ATP-HYDROLYZING ENZYME USEFUL FOR TREATING DYSBIOSIS

Abstract

The present invention provides an ATP hydrolyzing enzyme, a nucleic acid encoding an ATP hydrolyzing enzyme, or host cells, microorganisms, such as bacteria, or viral particles comprising such nucleic acids encoding an ATP hydrolyzing enzyme for use in the treatment of dysbiosis or a dysbiosis-related disease.

Claims

1. An ATP hydrolyzing enzyme, a nucleic acid comprising a polynucleotide encoding the ATP hydrolyzing enzyme, a host cell comprising the nucleic acid, a microorganism comprising the nucleic acid, a (recombinant) bacterium comprising the nucleic acid, or a viral particle comprising the nucleic acid for use in the treatment of dysbiosis or a dysbiosis-related disease.

2. An ATP hydrolyzing enzyme, a nucleic acid comprising a polynucleotide encoding the ATP hydrolyzing enzyme, a host cell comprising the nucleic acid, a microorganism comprising the nucleic acid, a (recombinant) bacterium comprising the nucleic acid, or a viral particle comprising the nucleic acid for use in restoring or improving the microbiome balance during or after dysbiosis.

3. An ATP hydrolyzing enzyme for use in the treatment of dysbiosis or a dysbiosis-related disease.

4. The ATP hydrolyzing enzyme for use according to any one of claims 1 to 3, wherein the ATP hydrolyzing enzyme is a soluble ATP hydrolyzing enzyme.

5. The ATP hydrolyzing enzyme for use according to any one of the previous claims, wherein the ATP hydrolyzing enzyme is apyrase.

6. The ATP hydrolyzing enzyme for use according to claim 5, wherein the apyrase is a bacterial apyrase or a plant apyrase.

7. The ATP hydrolyzing enzyme for use according to any one of the previous claims, wherein the ATP hydrolyzing enzyme comprises an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence variant thereof having at least 70%, 80% or 90% sequence identity.

8. A nucleic acid comprising a polynucleotide encoding the ATP hydrolyzing enzyme as defined in any one of claims 4-7 for use in the treatment of dysbiosis or a dysbiosis-related disease.

9. The nucleic acid for use according to claim 1, 2 or 7, wherein the nucleic acid comprising the polynucleotide encoding the ATP hydrolyzing enzyme is a vector.

10. The nucleic acid for use according to any one of claims 1, 2, 7 or 8, wherein the nucleic acid further comprises heterologous elements for (heterologous) expression of the ATP hydrolyzing enzyme.

11. A host cell comprising the nucleic acid as defined in any one of claims 8-10 for use in the treatment of dysbiosis or a dysbiosis-related disease.

12. The host cell for use according to claim 1, 2 or 11, wherein the host cell is a prokaryotic or a eukaryotic cell.

13. The host cell for use according to claim 1, 2, 11 or 12, wherein the host cell is a recombinant host cell heterologously expressing the ATP hydrolyzing enzyme.

14. A microorganism comprising the nucleic acid as defined in any one of claims 8-10 for use in the treatment of dysbiosis or a dysbiosis-related disease.

15. The microorganism for use according to claim 1, 2 or 14, wherein the microorganism is selected from archaea, bacteria and eukaryotes.

16. The microorganism for use according to claim 1, 2, 14 or 15, wherein the microorganism is selected from the group consisting of Escherichia spp., Salmonella spp., Yersinia spp., Vibrio spp., Listeria spp., Lactococcus spp., Shigella spp., Cyanobacteria, and Saccharomyces spp.

17. The microorganism for use according to any one of claims 1, 2 and 14 to 16, wherein the microorganisms are provided as probiotics.

18. The microorganism for use according to any one of claims 1, 2 and 14 to 17, wherein the virulence of the microorganism is attenuated.

19. The microorganism for use according to any one of claims 1, 2 and 14 to 17, wherein the microorganism is a recombinant microorganism heterologously expressing the ATP hydrolyzing enzyme.

20. A (recombinant) bacterium comprising the nucleic acid as defined in any one of claims 8-10 for use in the treatment of dysbiosis or a dysbiosis-related disease.

21. The bacterium for use according to claim 1, 2 or 20, wherein the bacterium heterologously expresses the ATP hydrolyzing enzyme.

22. The bacterium for use according to any one of claims 1, 2, 20 or 21, wherein the bacterium is selected from Gram-positive bacteria, Gram-negative bacteria and Cyanobacteria.

23. The bacterium for use according to any one of claims 1, 2 and 20 to 22, wherein the bacterium is selected from the group consisting of Escherichia coli, Salmonella typhi, Salmonella typhimurium, Yersinia enterocolitica, Vibrio cholerae, Listeria monocytogenes, Lactococcus lactis and Shigella flexneri.

24. The bacterium for use according to claim 23, wherein the bacterium is E. coli of the strain Nissle 1917.

25. A viral particle comprising the nucleic acid as defined in any one of claims 8-10 for use in the treatment of dysbiosis or a dysbiosis-related disease.

26. The viral particle for use according to claim 1, 2 or 25, wherein the viral particle is a bacteriophage.

27. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to any one of the previous claims, wherein dysbiosis is induced by an antibiotic agent, a chemotherapeutic agent, a diet or by maternal dysbiosis.

28. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to any one of the previous claims, wherein the ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle is comprised in a composition.

29. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to claim 28, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent and/or excipient.

30. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to any one of the previous claims, wherein the ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle is administered via an enteral route of administration, preferably via oral administration.

31. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to any one of the previous claims, wherein the dysbiosis-related disease is selected from inflammatory diseases, gastrointestinal tract-related disorders, metabolic disorders, CNS-related disorders, cancers and autoimmune diseases.

32. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to any one of the previous claims, wherein the dysbiosis-related disease is selected from inflammatory bowel disease, irritable bowel syndrome, obesity, diabetes, metabolic syndrome, coeliac disease, colorectal cancer, Clostridioides difficile infection, autism spectrum disorder, urinary stone disease (USD), lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Sjögren's syndrome, anti-phospholipid syndrome, cardiovascular syndrome, allergy, and asthma.

33. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to any one of the previous claims, wherein the ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle is administered in combination with a dysbiosis-inducing agent.

34. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to claim 33, wherein the dysbiosis-inducing agent is an antibiotic; preferably selected from the group consisting of penicllins, tetracyclines, cephalosporins, quinolones, lincosamides, macrolides, sulfonamides, glycopeptides, aminoglycosides, carbapenems, ansamycins, carbacephems, lipopeptides, monobactams, nitrofurans, oxazolidinones, and polypeptides; more preferably the antibiotic is selected from the group consisting of vancomycin, ampicillin, metronidazole and cefoperazone.

35. The ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism, the bacterium, or the viral particle for use according to claim 33 or 34, wherein the dysbiosis-inducing agent is a chemotherapeutic agent; preferably selected from alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I or II, kinase inhibitors, nucleotide analogs and precursor analogs, platinum-based agents, retinoids, and vinca alkaloids and derivatives; for example the chemotherapeutic agent is 5-fluorouracil (5-FU).

36. A combination of (i) a bacterium comprising a nucleic acid comprising a polynucleotide encoding an ATP hydrolyzing enzyme; and (ii) a dysbiosis-inducing agent.

37. The combination of claim 36, wherein the encoded ATP-hydrolyzing enzyme is as defined in any one of claims 4 to 7.

38. The combination of claim 36 or 37, wherein the nucleic acid comprised in the bacterium is as defined in any one of claims 8-10.

39. The combination of any one of claims 36-38, wherein the bacterium is as defined in any one of claims 20-24.

40. The combination of any one of claims 36-39, wherein the dysbiosis-inducing agent is an antibiotic; preferably selected from the group consisting of penicllins, tetracyclines, cephalosporins, quinolones, lincosamides, macrolides, sulfonamides, glycopeptides, aminoglycosides, carbapenems, ansamycins, carbacephems, lipopeptides, monobactams, nitrofurans, oxazolidinones, and polypeptides; more preferably the antibiotic is selected from the group consisting of vancomycin, ampicillin, metronidazole and cefoperazone.

41. The combination of any one of claims 36-40, wherein the dysbiosis-inducing agent is a chemotherapeutic agent; preferably selected from alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I or II, kinase inhibitors, nucleotide analogs and precursor analogs, platinum-based agents, retinoids, and vinca alkaloids and derivatives; for example the chemotherapeutic agent is 5-fluorouracil (5-FU).

42. The combination of any one of claims 36-41, wherein the bacterium and/or the dysbiosis-inducing agent is/are comprised in a composition.

43. The combination of any one of claims 36-42 for use in medicine.

44. The combination of any one of claims 36-43 for use in the treatment of dysbiosis.

45. The combination for use of any one of claims 33-35, 43 and 44, wherein (i) the dysbiosis-inducing agent; and/or (ii) the ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism or the viral particle are administered repeatedly.

46. The combination for use of any one of claims 33-35 and 43-45, wherein (i) the dysbiosis-inducing agent; and (ii) the ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism or the viral particle are administered on the same day.

47. The combination for use of any one of claims 33-35 and 43-46, wherein the ATP hydrolyzing enzyme, the nucleic acid, the host cell, the microorganism or the viral particle is administered after administration of the dysbiosis-inducing agent.

48. A kit comprising: (i) a bacterium comprising a nucleic acid comprising a polynucleotide encoding an ATP hydrolyzing enzyme; and (ii) a dysbiosis-inducing agent.

