pathogenesis using an in vivo model. We utilized C57BL/6 mice treated with streptomycin (to ensure a high colonization) then 24 h later infected with S. Typhimurium alone, or co-administered with S. Typhimurium and each EcN strain (3,4,66). The mice were monitored for clinical signs (weight loss, diarrhea, signs of abdominal pain) and the bacterial colonization was examined by enumeration of the feces, during 4 days (the point where the experiment must be arrested because of the lethality). When administered alone, S. Typhimurium readily colonized the intestine and this was associated with a high clinical score linked to a strong enteric salmonellosis (FIG. 8). In animals co-administered with the wild-type EcN, there was a marked reduction in the clinical scores and in S. Typhimurium fecal colonization (FIG. 8A-B). By day 2 following infection, EcN significantly outcompeted S. Typhimurium (FIG. 8C). In contrast, animals co-administered with the EcN ΔclbP mutant exhibited higher clinical scores and reduced antagonism of S. Typhimurium colonization, demonstrating the role of ClbP in EcN beneficial effect during acute Salmonella colitis. The EcN clbP-S95R strain reduced substantially the fecal shedding and outcompeted S. Typhimurium, and diminished the clinical scores, similarly to the wild-type EcN (FIG. 8).

[0134] Altogether, these results show that it is possible to decouple the genotoxic activity of EcN from its probiotic (antibacterial) activity, but also that the biosynthetic pathways of colibactin and siderophore-microcins are more entangled than initially thought.

[0135] The ClbP Dependent Antibacterial Activity is Observed in a Subset of E. coli Strains That Carry a Truncated Mcc Gene Cluster and the pks Island

[0136] Comparative genomic analyses have shown that EcN is closely related to E. coli pyelonephritis strain CFT073 and the asymptomatic bacteriuria strain ABU83972 (18). These three strains, as well as the reference strain ATCC®25922, carry the pks island, the iroA locus, and a truncated Mcc gene cluster deprived of genes mcmL/mchA and mcmK/mchS1. Therefore, we assessed whether the siderophore-Mcc antibacterial effect of these strains was ClbP-dependent, as observed in EcN. The inhibitory effect of two sets of E. coli strains was tested in co-culture experiments against LF82, as well as their respective ΔclbP mutants: i) strains similar to EcN that carry both a truncated Mcc gene cluster and the pks island: strains CFT073, ABU83972, and ATCC®25922; and ii) strains that carry the pks island but which are deprived of Mcc encoding genes: the human commensal strain M1/5, the meningitis-causing strain SP15, the murine commensal strain NC101, and the laboratory strain MG1655 that hosts a bacterial artificial chromosome (BAC) bearing the pks island. The three wild-type strains that carry both a truncated Mcc gene cluster and the pks island exhibited a marked inhibitory effect as observed in EcN (data not shown). The inhibitory effect of all three corresponding ΔclbP mutant strains was significantly reduced, whereas ClbP complementation restored the initial phenotype (data not shown). In contrast, in strains carrying only the pks island, there was no significant difference in LF82 growth whether it was cultivated with the wild-type strains or the ΔclbP mutants (data not shown). Cumulatively, these results show that the peptidase ClbP is involved in MccH47 and MccM antibacterial activity in E. coli strains that carry both the pks island and a truncated form of the Mcc gene cluster. Our results also show that this association is present in both pathogenic strains and probiotic strains.

[0137] Distribution of pks, Salmochelin and the MccH47 and MccM Ggene Clusters in an E. coli Population

[0138] We demonstrated that strains of E. coli that carry a truncated Mcc gene cluster exhibit a siderophore-Mcc-dependent antibacterial activity (data not shown). This antibacterial activity requires ClbP from the biosynthetic pathway that produces the genotoxin colibactin and IroB from the biosynthetic pathway that produces the siderophore salmochelin. Consequently, we checked this association between the pks island, the iro locus and the Mcc island in E. coli strains with genomes available in GenBank. Interestingly, all strains that lacked the mcmL and mcmK genes responsible for posttranslational modifications belonged to the B2 phylogroup and carried the pks island and iroA (data not shown), except for strain 1105 deprived of pks island. Conversely, the strains that carry mcmL/mchA and mcmK/mchSl belonged to B1, C or D phylogroups and lacked the pks island. These particular associations of genetic determinants led to the hypothesis that the truncated island is present almost exclusively in strains that carry pks and the iroA locus. It suggests that this interplay between colibactin, salmochelin, and the siderophore-Mcc biosynthetic pathways is due to a co-selection in strains that is either pathogenic or probiotic.

[0139] Production of Beneficial Compounds Likely Involved in the Probiotic Activity of Nissle 1917 by the Nissle clbPS95R Strain

[0140] Other metabolites than colibactin that are synthesized by the enzymes encoded on the pks island might have a role on the probiotic properties of Nissle 1917. It was recently shown that the metabolite C12AsnGABAOH is produced by Nissle 1917 wild-type (but not by a clbN mutant, indicating that the pks-encoded machinery has a role in its production) (34). C12-Asn-GABAOH was shown to inhibit nociceptors activation in neurons. Nociceptor neurons in the intestinal tract play an important role in protecting against enteropathogens and intestinal homeostasis, as they regulate M cell density and inflammation. Thus, it is important that we ensure that a non-genotoxic modified strain retain production of C12AsnGABAOH with a role in the anti-inflammatory property of Nissle.

[0141] We have quantified by HPLC-QQQ (34) N-acyl-Asn-GABAOH in bacterial cultures of Nissle 1917 wild type, Nissle clbN mutant and Nissle clbPS95R. The Nissle clbPS95R still produces the beneficial GABAOH lipopeptide similarly to the wild type strain (FIG. 9).

[0142] Genetic Stability of Nissle clbPS95R

[0143] To verify that the Nissle clbPS95R strain is genetically stable, we sequenced its genome DNA before and after oral gavage of a mouse (together with a pathogenic Salmonella (67)) and reisolation from the feces. The genomes were compared to that of the wild-type strain, which was also sequenced. The Nissle clbPS95R strain shown only 2 bp change (out of 5441200 bp) compared to the wild-type. No difference was found between Nissle clbPS95R before and after passage through the mouse intestine (data not shown). Thus, the Nissle clbPS95R appear genetically stable.

[0144] Discussion

[0145] Since Fleming discovered penicillin in 1928, antibiotics have contributed to the increase in human life expectancy. Many infections which were previously fatal became curable. Unfortunately, the overuse and misuse of antibiotics, in parallel with the lack of new antibacterial drugs enabled multi-resistant bacteria to emerge and spread (52). According to the World Health Organization (WHO), this phenomenon “poses a substantial threat to morbidity and mortality worldwide” (53). The trend is especially worrying for Gram-negative bacteria. For instance, the number of deaths attributable to 3r.sup.d generation cephalosporin-resistant or carbapenem-resistant E. coli increased by more than 4 times in Europe between 2007 and 2015 (54). Of the antibiotics that are currently being developed for intravenous administration, only a small proportion (15 out of 44) demonstrates some activity against Gram-negative bacteria, and all these molecules are derived from known antibiotic classes. Consequently, the WHO established that research and development of new antibiotics against Gram-negative bacteria was a “critical priority” (53).

[0146] In the search for new antimicrobials, microcins seem a promising alternative to “conventional” antibiotics. In fact, many microcins exhibit potent narrow-spectrum antimicrobial activity, whereas antibiotics can eliminate beneficial bacteria, alter the microbiota and promote the selection of resistant strains (55,56). A major challenge in using microcins is their delivery in sufficient quantities to the site of infection, especially after oral administration because they are often degraded in the upper digestive tract (57,58). Engineered probiotic bacteria were consequently proposed as in situ producers of microcins to fight against enteropathogens (59) or to reduce colonization by multi-resistant bacteria (60).