49. The kit of claim 48, wherein the encoded ATP-hydrolyzing enzyme is as defined in any one of claims 4-7.

50. The kit of claim 48 or 49, wherein the nucleic acid comprised in the bacterium is as defined in any one of claims 8-10.

51. The kit of any one of claims 48-50, wherein the bacterium is as defined in any one of claims 20-24.

52. The kit of any one of claims 48-51, wherein the dysbiosis-inducing agent is an antibiotic; preferably selected from the group consisting of penicllins, tetracyclines, cephalosporins, quinolones, lincosamides, macrolides, sulfonamides, glycopeptides, aminoglycosides, carbapenems, ansamycins, carbacephems, lipopeptides, monobactams, nitrofurans, oxazolidinones, and polypeptides; more preferably the antibiotic is selected from the group consisting of vancomycin, ampicillin, metronidazole and cefoperazone.

53. The kit of any one of claims 48-52, wherein the dysbiosis-inducing agent is a chemotherapeutic agent; preferably selected from alkylating agents, anthracyclines, cytoskeletal disruptors, epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I or II, kinase inhibitors, nucleotide analogs and precursor analogs, platinum-based agents, retinoids, and vinca alkaloids and derivatives; for example the chemotherapeutic agent is 5-fluorouracil (5-FU).

54. The kit of any one of claims 48-53, wherein the bacterium and/or the dysbiosis-inducing agent is/are comprised in a composition.

55. The kit of any one of claims 48-54, wherein the kit further comprises a package insert or label with directions to treat dysbiosis or a dysbiosis-related disease by using a combination of (i) the dysbiosis-inducing agent and (ii) the bacterium comprising the nucleic acid comprising the polynucleotide encoding the ATP hydrolyzing enzyme.

56. The kit of any one of claims 48-55 for use in medicine, preferably in the treatment of dysbiosis.

57. A method for reducing the risk of occurrence, treating, ameliorating, or reducing dysbiosis or a dysbiosis-related disease in a subject in need thereof, comprising administering to the subject (a) an ATP hydrolyzing enzyme, (b) a nucleic acid comprising a polynucleotide encoding the ATP hydrolyzing enzyme, (c) a host cell comprising the nucleic acid, (d) a microorganism comprising the nucleic acid, or (e) a viral particle comprising the nucleic acid.

58. A method for restoring or improving the balance of intestinal microbiota in a subject in need thereof, comprising administering to the subject (a) an ATP hydrolyzing enzyme, (b) a nucleic acid comprising a polynucleotide encoding the ATP hydrolyzing enzyme, (c) a host cell comprising the nucleic acid, (d) a microorganism comprising the nucleic acid, or (e) a viral particle comprising the nucleic acid.

59. The method according to claim 58, wherein the microbiota balance is restored or improved during or after dysbiosis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0287] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0288] FIG. 1 shows a map of the pHND10 plasmid carrying the phoN2 gene encoding periplasmic ATP-diphosphohydrolase (apyrase).

[0289] FIG. 2 shows the amino acid sequence of wild-type phon2 protein (apyrase; SEQ ID NO: 1) and indicates the position of the R192P substitution in the loss-of-function isoform (SEQ ID NO: 2).

[0290] FIG. 3 shows the nucleotide sequence of the phoN2 gene (SEQ ID NO: 3) used for generating pHND10 plasmid.

[0291] FIG. 4 shows for Example 2 the treatment schedule in a mouse model of antibiotics-induced dysbiosis.

[0292] FIG. 5 shows for Example 2 the metagenomic analysis by 16S sequencing of caecal samples from mice treated as described herein. Shannon diversity index at the bacterial family level in caecal samples from control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pAPyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. **p<0.01.

[0293] FIG. 6 shows for Example 2 that treatment with bacteria expressing apyrase preserves beta-diversity after induction of dysbiosis. Principle coordinate analysis (PCoA) of bacterial beta-diversity based on an unweighted Unifrac dissimilarity matrix. PERMANOVA was used. p<0.001.

[0294] FIG. 7 shows for Example 2 that treatment with bacteria expressing apyrase promotes microbiome recovery from dysbiosis. The heatmap shows bacterial species in caecal microbiota that discriminate the experimental groups: not treated (control); ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr treated C57BL/6 mice. Species were selected according to p<0.05 with Wald test using FDR p-value correction following DESeq2 read counts normalization. Each line represents one species, and each column represents an individual mouse. Mean relative abundances (log 10) of species detected in not treated (control), ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr are shown.

[0295] FIG. 8 shows for Example 2 the p values related to the heatmap shown in FIG. 7, calculated with Wald test using FDR p-value correction following DESeq2 read counts normalization.

[0296] FIG. 9 shows for Example 3 the treatment schedule in a mouse model of Citrobacter rodentium infection after induction of dysbiosis.

[0297] FIG. 10 shows for Example 3 the percentage body weight variation in mice not treated (control), infected with C. rodentium or pretreated with E. coli.sup.pHND19 or E. coli.sup.pApyr and then infected with C. rodentium. Means±SEM are shown. Two-way ANOVA was used. **p<0.01, ***p<0.001.

[0298] FIG. 11 shows for Example 3 the statistical analysis of PMN cells (gated as CD45.sup.+Gr1.sup.+CD11b.sup.+) infiltrates in caecum lamina propria six days after infection, in mice not treated (control), infected with C. rodentium or pretreated with E. coli.sup.pHND19 or E. coli.sup.pApyr and then infected with C. rodentium. Accordingly, PMN cells infiltration in the caecum lamina propria of mice treated with E. coli.sup.pApyr and infected with C. rodentium is reduced. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used *p<0.05.

[0299] FIG. 12 shows for Example 3 the statistical analysis of inflammatory monocytes (gated as CD45.sup.+CD11b.sup.+Ly6c.sup.+Ly6g.sup.−) infiltrates in caecum lamina propria 6 days after infection, in mice not treated (control), infected with C. rodentium or pretreated with E. coli.sup.pHND19 or E. coli.sup.pApyr and then infected with C. rodentium. Accordingly, monocytes infiltration in the caecum lamina propria of mice treated with E. coli.sup.pApyr and infected with C. rodentium is reduced. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used *p<0.05, **p<0.01.

[0300] FIG. 13 shows for Example 4 the treatment schedule in a mouse model of Clostridioides difficile infection after induction of dysbiosis. Dysbiosis was induced by daily oral gavage of ABX for 4 consecutive days. After the antibiotic treatment, during the recovery phase, mice were orally gavaged for 4 days with PBS (control) or 10.sup.10 CFU of E. coli.sup.pHND19 or E. coli.sup.PApyr. At day 4, mice were orally infected with 10.sup.5 of C. difficile VPI 10463 spores

[0301] FIG. 14 shows for Example 4 the percentage body weight loss in mice not treated (control), infected with C. difficile or pretreated with E. coli.sup.pHND19 or E. coli.sup.pApyr and then infected with C. difficile. Accordingly, E. coli.sup.pApyr treatment attenuates body weight loss induced by C. difficile intestinal infection. Means±SEM are shown. Two-way ANOVA was used. *p<0.05, ***p<0.001.

[0302] FIG. 15 shows for Example 4 the clinical score variations in mice not treated (control), infected with C. difficile or pretreated with E. coli.sup.pHND19 or E. coli.sup.pApyr and then infected with C. difficile. Accordingly, E. coli.sup.pApyr treatment ameliorates the clinical score in mice infected with C. difficile. Means±SEM are shown. Two-way ANOVA was used. ***p<0.001

[0303] FIG. 16 shows for Example 4 the percent survival of mice not treated (control), infected with C. difficile or pretreated with E. coli.sup.pHND19 or E. coli.sup.pAPyr and then infected with C. difficile. Accordingly, E. coli.sup.pApyr treatment improves the survival of mice infected with C. difficile. Log-rank (Mantel-Cox) test was used. *p<0.05, **p<0.01.

[0304] FIG. 17 shows for Example 4 the colon length in mice not treated (control), infected with C. difficile or pretreated with E. coli.sup.pHND19 or E. coli.sup.pApyr and then infected with C. difficile. Accordingly, E. coli.sup.pApyr treatment attenuates colitis induced by C. difficile infection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0305] FIG. 18 shows for Example 4 fecal Lipocalin-2 levels 72 h post infection in mice not treated (control), infected with C. difficile or pretreated with E. coli.sup.pHND19 or E. coli.sup.pAPyr and then infected with C. difficile. Accordingly, E. coli.sup.pApyr treatment attenuates intestinal inflammation induced by C. difficile infection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0306] FIG. 19 shows for Example 4 the serum Lipocalin-2 levels 72 h post infection in mice not treated (control), infected with C. difficile or pretreated with E. coli.sup.pHND19 or E. Coli.sup.pApyr and then infected with C. difficile. Accordingly, E. coli.sup.pApyr treatment attenuates the systemic inflammation induced by C. difficile infection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0307] FIG. 20 shows for Example 5 the treatment schedule in a mouse model of Clostridioides difficile infection after induction of dysbiosis with cefoperazone. Dysbiosis was induced by oral gavage of cefoperazone (2.5 mg/mouse) in the evening for 5 days. 10.sup.10 CFU of E. coli.sup.pHND19 or E. coli.sup.pApyr were orally gavaged in the morning concomitantly to cefoperazone treatment. At day 6 the cefoperazone treatment was stopped and E. coli.sup.pHND19 or E. coli.sup.pApyr gavaging was protracted for 3 additional days. Mice were then orally infected with 10.sup.5 C. difficile VPI 10463 spores. Mice were analysed 72 h post infection in order to evaluate intestinal inflammation.