[0147] EcN has been used as a probiotic for over a century, with numerous therapeutic benefits described. However, serious concerns about the safety of EcN administration have emerged over the years. EcN was reported to be responsible for severe sepsis in an infant (61) and its genome was shown to have the pathogenicity island pks (21,35), which codes for colibactin, a bona fide virulence factor for E. coli strains responsible for extraintestinal infections (26,27). In addition, the carriage of colibactin producing E. coli could also be deleterious to gut homeostasis. In adult rats, it increased intestinal epithelial permeability, led to signs of genotoxic damages in intestinal cells, such as crypt fission, and increased cell proliferation (28). In mice predisposed to colorectal cancer, pks-positive E. coli increased the size and the number of tumors (31,62). In human beings, several studies reported that pks-positive E. coli were over-represented in colorectal cancer biopsies compared to controls (31,32,63). On a whole, these studies suggest that colibactin-producing bacteria could promote tumorigenesis. Therefore, our goal was to understand the interplay between the production of the genotoxin colibactin and the beneficial effects related to the pks island in the probiotic activity of EcN. Consequently, we attempted to disarm EcN while keeping its probiotic properties.

[0148] In a previous attempt, our team constructed a non-genotoxic EcN PPTase ClbA mutant, which also lost its probiotic activity (35). Subsequently, it was discovered that the PPTase ClbA contributes to the synthesis of enterobactin (and therefore salmochelin) and yersiniabactin (36). In this study, we demonstrated that there is collaboration between the salmochelin (iroB) and the Mcc gene clusters, both of which are located on EcN genomic island I, and the pks island (clbP) (FIG. 6). The interweaving is so strong between these determinants, that a single protein, ClbP is involved both in colibactin and Mcc production. Up until now, ClbP had only been described as a peptidase that removes the N-acyl-D-asparagine prodrug scaffold from precolibactin (24,25). Although the complete C-terminal domain with the three transmembrane helices is required for the bioactivity of ClbP, the catalytic activity is performed by the N-terminal periplasmic domain (25,38). In this study, we demonstrated that the C-terminal domain of ClbP, deprived of the known enzymatic function, is necessary for EcN antibacterial activity due to MccH47 and MccM. It suggests that the ClbP C-terminal transmembrane domain could facilitate the export of the MccH47 and MccM of EcN through the MchE-MchF efflux pump (FIG. 7).

[0149] Using both functional and bioinformatic analyses, we demonstrated interplay between siderophore-Mcc, salmochelin, and colibactin assembly lines. Strikingly, two groups of E. coli strains emerged. On one hand, all strains that carry a “truncated” MccH47 and MccM gene cluster (i.e. strains such as EcN lacking mcmL/mchA and mcmK/mchS1) are B2 strains that also bear the pks island and the iroA locus. It should be noted that isolates from urine were over-represented in this group of strains (CFT073, clones D i14 and D i2, UPEC 26-1, and ABU 83972). On the other hand, the pks island and the iroA locus are absent in the non-B2 strains that carry a “complete” MccH47 and MccM gene cluster. All these strains were isolated from stools (except ACNO02 for which the origin is unknown). Therefore, we can hypothesize that these strains with a “complete” Mcc gene cluster are specialized in Mcc production in order to survive in the competitive intestinal environment, which is their exclusive niche. In contrast, extraintestinal pathogenic E. coli (ExPEC) must be efficient gut colonizers in order to emerge from the intestinal niche and infect other body sites (such as the urinary tract) to which they must subsequently adapt. That is why it has been suggested that ExPEC are “generalists” rather than specialized strains (64). The strains we examined in our study fit this model. They can express various virulence factors depending on their environment: MccH47 and M, siderophores and analgesic lipopeptides derived from the colibactin pathway, for instance. To be able to produce so many virulence or fitness factors with a genome of limited size (65), the elements of the assembly lines that produce these determinants must be versatile and intervene in several apparently independent metabolic pathways.