[0308] FIG. 21 shows for Example 5 the survival rates of mice in the dysbiosis/Clostridioides difficile infection challenge model shown in FIG. 20. Prior to infection, mice were daily gavaged with 2.5 mg/mouse of cefoperazone in the evening and 10.sup.10 CFU of E. coli.sup.pHND19 or E. coli.sup.pApyr in the morning. At day 6 the cefoperazone treatment was stopped and only bacterial treatment was performed for additional three consecutive days. Mice were then orally infected with 10.sup.5 C. difficile VPI 10463 spores. Percent survival of mice not treated (control), infected with C. difficile or pretreated with E. coli.sup.pHND19 or E. coli.sup.pApyr and then infected with C. difficile. The figure shows that E. coli.sup.pApyr treatment improves the survival in mice infected with C. difficile. Log-rank (Mantel-Cox) test was used. *p<0.05, ***p<0.001.

[0309] FIG. 22 shows for Example 5 the clinical score at 24 h post infection in mice not treated (control), infected with C. difficile or pretreated with E. coli.sup.pHND19 or E. coli.sup.pAPyr and then infected with C. difficile. The data show that E. coli.sup.pApyr treatment ameliorates the clinical score in mice infected with C. difficile. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. Two-tailed Mann-Whitney U-test * p<0.05, ** p<0.01, **** p<0.0001.

[0310] FIG. 23 shows for Example 6 the schedule for monocolonization of C57BL/6 GF mice with E. coli.sup.pBAD28 or E. coli.sup.pApyr. Germ free mice were orally gavaged with 5×10.sup.9 CFU/mouse of E. coli.sup.pBAD28 or E. coli.sup.pApyr and 28 days later small intestine epithelial cells were purified in order to perform transcriptomic analysis.

[0311] FIG. 24 shows for Example 6 the gene transcription in intestinal epithelial cells of monocolonized animals. The Volcano plot shows for each gene (dot) the differential expression [log.sub.2 fold-change (log.sub.2FC)] and its associated statistical significance (log.sub.10 p-value) in gnotobiotic E. coli.sup.pApyr vs E. coli.sup.pBAD28 WT mice. The dark gray dots indicate those genes with an FDR-corrected p-value<0.05 and |log.sub.2FC|>1. The 79 down- and 53 up-regulated genes (FDR-corrected p-value<10.sup.−5 and |log.sub.2FC|>1.5) are also highlighted by the two rectangles.

[0312] FIG. 25 shows for Example 6 the relative expression level (Z-score) of the differentially expressed genes by Gene Ontology (GO) analysis (FDR-corrected p-value<0.05 and log.sub.2FC>1) in E. coli.sup.pApyr vs E. coli.sup.pBAD28 monocolonized mice. z-score was calculated as the number of genes upregulated minus the number of genes downregulated divided for the square root of the total number of genes analyzed;

[00001]$z=\frac{\left(n\mathrm{up}-n\mathrm{down}\right)}{\sqrt{n\mathrm{tot}}}.$

[0313] FIG. 26 shows for Example 6 Gene Ontology analysis of the differentially expressed genes (FDR-corrected p-value<0.05 and log.sub.2FC>1) in E. coli.sup.pApyr vs E. coli.sup.pBAD28 intestinal epithelial cells.

[0314] FIG. 27 shows for Example 7 blood glucose variation after 4 days of antibiotic treatment and 4 days of recovery (see diagram in FIG. 4) in control (PBS treated), antibiotic (ABX) treated, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pAPyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0315] FIG. 28 shows for Example 8 the experimental schedule in a mouse model of cefoperazone mediated dysbiosis and recovery protocol. Dysbiosis was induced by oral gavage of cefoperazone (2.5 mg/mouse) in the evening for 5 days. Concomitantly, 10.sup.10 CFU of E. coli.sup.pHND19 or E. coli.sup.pApyr were orally gavaged in the morning. E. coli.sup.pHND19 and E. coli.sup.pApyr treatments were protracted after cefoperazone treatment as indicated.

[0316] FIG. 29 shows for Example 8 the body weight variation at the end of the experiment in control (PBS treated), cefoperazone treated, cefoperazone+E. coli.sup.pHND19 and cefoperazone+E. coli.sup.pApyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0317] FIG. 30 shows for Example 8 Percentage of white adipose tissue deposition normalized for the mouse body weight, in control (PBS treated), cefoperazone treated, cefoperazone+E. coli.sup.pHND19 and cefoperazone+E. coli.sup.pApyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0318] FIG. 31 shows for Example 9 the experimental schedule in a mouse model of antibiotics-induced dysbiosis. Except for control mice, dysbiosis was induced by daily oral gavage of ABX for 4 consecutive days. On the same days as the antibiotic treatment, mice were orally gavaged with PBS or 40 μg of purified recombinant apyrase every 12 h.

[0319] FIG. 32 shows for Example 9 blood glucose variation in control (PBS treated), antibiotic (ABX) treated, ABX+apyrase treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. **p<0.01.

[0320] FIG. 33 shows for Example 9 percent WAT deposition in control (PBS treated), antibiotic (ABX) treated, ABX+apyrase treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. **p<0.01.

[0321] FIG. 34 shows for Example 6 the gene transcription in intestinal epithelial cells of monocolonized Igh-J.sup.−/− animals. The Volcano plot shows for each gene (dot) the differential expression [log 2 fold-change (log 2FC)] and its associated statistical significance (logic p-value) in gnotobiotic E. coli.sup.pApyr vs E. coli.sup.pBAD28 Igh-J.sup.−/− mice. The two quadrants delineate the regions corresponding to FDR-corrected p value<10-5 and |log.sub.2FC|>1.5 used to highlight most prominently regulated genes in the same experiment performed with WT mice shown in FIG. 24.

[0322] FIG. 35 shows for Example 6 body weight variation in wild-type C57BL/6 mice monocolonized with E. coli.sup.pApyr or E. coli.sup.pBAD28. Means±SEM are shown. Two-way ANOVA test was used. **p<0.01.

[0323] FIG. 36 shows for Example 6 fasting blood glucose measured in wild-type C57BL/6 GF mice or monocolonized with E. coli.sup.pApyr or E. coli.sup.pBAD28. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. **p<0.01, ***p<0.001.

[0324] FIG. 37 shows for Example 6 serum insulin quantification in wild-type C57BL/6 GF mice or monocolonized with E. coli.sup.pApyr or E. coli.sup.pBAD28. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05.

[0325] FIG. 38 shows for Example 6 the quantification of white adipose tissue (WAT) deposition in wild-type C57BL/6 GF mice or monocolonized with E. coli.sup.PAPYr or E. coli.sup.pBAD28. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05; **p<0.01.

[0326] FIG. 39 shows for Example 6 glucose homeostasis determined by glucose tolerance test (GTT) in wild-type C57BL/6 GF mice or monocolonized with E. coli.sup.pApyr or E. coli.sup.pBAD28. Means±SEM are shown. Two-way ANOVA was used. *p<0.05, **p<0.01, **** p<0.0001.

[0327] FIG. 40 shows for Example 6 body weight variation in C57Bl/6 Igh-J.sup.−/− mice monocolonized with E. coli.sup.pApyr or E. coli.sup.pBAD28. Means±SEM are shown. Two-way ANOVA test did not reveal any statistically significant difference between the 2 groups.

[0328] FIG. 41 shows for Example 6 fasting blood glucose measured in C57Bl/6 Igh-J.sup.−/− mice monocolonized with E. coli.sup.pApyr or E. coli.sup.pBAD28. Means±SEM are shown. Two-tailed Mann-Whitney U-test did not reveal any statistically significant difference between the 2 groups.

[0329] FIG. 42 shows for Example 6 glucose homeostasis determined by glucose tolerance test (GTT) in C57Bl/6 Igh-J.sup.−/− mice monocolonized with E. coli.sup.pApyr or E. coli.sup.pBAD28. Means±SEM are shown. Two-way ANOVA test did not reveal any statistically significant difference between the 2 groups.

[0330] FIG. 43 shows for Example 7 WAT deposition normalized by total body weight in control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0331] FIG. 44 shows for Example 7 blood glucose variation after 4 days of antibiotic treatment and 4 days of recovery in control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr C57BL/6 Igh-J.sup.−/− mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05 FIG. 45 shows for Example 7 WAT deposition normalized by total body weight in control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. Coli.sup.pApyr C57BL/6 Igh-J.sup.−/− mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. **p<0.01.

[0332] FIG. 46 shows for Example 10 caecum weight normalized by total body weight in control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr treated C57BL/6 wild-type mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0333] FIG. 47 shows for Example 10 the colony forming units (CFU) of aerobic bacteria recovered from the MLN of control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr treated C57BL/6 wild-type mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0334] FIG. 48 shows for Example 10 the CFU of anaerobic bacteria recovered from the MLN of control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr treated C57BL/6 wild-type mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0335] FIG. 49 shows for Example 10 caecum weight normalized by total body weight in control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr treated C57BL/6 Igh-J.sup.−/− mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. **p<0.01.

[0336] FIG. 50 shows for Example 10 the CFU of aerobic bacteria recovered from the MLN of control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr treated C57Bl/6 Igh-J.sup.−/− mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. **p<0.01.

[0337] FIG. 51 shows for Example 10 the CFU of anaerobic bacteria recovered from the MLN of control, ABX, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pApyr treated C57Bl/6 Igh-J.sup.−/− mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0338] FIG. 52 shows for Example 11 the DNA fragment insertion for the integration of S. flexneri phoN2 gene in EcN genome. malP: EcN gene for maltodextrin phosphorylase; cat: E. coli gene for chloramphenicol acetyltransferase; phoN2: S. flexneri gene for apyrase; malT: EcN gene for the transcriptional activator of the maltose and maltodextrins operon; FRT: Flippase Recognition Target sequence; P.sub.cot: promoter of the cat gene; P.sub.proD: promoter of the phoN2 gene; BBa_BB0032 RBS: Ribosome Binding Site of the phoN2 gene; T.sub.phoN2: transcriptional terminator of the phoN2 gene.

[0339] FIG. 53 shows for Example 11 the nucleotide sequence of the EcN malP gene portion (SEQ ID NO: 6). The malP stop codon is indicated in bold.

[0340] FIG. 54 shows for Example 11 the nucleotide sequence of the EcN malTgene portion (SEQ ID NO: 7). The malT start codon is indicated in bold.

[0341] FIG. 55 shows for Example 11 the nucleotide sequence (SEQ ID NO: 8) of the DNA fragment including the P.sub.proD promoter, the BBa_BB0032 RBS, the S. flexneri phoN2 gene and the phoN2 transcriptional terminator. The P.sub.proD sequence is underlined. The BBa_BB0032 RBS is shown in italics. The phoN2 start and stop codons are indicated in bold. The phoN2 transcriptional terminator is shown in bold italics.

[0342] FIG. 56 shows for Example 11 the nucleotide sequence of the DNA fragment including the E. coli cat gene flanked by the FRT sequences (SEQ ID NO: 9). The cat start and stop codons are indicated in bold. The FRT sequences are shown in italics.

[0343] FIG. 57 shows for Example 11 the malP-phoN2-malT recombinant genomic region of EcN::phoN2. malP: EcN gene for maltodextrin phosphorylase; phoN2: S. flexneri gene for apyrase; malT: EcN gene for the transcriptional activator of the maltose and maltodextrins operon; FRT: Flippase Recognition Target sequence; P.sub.proD: promoter of the phoN2 gene; BBa_BB0032 RBS: Ribosome Binding Site of the phoN2 gene; T.sub.phoN2: transcriptional terminator of the phoN2 gene.

[0344] FIG. 58 shows for Example 11 Apyrase detection in recombinant E. coli Nissle (EcN) EcN::phoN2 periplasmic extracts compared to non-recombinant E. coli Nissle (EcN) extracts. EcN and EcN::phoN2 clone 1 (cl 1) bacterial cultures were grown for 2.5 h, in LB medium, at 37° C. and harvested by centrifugation. The periplasmic fraction of each culture was isolated, precipitated with trichloroacetic acid (TCA), solubilized in Laemmli buffer and analyzed by Western blot using a polyclonal anti-apyrase rabbit serum.

[0345] FIG. 59 shows for Example 11 the dose-dependent degradation of ATP by EcN::phoN2 periplasmic extract. EcN and EcN::phoN2 clone 1 (cl 1) bacterial cultures were grown for 6 h, in LB medium, at 37° C. and harvested by centrifugation. The periplasmic fraction of each culture was isolated, dialyzed against PBS 1× and serially diluted with PBS 1×. The apyrase activity in periplasmic extracts (PE) was measured as percentage of degradation of 50 μM ATP relative to PBS 1×. Apyrase activity in PE was evaluated by an ATP-dependent bioluminescence assay with recombinant firefly luciferase and its substrate D-luciferin according to the manufacturer's protocol (Life Technologies Europe B.V.).

[0346] FIG. 60 shows for Example 12 the experimental layout showing the model of antibiotics-induced dysbiosis. 8-week old C57BL/6 male mice were randomly assigned to 4 different experimental groups: not treated (control), treated with antibiotics (ABX: Vancomycin 1.25 mg, ampicillin 2.5 mg and metronidazole 1.25 mg in 200 μl sterile water per mouse), treated with ABX and 10.sup.10 CFU of EcN and treated with ABX and 10.sup.10 CFU of EcN::phoN2.

[0347] FIG. 61 shows for Example 12 blood glucose variation after 4 days of antibiotic treatment and 4 days of recovery (see diagram in FIG. 60) in control, ABX, ABX+EcN or EcN::phoN2 treated C57BL/6 male mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0348] FIG. 62 shows for Example 12 WAT deposition normalized by total body weight in control, ABX, ABX+EcN or EcN::phoN2 treated C57BL/6 male mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01, ***p<0.001.

[0349] FIG. 63 shows for Example 13 the caecum weight normalized by total body weight in control, ABX, ABX+EcN or EcN::phoN2 treated C57BL/6 male mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0350] FIG. 64 shows for Example 13 CFU of aerobic bacteria recovered from the MLN of control, ABX, ABX+EcN or EcN::phoN2 treated C57BL/6 male mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0351] FIG. 65 shows for Example 13 CFU anaerobic bacteria recovered from the MLN of control, ABX, ABX+EcN or EcN::phoN2 treated C57BL/6 male mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01, ***p<0.001.

[0352] FIG. 66 shows for Example 14 the map of the pNZ-Apyr plasmid carrying the phoN2 gene encoding apyrase used to transform Lactococcus lactis. P.sub.nisA, nisin A inducible promoter; SP usp45: signal sequence of usp45 gene; phoN2: S. flexneri apyrase gene; repC: replication gene C; repA: replication gene A; camR (cat): chloramphenicol resistance gene.

[0353] FIG. 67 shows for Example 15 a schematic of the components of each diet, expressed as percentages of total calories: normal diet (ND: 20% protein and 15% fat) and a modified diet able to induce dysbiosis (DID: 7% protein and 5% fat).

[0354] FIG. 68 shows for Example 15 the experimental layout for DID in 5 weeks old mice. At 5 weeks of age, female C67BL/6 mice were randomized into receiving either a normal diet (ND: 20% protein and 15% fat) or a modified diet able to induce dysbiosis (DID: 7% protein and 5% fat). During this period, DID mice were orally gavaged every day with PBS or 10.sup.10 of L. lactis.sup.pNZ or L. lactis.sup.pNZ-Apyr. After 8 weeks, mice were sacrificed and analyzed in order to evaluate apyrase effects on DID.

[0355] FIG. 69 shows for Example 15 the concentration of FITC in the serum assessed 4 h post dextran-FITC oral administration, after mice were fed the indicated diet for 8 weeks and daily gavaged with PBS or 10.sup.10 of L. lactis.sup.pNZ or L. lactis.sup.pApyr as indicated. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0356] FIG. 70 shows for Example 15 CFU of aerobic bacteria recovered from the MLN of ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr treated adult C57BL/6 mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05.

[0357] FIG. 71 shows for Example 15 CFU of anaerobic bacteria recovered from the MLN of ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr treated adult C57BL/6 mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05.

[0358] FIG. 72 shows for Example 15 fecal LCN-2 concentration measured after mice were fed the indicated diet for 8 weeks and daily gavaged with PBS or 10.sup.10 of L. lactis.sup.pNZ or L. lactis.sup.pApyr. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. * p<0.05.

[0359] FIG. 73 shows for Example 16 the experimental layout for the neonatal model of DID. At eight weeks of age, female C57BL/6 mice were randomized into receiving either a normal diet (ND: 20% protein and 15% fat) or a modified diet able to induce dysbiosis (DID: 7% protein and 5% fat). After 15 days, ND and DID mice were mated with male mice. Starting immediately after birth, DID pups were orally gavaged with PBS or 10.sup.8 of L. lactis.sup.pNZ or L. lactis.sup.pNZ-Apyr two times a week until 21 days after birth. Pups were daily monitored for body weight, tail length and behavior.

[0360] FIG. 74 shows for Example 16 the concentration of FITC in the serum assessed 4 h post dextran-FITC oral administration in ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr mice at 21 days after birth. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0361] FIG. 75 shows for Example 16 CFU of aerobic bacteria recovered from the MLN of ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr treated 21 days old C57BL/6 mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01.

[0362] FIG. 76 shows for Example 16 CFU of anaerobic bacteria recovered from the MLN of ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr treated 21 days old C57BL/6 mice. Dashed line indicates lower limit of detection. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05.

[0363] FIG. 77 shows for Example 17 body weight variation assessed at 21 days after birth in ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. **p<0.01, ****p<0.0001.

[0364] FIG. 78 shows for Example 17 tail length measured at 21 days after birth in ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, ****p<0.0001.

[0365] FIG. 79 shows for Example 17 small intestine length measured at 21 days after birth in ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, ***p<0.001, ****p<0.0001.

[0366] FIG. 80 shows for Example 17 colon length measured at 21 days after birth in ND, DID, DID+L. lactis.sup.pNZ and DID+L. lactis.sup.pNZ-Apyr treated C57BL/6 mice. Means±SEM are shown. Two-tailed Mann-Whitney U-test was used. *p<0.05, **p<0.01, ****p<0.0001.