[0150] In conclusion, we discovered that the pks island is even more intimately connected to EcN probiotic activity than expected. This entanglement reflects the co-evolution of probiotic and pathogenic determinants to adapt to various environments. Decoupling the probiotic from the genotoxic activities by specifically targeting the enzymatic domain of ClbP opens the way to safe use of EcN.

TABLE-US-00003 TABLE 3 Strains and plasmids used in this study. Source or Strain or plasmid Genotype or phenotype reference E. coli Nissle (EcN) Probiotic strain; colibactin genotoxin DSM 6601, producer; enterobactin and salmochellin Mutaflor ® siderophores producer; microcins H47 and M producer EcN WT EcN mutant in rpsl, Str.sup.R [1, 2] EcN ΔclbA clbA mutant of strain EcN WT, Str.sup.R, Kan.sup.R  [1] EcN ΔclbB clbB mutant of strain EcN WT, Str.sup.R, Kan.sup.R  [3] EcN ΔclbC clbC mutant of strain EcN, Str.sup.R, Chl.sup.R  [3] EcN ΔclbH clbH mutant of strain EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔclbL clbL mutant of strain EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔclbM clbM mutant of strain EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔclbN clbN mutant of strain EcN WT, Str.sup.R, Kan.sup.R  [3] EcN ΔclbO clbO mutant of strain EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔclbP clbP mutant of strain EcN WT, Str.sup.R, Kan.sup.R  [3] EcN ΔclbP pclbP EcN ΔclbP mutant complemented with This study pBRSKpclbP, Str.sup.R, Kan.sup.R, Carb.sup.R EcN ΔclbP pclbP-S95A EcN ΔclbP mutant complemented with This study pclbP-S95A, Str.sup.R, Kan.sup.R, Carb.sup.R EcN ΔclbP pclbP-K98T EcN ΔclbP mutant complemented with This study pclbP-K98T, Str.sup.R, Kan.sup.R, Carb.sup.R EcN ΔclbP pclbP-3H EcN ΔclbP mutant complemented with This study pclbP-3H EcN clbP-S95R EcN clbP-S95R chromosomal isogenic This study mutant EcN ΔclbQ clbQ mutant of strain EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔmcmA mcmA mutant of EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔmchB mchB mutant of EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔmcmAΔmchB mcmA mchB mutant of EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔmchCD mchC mchD mutant of EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔmchCD::FRT mchCD::FRT mutant of strain EcN WT, Str.sup.R This study EcN ΔmchCD pmchCD EcN ΔmchCD::FRT complemented with This study TopoXL mchCD, Str.sup.R, Kan.sup.R EcN ΔmchEF mchE mchF mutant of EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔmchEF::FRT mchEF::FRT mutant of strain EcN WT, Str.sup.R This study EcN ΔmchEF pmchEF EcN ΔmchEF::FRT complemented with This study TopoXL mchEF, Str.sup.R, Kan.sup.R EcN ΔentE entE mutant of EcN WT, Str.sup.R, Chl.sup.R This study EcN ΔentD entE mutant of EcN WT, Str.sup.R, Chl.sup.R This study EcN ΔentDΔclbA clbA, entE mutant of EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔiroB iroB mutant of EcN WT, Str.sup.R, Chl.sup.R This study EcN ΔiroB piroB EcN ΔiroB mutant complemented with This study pASK75 iroB EcN ΔiroC iroC mutant of EcN WT, Str.sup.R, Kan.sup.R This study EcN ΔiroD iroD mutant of EcN WT, Str.sup.R, Chl.sup.R This study EcN ΔiroE iroE mutant of EcN WT, Str.sup.R, Kan.sup.R This study E. coli LF82 Strain isolated from an ileal biopsy of a  [4] patient with Crohn's disease; adherent- invasive E. coli, Rif.sup.R LF82 pMcMi LF82 carrying