EXAMPLES

[0367] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Design and Production of Apyrase-Expressing Bacteria

[0368] To obtain bacteria expressing apyrase, full length phoN2::HA fusion, encoding periplasmic ATP-diphosphohydrolase (apyrase) of Shigella flexneri (SEQ ID NO: 1) with a hemagglutinin (HA) fragment as tag were cloned into the polylinker site of plasmid pBAD28 (ATCC 8739387402), under the control of the PBAD L-arabinose inducible promoter. Thereby, plasmid pHND10 was generated, essentially as described in Santapaola, D., Del Chierico, F., Petrucca, A., Uzzau, S., Casalino, M., Colonna, B., Sessa, R., Berlutti, F., and Nicoletti, M. (2006). Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene, is necessary for proper unipolar IcsA localization and for efficient intercellular spread. Journal of bacteriology 188, p. 1620-1627.

[0369] As control, plasmid pHND19 was produced essentially as described in Scribano, D., Petrucca, A., Pompili, M., Ambrosi, C., Bruni, E., Zagaglia, C., Prosseda, G., Nencioni, L., Casalino, M., Polticelli, F., et al. (2014). Polar localization of PhoN2, a periplasmic virulence-associated factor of Shigella flexneri, is required for proper IcsA exposition at the old bacterial pole. PloS one 9, e90230. In contrast to the pHND10 plasmid, the pHND19 plasmid (control) contains a phoN2.sub.R192P::HA fusion, which encodes a loss-of-function isoform of apyrase carrying the R192P substitution.

[0370] FIG. 1 shows a map of the pHND10 plasmid carrying the phoN2 gene encoding periplasmic ATP-diphosphohydrolase (apyrase). This map applies in general also to the pHND19 control plasmid, with the only difference that the loss-of-function isoform of apyrase carrying the R192P substitution is encoded instead of wild-type apyrase. FIG. 2 shows the amino acid sequence of wild-type phon2 protein (apyrase; SEQ ID NO: 1) and indicates the position of the R192P substitution in the loss-of-function isoform (SEQ ID NO: 2). The nucleotide sequence of the phoN2 gene used for generating pHND10 plasmid is shown in FIG. 3 (SEQ ID NO: 3).

[0371] Escherichia coli DH10B were transformed with pHND10 (E. coli.sup.pApyr) or pHND19.sub.R192P (E. coli.sup.pHND19) and grown in LB medium supplemented with L-arabinose (0.03%) and ampicillin (100 μg/ml).

Example 2: Administration of Bacteria Expressing Apyrase Reduces Dysbiosis-Induced Decreases in Microbiota Diversity

[0372] In order to investigate a possible beneficial effect of apyrase in the recovery from dysbiosis, a mouse model of induced dysbiosis was used. Dysbiosis was induced by daily oral gavage of a mix of antibiotics (ABX: Vancomycin 1.25 mg, ampicillin 2.5 mg and metronidazole 1.25 mg) for 4 days. After the antibiotic treatment, during the recovery phase, mice were orally gavaged for 4 days with PBS (control) or 10.sup.10 CFU (colony forming unit) of E. coli.sup.pApyr or E. coli expressing the loss-of-function isoform of apyrase with the R192P amino acid substitution as described in Example 1 (E. coli.sup.pHND19)

[0373] The treatment schedule is shown in FIG. 4. 8-week old C57BL/6 mice were randomly assigned to 4 different experimental groups: not treated (control), treated with antibiotics (ABX: Vancomycin 1.25 mg, ampicillin 2.5 mg and metronidazole 1.25 mg; in 200 μl sterile water per mouse), treated with ABX and 10.sup.10 CFU of E. coli.sup.pHND19; and treated with ABX and 10.sup.10 CFU of E. coli.sup.pApyr. At the end of the experiment, mice were sacrificed by CO.sub.2 inhalation and caecal samples were collected.

[0374] Extraction, lysis and DNA isolation was done by using the Fast DNA Stool Mini Kit (Qiagen) according to manufacturer's recommendation. Bead beating was run on a fastprep24 instrument (MPBiomedicals; 4 cycles of 45 s at speed 4 followed) in 2 ml screwcap tubes containing 0.6 g 0.1 mm glass beads. 200 μl of raw extract was prepared for DNA-isolation. Concentration of the isolated DNA was assessed with PicoGreen measurement (Quant-iTT PicoGreenT dsDNA Assay Kit, Thermo Fisher) and integrity was checked by agarose gel electrophoresis for a random sample.

[0375] For the amplification of the bacterial 16S rRNA gene, a primer set specific for the V3-V4 hypervariable regions was used (Fw: 5′-CCT ACG GGN GGC WGC AG-3′ (SEQ ID NO: 4); and Rev: 5′-GAC TAC HVG GGT ATC TAA TCC-3′ (SEQ ID NO: 5)). Subsequently, the Illumina MiSeq platform and a v2 500 cycles kit were used to sequence the PCR libraries. The produced paired-end reads, which passed Illumina's chastity filter, were subjected to de-multiplexing and trimming of Illumina adaptor residuals using Illumina's real time analysis software included in the MiSeq reporter software v2.6 (no further refinement or selection). The quality of the reads was checked with the software FastQC version 0.11.8. The sequences were analyzed through the Qiime2 virtual environment (Bolyen E, Rideout J R, Dillon M R, et al. 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnology 37: 852-857. doi.org/10.1038/s41587-019-0209-9). The raw sequences were in total 4′896′770 (median=71942, mean=72′011.3, SD=15′891.2). The trimming step on the first 7 and the last 25 bases and the reads filtration have allowed to obtain excellent quality sequences (Phred>30). A denoising algorithm (DADA2 algorithm; Callahan B J, McMurdie P J, Rosen M J, Han A W, Johnson A J, Holmes S P. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016; 13(7):581-583. doi:10.1038/nmeth.3869) was performed on these high quality sequences. The overlapping regions R1 and R2 were joined to obtain the non-chimeric reads used in the project. These were in total 1′145′671 (median=16′277, mean=16′848.1, SD=3′897.6).

[0376] The taxonomy assignment was performed by BLAST feature-classifier. It performs BLAST+ local alignment between query and reference reads. Then, it assigns consensus taxonomy to each query sequence on the last database version of Greengene (gg_12_10).

[0377] A rooted tree was constructed based on IQ-TREE stochastic algorithm that allows maximum likelihood analysis of large phylogenetic data (Nguyen L T, Schmidt H A, von Haeseler A, Minh B Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015; 32(1):268-274. doi:10.1093/molbev/msu300).

[0378] Alpha diversity (Shannon-index; within-sample richness) was calculated using the main indexes to allow an exploration of data in term of richness and evenness. Alpha-diversity estimates were computed using the phyloseq R package (McMurdie P J, Holmes S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013; 8(4):e61217. Published 2013 Apr. 22. doi:10.1371/journal.pone.0061217). Statistically significant changes in the alpha diversity were determined through the Mann-Whitney signed-rank test.

[0379] Results are shown in FIG. 5. The metagenomic analysis revealed a strong reduction of alpha diversity, expressed as Shannon index, in control mice treated with ABX and ABX+E. coli.sup.pHND19, indicating strong dysbiosis. However, treatment with E. coli.sup.pApyr after dysbiosis induction resulted in a significant improvement of this parameter.

[0380] In order to determine similarity in bacterial composition in the different experimental groups, beta-diversity (between-sample dissimilarity) was analyzed in a principle coordinate analysis (PCoA) using a dissimilarity table obtained by Unweighted Unifrac algorithms (Lozupone C, Knight R. UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol. 2005; 71(12):8228-8235. doi:10.1128/AEM.71.12.8228-8235.2005). Beta-diversity estimates were computed using the phyloseq R package (McMurdie P J, Holmes S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013; 8(4):e61217. Published 2013 Apr. 22. doi:10.1371/journal.pone.0061217). Permutational MANOVA (PERMANOVA) was performed on the unweighted UniFrac distance using the adonis( ) function of the vegan R package with 999 permutations.

[0381] Results are shown in FIG. 6. Despite each antibiotic treatment group clustered separately from the untreated control (PERMANOVA<0.001), E. coli.sup.pAPyr treated mice clustered closer to the control group, indicating an improved recovery of the physiological microbiota composition.

[0382] Differences in microbiota composition between all the populations were determined using Wald test using FDR p-value correction following DESeq2 read counts normalization (counts divided by sample-specific size factors determined by median ratio of gene counts relative to geometric mean per gene; Anders, S., Huber, W. Differential expression analysis for sequence count data. Genome Biol 11, R106 (2010). doi.org/10.1186/gb-2010-11-10-r106). Microbiota species were selected according to p<0.05 with Wald test using FDR p-value correction following DESeq2 read counts normalization. FIG. 7 shows a heatmap of differentially represented amplicon sequence variants (ASVs) that discriminate the caecal microbiota of the distinct experimental groups: control, ABX treated, ABX+E. coli.sup.pHND19 and ABX+E. coli.sup.pAPyr treated C57BL/6 mice. FIG. 8 shows the p values of differentially represented ASVs, calculated with Wald test using FDR p-value correction following DESeq2 read counts normalization.

[0383] These results reveal that administration of apyrase expressing bacteria resulted in an improved microbial community structure with the selective preservation of 41 species belonging to the families of Bacteroidales, Clostridiales, Lactobacillales and Burkholderiales. Among Bacteroidales, Muribaculum intestinale was detected by multiple ASVs. The reduction of this bacterial species was shown to correlate with higher susceptibility to ileitis (Dobranowski, P. A., Tang, C., Sauve, J. P., Menzies, S. C., and Sly, L. M. (2019). Compositional changes to the ileal microbiome precede the onset of spontaneous ileitis in SHIP deficient mice. Gut Microbes 10, 578-598). E. coli.sup.pApyr administration favored the preservation of Clostridium scindens, a bacterium that was shown to protect from C. difficile infection through the generation of secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA). Reconstitution with C. scindens alone or within a bacterial consortium protected antibiotic treated mice from C. difficile intestinal colonization (Buffie, C. G., Bucci, V., Stein, R. R., McKenney, P. T., Ling, L., Gobourne, A., No, D., Liu, H., Kinnebrew, M., Viale, A., et al. (2015). Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517, 205-208). Different species belonging to Lactobacillales family were also significantly enriched in E. coli.sup.pApyr treated mice. In particular, Lactobacillus johnsonii and Lactobacillus reuteri were significantly enriched. These two strains are commonly used as probiotics and were shown to confer protection against Citrobacter rodentium (Mackos, A. R., Eubank, T. D., Parry, N. M., and Bailey, M. T. (2013). Probiotic Lactobacillus reuteri attenuates the stressor-enhanced severity of Citrobacter rodentium infection. Infect Immun 81, 3253-3263) and Campylobacter jejuni (Bereswill, S., Ekmekciu, I., Escher, U., Fiebiger, U., Stingl, K., and Heimesaat, M. M. (2017). Lactobacillus johnsonii ameliorates intestinal, extra-intestinal and systemic pro-inflammatory immune responses following murine Campylobacter jejuni infection. Sci Rep 7, 2138) infection.

Example 3: Administration of Bacteria Expressing Apyrase Reduces Effects of C. rodentium Infection after Induction of Dysbiosis

[0384] The gastrointestinal tract of mammals is colonized by hundreds of microbial species that confer colonization resistance against intestinal pathogens. Dysbiosis results in the loss of colonization resistance and susceptibility to enteric infections. Enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC) and Citrobacter rodentium are Enterobacteriaceae that belong to the family of attaching and effacing (A/E) lesion-forming bacteria. EHEC and EPEC can cause severe intestinal inflammation and diarrhea. In addition, EHEC strains expressing the highly potent Shiga toxin (Stx) cause nephrotoxicity resulting in severe cases in the death of infected individuals (Collins, J. W., Keeney, K. M., Crepin, V. F., Rathinam, V. A., Fitzgerald, K. A., Finlay, B. B., and Frankel, G. (2014). Citrobacter rodentium: infection, inflammation and the microbiota. Nat Rev Microbiol 12, 612-623). Since human EHEC and EPEC only induce modest pathogenicity in antibiotic treated adult mice, C. rodentium is frequently used to mimic these infections in mice (Collins, J. W., Keeney, K. M., Crepin, V. F., Rathinam, V. A., Fitzgerald, K. A., Finlay, B. B., and Frankel, G. (2014). Citrobacter rodentium: infection, inflammation and the microbiota. Nat Rev Microbiol 12, 612-623; Bhinder, G., Sham, H. P., Chan, J. M., Morampudi, V., Jacobson, K., and Valiance, B. A. (2013). The Citrobacter rodentium mouse model: studying pathogen and host contributions to infectious colitis. J Vis Exp, e50222; Mallick, E. M., McBee, M. E., Vanguri, V. K., Melton-Celsa, A. R., Schlieper, K., Karalius, B. J., O'Brien, A. D., Butterton, J. R., Leong, J. M., and Schauer, D. B. (2012). A novel murine infection model for Shiga toxin-producing Escherichia coli. J Clin Invest 122, 4012-4024).

[0385] To investigate if the microbiota community structure induced by apyrase expressing bacteria could confer protection from C. rodentium infection, ABX was administered to C57BL/6 mice for 4 days, as described in Example 2. The treatment schedule is shown in FIG. 9. 8-week old C57BL/6 mice were randomly assigned to 4 different experimental groups: not treated (control), treated with antibiotics (ABX: Vancomycin 1.25 mg, ampicillin 2.5 mg and metronidazole 1.25 mg; in 200 μl sterile water per mouse), treated with ABX and 10.sup.10 CFU of E. coli.sup.pHND19; and treated with ABX and 10.sup.10 CFU of E. coli.sup.pApyr. After the antibiotic treatment, mice were orally gavaged for 4 days with PBS (control); or 10.sup.10 CFU of E. coli.sup.pHND19; or E. coli.sup.pApyr, similarly as in Example 2.

[0386] Thereafter, mice were orally infected with 10.sup.8 CFU/mouse of Citrobacter rodentium (except for the untreated control group). For infection experiments, Citrobacter rodentium ATCC®51459 (DBS100 strain) was cultured on LB agar plates and then expanded in Luria broth overnight at 37° C.

[0387] On days 0, 1, 2, 3, 4 and 5 post infection, the body weight of the animals was assessed. Results are shown in FIG. 10. Analysis of the percentage of body weight loss following C. rodentium infection revealed reduced body weight loss in mice orally gavaged with E. coli.sup.pAPyr as compared to the groups treated with ABX alone or in combination with E. coli.sup.pHND19, thus showing that administration of apyrase expressing bacteria improves mice resistance to C. rodentium infection.

[0388] In order to evaluate intestinal inflammation, mice were sacrificed 6 days post infection. Caecum was removed, opened longitudinally, delicately separated by caecal content and washed trice with ice cold PBS. The caecum was digested at 37° C. for 30 min with RPMI added with 5 mM EDTA for two times. The filtrated fragments were then digested in RPMI 5% FBS (fetal bovine serum), 1 mg/ml collagenase type II, 40 μg/ml DNase-I for 40 minutes. The filtered suspension, containing the caecum lamina propria cells, was centrifuged for 5 min at 1500 rpm and resuspended in RPMI complete medium. Single-cell suspensions from caecal lamina propria were stained with labelled antibodies diluted in PBS with 2% FBS for 20 min on ice. The following mouse antibodies (mAbs) were used for experiments: FITC conjugated anti mouse CD45 (1:200, clone: 30F11, Cat. #: 103108, Biolegend), PeCy7 conjugated anti-mouse CD11B (1:200, clone: N418, Cat. #:117318, Biolegend), biotinylated anti-mouse Ly6C (1:200, clone: HK1.4, Cat. #:128004, Biolegend), Pacific Blue conjugated anti-mouse Ly6G (1:200, clone: 1A8, Cat. #:127612 Biolegend), PE conjugated anti-mouse Gr1 (1:200, clone: RB6-8C5, Cat. #:50-5931-U100, TOMBO Bioscience), Streptavidin APC (1:200, Cat. #:405207, Biolegend). Samples were acquired on an LSR Fortessa (BD Biosciences, Franklin Lakes N.J., USA) flow cytometer. Data were analyzed using the FlowJo software (TreeStar, Ashland, Oreg., USA) or FACS Diva software (BD Biosciences, Franklin Lakes N.J., USA).

[0389] Statistical analysis of the frequencies of CD45.sup.+Gr1.sup.+CD11b.sup.+ polymorphonucleated (PMN) cells in caecum lamina propria of the different experimental groups was performed. Results are shown in FIG. 11. These data show a significant reduction of PMN cells in mice treated with ABX and gavaged with E. coli.sup.pApyr as compared to the groups treated with ABX alone or ABX in combination with E. coli.sup.pHND19. This result indicates that E. coli.sup.pApyr administration attenuates dysbiosis-mediated infiltration of PMN in response to C. rodentium infection.

[0390] In addition, statistical analysis of the frequencies of CD45.sup.+CD11b.sup.+Ly6c.sup.+Ly6g.sup.− inflammatory monocytes in caecum lamina propria of the different experimental groups was performed. Results are shown in FIG. 12. These data show a significant reduction of inflammatory monocytes in mice treated with ABX and gavaged with E. coli.sup.pApyr as compared to the groups treated with ABX alone or ABX in combination with E. coli.sup.pHND19. Analogously to the observation of reduced PMN infiltration, this result indicates that E. coli.sup.pApyr administration attenuates dysbiosis-mediated inflammatory infiltration of intestinal lamina propria in response to C. rodentium infection.

Example 4: Administration of Bacteria Expressing Apyrase Reduces Effects of C. difficile Infection after Induction of Dysbiosis

[0391] Clostridioides difficile is a major cause of antibiotic-associated diarrhea and has been shown to be associated with gut microbial dysbiosis, including reduced bacterial community diversity and depletion of key taxa.

[0392] To investigate whether the microbiota community structure induced by apyrase expressing bacteria could counteract intestinal invasion by C. difficile, ABX was administered to C57BL/6 mice for 4 days to induce microbiota depletion and dysbiosis, as described in Examples 2 and 3. The treatment schedule is shown in FIG. 13. 8-week old C57BL/6 mice were randomly assigned to 4 different experimental groups: not treated (control), treated with antibiotics (ABX: Vancomycin 1.25 mg, ampicillin 2.5 mg and metronidazole 1.25 mg; in 200 μl sterile water per mouse), treated with ABX and 10.sup.10 CFU of E. coli.sup.pHND19; and treated with ABX and 10.sup.10 CFU of E. coli.sup.pApyr. After the antibiotic treatment, mice were orally gavaged for 4 days with PBS (control); or 10.sup.10 CFU of E. coli.sup.pHND19; or E. coli.sup.pApyr, similarly as in Examples 2 and 3.