pMcMi, Rif.sup.R, Carb.sup.R This study LF82 pMcHi LF82 carrying pMcHi, Rif.sup.R, Carb.sup.R This study E. coli M1/5 Commensal E. coli strain isolated from feces  [5] of a healthy adult; B2 phylogenetic group; colibactin genotoxin producer; aerobactin, enterobactin and yersiniabactin siderophores producer M1/5 ΔclbP clbP mutant of strain M1/5, Kan.sup.R  [6] E. coli SP15 Strain isolated from spinal fluid of a neonate  [7] with meningitis; O18:K1 serotype; colibactin genotoxin producer; aerobactin, enterobactin, salmochelins and yersiniabactin siderophores producer SP15 ΔclbP clbP mutant of strain SP15, Kan.sup.R This study E. coli NC101 Non-pathogenic murine E. Coli strain; [8, 9] colibactin genotoxin producer; NC101 ΔclbP clbP mutant of strain NC101, Kan.sup.R [10] E. coli MG1655 bacpks Enterobactin siderophore producer E. coli [11] strain carrying a bacterial artificial chromosome bearing the entire pks island, Chl.sup.R MG1655 bacpks ΔclbP clbP mutant of strain MG1655 bacpks, Chl.sup.R This study E. coli CFT073 Strain isolated from a patient with [12] pyelonephritis, colibactin genotoxin producer; enterobactin siderophores producer; microcins H47 and M producer CFT073 ΔclbP clbP mutant of strain CFT073, Kan.sup.R This study CFT ΔclbP pclbP CFT073 ΔclbP mutant complemented with This study pBRSKpclbP, Kan.sup.R, Carb.sup.R E. coli ABU83972 Strain isolated from a patient with [13, 14] asymptomatic bacteriuria, colibactin genotoxin producer; enterobactin siderophores producer; microcins H47 and M producer ABU83972 ΔclbP clbP mutant of strain ABU83972, Kan.sup.R This study ABU83972 ΔclbP pclbP ABU83972 ΔclbP mutant complemented This study with pBRSKpclbP, Kan.sup.R, Carb.sup.R E. coli ATCC ®25922 Strain isolated from a patient in Seattle DSM1103 (1946), colibactin genotoxin producer; enterobactin siderophores producer; microcins H47 and M producer ATCC ®25922 ΔclbP clbP mutant of strain ATCC ®25922, Kan.sup.R This study ATCC ®25922 ΔclbP ATCC ®25922 ΔclbP mutant complemented This study pclbP with pBRSKpclbP, Kan.sup.R, Carb.sup.R E. coli ST131 isolate Strain isolated in the USA (2007) from a [15] JJ1886 patient with fatal urosepsis, Str.sup.R, Kan.sup.R, Carb.sup.R, Chl.sup.R E. coli NRG857c Strain isolated from the ileum of a Crohn's [16] Disease patient, Carb.sup.R, Chl.sup.R Salmonella enterica Nal.sup.R derivative of S. enterica serovar [17] serovar Typhimurium Typhimurium ATCC14028 IR715 Enterobacter aerogenes Strain isolated from sputum in the USA ATCC ®13048 ATCC ®13048 (Center for Disease Control and Prevention) Klebsiella oxytoca Strain isolated from a pharyngeal tonsil ATCC ®13182 ATCC ®13182 pclbP pBRSK encoding clbP sequence [18] pclbP-S95A pBRSK encoding the mutant S95A of ClbP [18] (pOB902), Carb.sup.R pclbP-K98T pBRSK encoding the mutant K98T of ClbP [18] (pOB903), Carb.sup.R pclbP-3H pASK74 carrying the fusion between ClbP This study N-terminal signal sequence, the alkaline phosphatase PhoA, and the 3 transmembrane helices of ClbP, Carb.sup.R pmchCD pCR XL-TOPO vector encoding mchC and This study mchD from EcN, Kan.sup.R pmchEF pCR XL-TOPO vector encoding mchE and This study mchF from EcN, Kan.sup.R pMcMi Carrying mcmI from MccM gene cluster, F. Moreno, Carb.sup.R unpublished data [19] pMcHi Carrying mchI from MccH47 gene cluster, F. Moreno, ChlR unpublished data, [19]

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