[0393] Thereafter, mice were orally infected with 10 s C. difficile VPI 10463 spores (except for the untreated control group). To this end, Clostridioides difficile ATCC® 43255TM (VPI 10463 A.sup.+B.sup.+CDT.sup.−) spores were stocked at 10.sup.8/ml at −80° C. in PBS+1% BSA. Spores titres were confirmed by plating serial dilutions of the stocks on brain heart infusion (BD Biosciences) agar plates supplemented with 5 g/I yeast extract and 0.1% taurocholate to induce germination. Plates were kept at least 24 h in airtight canisters equipped with Oxoid Anaerogen™.

[0394] On days 0, 1, 2 and 3 post infection, the body weight, clinical scores and survival of the animals was assessed. The clinical score used in Clostridioides difficile infection is shown in Table 1:

TABLE-US-00001 TABLE 1 clinical score used in Clostridioides difficile infection. body weight loss from 0% to 5% 0 from 6% to 10% 1 from 11% to 15% 2 more than 15% 3 dehydration no 0 mild 1 severe 2 perianal appearance clean 0 lightly dirty 1 wet tail 2 activity normal 0 reduced (hunched) 1 absent (hunched) 2

[0395] Results are shown in FIGS. 14 (body weight), 15 (clinical score) and 16 (survival). Analysis of the percentage of body weight loss following C. difficile infection revealed a significant reduction of body weight loss in mice orally gavaged with E. coli.sup.pApyr as compared to the groups treated with ABX alone or in combination with E. coli.sup.pHND19, thus indicating that administration of apyrase expressing bacteria protects mice from C. difficile infection. Evaluation of the clinical score over time during C. difficile infection confirmed that mice treated with ABX and gavaged with E. coli.sup.pApyr were less affected by C. difficile infection compared to mice treated with ABX alone or ABX in combination with E. coli.sup.pHND19. Analysis of survival in the different experimental groups revealed improved survival in mice treated with ABX and gavaged with E. coli.sup.pApyr compared to mice treated with ABX alone or ABX in combination with E. coli.sup.pHND19. This result indicates that E. coli.sup.pApyr treatment profoundly attenuates the invasivity of C. difficile following antibiotic treatments.

[0396] In order to evaluate intestinal inflammation, mice were sacrificed 72 h post infection and colon length was measured. Results are shown in FIG. 17. The measurement of colon length, an important parameter to score colitis, in mice treated with ABX and gavaged with E. coli.sup.pApyr showed similar values to non-infected mice (control), whereas in mice treated with ABX alone or in combination with E. coli.sup.pHND19, the colon length was drastically reduced. These data indicate that treatment with apyrase expressing bacteria attenuates colitis induced by C. difficile infection. C. difficile induced colitis was evaluated also by measuring fecal and serum lipocalin 2 (LCN-2), a marker of intestinal inflammation linked to epithelial damage and neutrophil infiltration, at 72 h post-infection, before sacrifice. The inflammation status of mice was evaluated by measuring the levels of Lipocalin-2 (LCN-2) in fecal supernatants via ELISA assay (R&D systems, DuoSet ELISA Mouse Lipocalin-2/NGAL). Briefly, feces were resuspended 0.01 g in 100 μl in PBS, centrifuged for 10 min at maximum speed and diluted before performing the ELISA assay, according to manufacturer's instructions. For the measurement of serum lipocalin 2 (LCN-2) levels, mice were bled and LCN-2 levels in the serum were assessed by ELISA assay as above.

[0397] Results for fecal LCN-2 levels are shown in FIG. 18. Mice treated with ABX and gavaged with E. coli.sup.pApyr were characterized by lower levels of LCN-2 as compared to mice treated with ABX alone or ABX in combination with E. coli.sup.pHND19 further confirming that E. coli.sup.pApyr can mitigate C. difficile mediated intestinal inflammation.

[0398] Results for serum LCN-2 levels are shown in FIG. 19. Quantification of serum LCN-2 in the different experimental groups revealed lower levels of LCN-2 in mice treated with ABX and gavaged with E. coli.sup.pApyr as compared to mice treated with ABX alone or in combination with E. coli.sup.pHND19 This result indicates that E. coli.sup.pApyr treatment limits the systemic dissemination of the pathogen.

Example 5: Administration of Bacteria Expressing Apyrase Reduces Effects of C. difficile Infection after Induction of Dysbiosis in a Distinct Challenge Model

[0399] To address the possible effect of bacteria expressing apyrase also in distinct challenge model of C. difficile infection, dysbiosis was induced by daily gavaging the antibiotic cefoperazone in the evening (2.5 mg/mouse) for 5 consecutive days. On the same days, 10.sup.10 CFU of E. coli.sup.pHND19 or E. coli.sup.pApyr were orally gavaged in the morning. At day 6 the cefoperazone treatment was stopped and E. coli.sup.pHND19 or E. coli.sup.pApyr treatment was continued for three additional days. Mice were then orally infected with 10′ of C. difficile VPI 10463 spores essentially as described above (Example 4). The treatment schedule is shown in FIG. 20.

[0400] FIG. 21 shows the survival rates of mice treated as shown in FIG. 20. Analysis of mice survival in the different experimental groups revealed that oral gavaging with E. coli.sup.pApyr resulted in the reduction of mortality compared to mice treated with cefoperazone alone or in combination with E. coli.sup.pHND19. This result further supports the idea that administration of bacteria expressing apyrase effectively protects from C. difficile mediated mortality.

[0401] Clinical scores were assessed at 24 h post C. difficile infection as described above (Example 4, Table 1). Results are shown in FIG. 22. The analysis of clinical scores at 24 h post C. difficile infection showed that mice gavaged with E. coli.sup.pApyr were characterized by less severe signs of infection compared to mice treated with cefoperazone alone or in combination with E. coli.sup.pHND19 Accordingly, administration of bacteria expressing apyrase ameliorates the clinical score in mice infected with C. difficile.

Example 6: Monocolonization of Germ-Free Mice with Bacteria Expressing Apyrase

[0402] The transcriptional regulation in intestinal epithelial cells (IECs) plays a prominent role in the modulation of the composition of microbiota and host metabolic homeostasis, in a complex interplay between the immune system, the intestinal epithelium and the gut microbiota (Shulzhenko, N., Morgun, A., Hsiao, W., Battle, M., Yao, M., Gavrilova, O., Orandle, M., Mayer, L., Macpherson, A. J., McCoy, K. D., et al. (2011). Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut. Nat Med 17, 1585-1593).

[0403] Germ-free (GF) mice monocolonized with E. coli.sup.pApyr showed significantly reduced ATP in the intestine compared with mice monocolonized with bacteria carrying an empty vector (Perruzza, L., Gargari, G., Proietti, M., Fosso, B., D'Erchia, A. M., Faliti, C. E., Rezzonico-Jost, T., Scribano, D., Mauri, L., Colombo, D., et al. (2017). T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP. Cell Rep 18, 2566-2575). Consistent with a role of endoluminal ATP in regulating T follicular helper (Tfh) cell number and germinal center (GC) reaction in the Peyer's patches (PPs) of the small intestine (Proietti, M., Cornacchione, V., Rezzonico Jost, T., Romagnani, A., Faliti, C. E., Perruzza, L., Rigoni, R., Radaelli, E., Caprioli, F., Preziuso, S., et al. (2014). ATP-gated ionotropic P2X7 receptor controls follicular T helper cell numbers in Peyer's patches (PP) to promote host-microbiota mutualism. Immunity 41, 789-801), both Tfh and GC B cells were increased in animals colonized with apyrase-expressing bacteria. Moreover GF mice monocolonized with E. coli.sup.pApyr showed higher amounts of E. coli specific IgA compared to E. coli.sup.pBAD28 monocolonized mice (Perruzza, L., Gargari, G., Proietti, M., Fosso, B., D'Erchia, A. M., Faliti, C. E., Rezzonico-Jost, T., Scribano, D., Mauri, L., Colombo, D., et al. (2017). T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP. Cell Rep 18, 2566-2575.). These data indicate that extracellular ATP released by commensal microbiota limits the secretory IgA response in the small intestine.

[0404] To investigate whether apyrase could affect host metabolism by regulating gene transcription in intestinal epithelial cells (IECs), monocolonized mice conditioned by apyrase were generated. To this end, germ-free (GF) mice were orally gavaged with E. coli.sup.pApyr or E. Coli.sup.pBAD28 once in order to generate monocolonized mice either conditioned by apyrase or not. Twenty-eight days later, mice were sacrificed and a genome-wide expression profiling was performed to compare ex vivo isolated IECs of differently colonized animals. The experimental schedule is shown in FIG. 23.

[0405] IECs were isolated by the method as described in Romagnani, A., Vettore, V., Rezzonico-Jost, T., Hampe, S., Rottoli, E., Nadolni, W., Perotti, M., Meier, M. A., Hermanns, C., Geiger, S., et al. (2017). TRPM7 kinase activity is essential for T cell colonization and alloreactivity in the gut. Nat Commun 8, 1917. Total RNA was extracted from IECs through Trizol precipitation (Invitrogen, Carlsbad, Calif.) and then digested with DNase I at 37° C. for 15 min to remove any contaminating DNA. The quality of total RNA was first assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, Calif.). Biotin-labelled cDNA targets were synthesized starting from 150 ng of total RNA. Double stranded cDNA synthesis and related cRNA was performed with GeneChip® WT Plus Kit (Affymetrix, Santa Clara, Calif.). The sense strand cDNA was synthesized before with the same kit to be fragmented and labelled. All steps of the labelling protocol were performed as described by the kit's manufacturer, starting from 5.5 μg of ssDNA. Each eukaryotic GeneChip® probe array contained probe sets for several B. subtilis genes that were absent in the samples analyzed (lys, phe, thr, and dap). The GeneChip® Poly-A RNA Control Kit contained in vitro synthesized, polyadenylated transcripts for these B. subtilis genes that are pre-mixed at staggered concentrations to allow GeneChip® probe array users to assess the overall success of the assay. Poly-A RNA controls final concentration in each target were lys 1:100,000, phe 1:50,000, thr 1:25,000 and dap 1:6,667. Hybridization was performed using the GeneChip® Hybridization, Wash and Stain Kit containing a mix for target dilution, DMSO at a final concentration of 7% and pre-mixed biotin-labelled control oligo B2 and bioB, bioC, bioD and cre controls (Affymetrix cat #900299) at a final concentration of 50 pM, 1.5 pM, 5 pM, 25 pM and 100 pM, respectively. Fragmented and labelled sscDNA were diluted in hybridization buffer at a concentration of 23 ng/μl for a total of 2.3 μg and denatured at 99° C. for 5 min incubated at 45° C. for 5 min and centrifuged at maximum speed for 1 min prior to introduction into the GeneChip® cartridge. A single GeneChip® Mouse Clariom S was then hybridized with each biotin-labelled sense target. Hybridizations were performed for 16 h at 45° C. in a rotisserie oven. GeneChip® cartridges were washed and stained with GeneChip® Hybridization Wash and Stain Kit in the Affymetrix Fluidics Station 450 following the FS450_0007 standard protocol. The GeneChip® arrays were scanned using an Affymetrix GeneChip® Scanner 3000 7G using default parameters. Affymetrix GeneChip® Command Console software (AGCC) was used to acquire GeneChip® images and generate .DAT and .CEL files, which were used for subsequent analysis with proprietary software. Raw data was normalized using the quantile normalization of robust multiarray average (RMA) method. The identification of the differentially expressed transcripts and the hierarchical cluster analysis with Euclidean distance was performed using the commercial software Partek Genomics Suite (v6.6).

[0406] Results of the differential expression analysis are shown in FIG. 24. Differential expression analysis performed in gnotobiotic mice monocolonized with E. coli.sup.pApyr or E. coli.sup.pBAD28 resulted in a transcriptional signature of 53 upregulated and 79 downregulated genes (highlighted by the rectangles in FIG. 24) in IECs isolated from E. coli.sup.pApyr with respect to E. coli.sup.pBAD28 WT mice (FDR≤5% and absolute fold change≥1.5), suggesting that the absence of extracellular ATP (eATP) significantly influenced gene transcription in IECs.

[0407] Next, relative expression level (Z-score) of the differentially expressed genes were determined by Gene Ontology (GO) analysis. To this end, the list of differentially expressed genes were loaded into DAVID Bioinformatics Resources (v6.8; Huang da, W., Sherman, B. T., and Lempicki, R. A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44-57) for GO enrichment analysis and visualized using the R package GOplot Walter, W., Sanchez-Cabo, F., and Ricote, M. (2015). GOplot: an R package for visually combining expression data with functional analysis. Bioinformatics 31, 2912-2914) and gplots. Z-score was calculated as the number of genes upregulated minus the number of gene downregulated divided for the square root of the total number of genes analysed;

[00002]$z=\frac{\left(n\mathrm{up}-n\mathrm{down}\right)}{\sqrt{n\mathrm{tot}}}.$

[0408] Results are shown in FIG. 25. Genes relative expression levels measured as Z-score, showed that E. coli.sup.pAPyr treatment induced the downregulation of genes related to cell cycle and division and upregulation of genes related to lipid metabolic and oxidation-reduction processes. These two groups of genes are important for absorption of nutrients, and protection against chemical-induced oxidant injury. Moreover, upregulation of genes related to oxidation-reduction processes are important to preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress (Circu, M. L., and Aw, T. Y. (2011). Redox biology of the intestine. Free Radic Res 45, 1245-1266).

[0409] FIG. 26 shows the Gene Ontology analysis of differentially expressed genes in E. coli.sup.pApyr vs E. coli.sup.pBAD28 intestinal epithelial cells of monocolonized mice. Functional over-representation analysis revealed that gene sets associated to DNA replication were enriched in IECs from E. Coli.sup.pBAD28 mice, whereas signature of metabolic functions governing principally lipids, fatty acids and vitamin A metabolism, solute carriers for carnitine, small peptides and ions were enriched in E. Coli.sup.pApyr mice. E. Coli.sup.pApyr monocolonized mice showed also an upregulation of genes belonging to the CYP family, that were shown to be important not only for drug metabolism but also in the oxidative, peroxidative and reductive metabolism of endogenous compounds such as steroids, bile acids, fatty acids, prostaglandins, biogenic amines and retinoids (Chang, G. W., and Kam, P. C. (1999). The physiological and pharmacological roles of cytochrome P450 isoenzymes. Anaesthesia 54, 42-50; Thelen, K., and Dressman, J. B. (2009). Cytochrome P450-mediated metabolism in the human gut wall. J Pharm Pharmacol 61, 541-558). These results suggest that depletion of eATP in the intestinal lumen modifies the interaction of colonizing bacteria with enterocytes, thereby conditioning their function and host metabolism.

[0410] To evaluate the role of apyrase-mediated enhanced IgA generation on the observed conditioning of the transcriptional activity of the intestinal epithelium, genome wide transcription in the intestinal epithelium from monocolonized mice deficient in the generation of antibody response because of J segment deletion in Ig heavy chain locus (Igh-J.sup.−/− mice) was analyzed. Strikingly, as shown by the volcano plot of FIG. 34, minimal differences in gene expression were detected between E. coli.sup.pApyr and E. coli.sup.pBAD28 monocolonized Igh-J.sup.−/− mice. These data indicate that enhanced IgA generation in wild-type mice monocolonized with E. coli.sup.pApyr was responsible of favouring metabolic over immune functions in the enterocyte.

[0411] Accordingly, wild-type mice monocolonized with E. coli.sup.PAPYr showed increased body weight variation (FIG. 35), blood glucose (FIG. 36), serum insulin (FIG. 37) and white adipose tissue (WAT) deposition (FIG. 38), as well as improvement of glucose tolerance test (FIG. 39) compared to mice monocolonised with E. coli.sup.pBAD28. In contrast, no differences in body weight variation (FIG. 40), blood glucose (FIG. 41) and response in glucose tolerance test (FIG. 42) were observed in Igh-J.sup.−/− mice monocolonized either with E. coli.sup.pBAD28 or E. Coli.sup.pApyr. These data suggest that modulation of endoluminal ATP by apyrase can promote metabolic adaptation of the host via secretory IgA conditioning of commensal microbes.

Example 7: Administration of Bacteria Expressing Apyrase Reduces Dysbiosis-Induced Hypoglycemia

[0412] To investigate the effects of apyrase on hypoglycemia due to antibiotics-mediated dysbiosis, dysbiosis was induced and bacteria expressing apyrase were administered as described in Example 2 (experimental schedule as shown in FIG. 4). Blood glucose was analyzed after 4 days of antibiotic treatment and 4 days of recovery (see FIG. 4).

[0413] Results are shown in FIG. 27. Dysbiosis induced by antibiotics treatment resulted in a pronounced decrease in blood glucose (hypoglycemia). However, mice treated with apyrase expressing bacteria showed higher serum glucose levels as compared to ABX or ABX+E. coli.sup.pHND19 treated mice. These data indicate that E. coli.sup.pApyr administration mitigates the antibiotic-mediated induction of hypoglycemia.

[0414] Moreover, white adipose tissue (WAT) was collected and quantified in order to evaluate the effect of apyrase on antibiotics-mediated WAT loss. Results are shown in FIG. 43. Quantification of WAT as percentage of total body weight revealed a significant reduction of WAT in ABX-induced dysbiosis (in both mice treated with ABX alone or in association with E. coli.sup.pHND19) that was significantly attenuated by administration of E. coli.sup.PAPYr.

[0415] No improvements in blood glucose levels (FIG. 44) and WAT deposition (FIG. 45) after ABX treatment were observed in Igh-J.sup.−/− mice treated with E. Coli.sup.pApyr as compared to the counterpart treated with ABX or the combination of ABX and E. coli.sup.pHND19, consistent with the function of apyrase in conditioning host metabolism during antibiotic treatment via regulation of the secretory IgA response induced by commensal bacteria.

Example 8: Mouse Model of Cefoperazone-Mediated Dysbiosis and Recovery

[0416] To further assess the efficacy of apyrase treatment in counteracting the metabolic impairment due to dysbiosis, dysbiosis was induced in a mouse model by daily administration of cefoperazone for five consecutive days. The experimental schedule is shown in FIG. 28.

[0417] To induce dysbiosis, mice were treated in the evening with cefoperazone (2.5 mg/mouse) for 5 consecutive days. Concomitantly, 10.sup.10 CFU of E. coli.sup.pHND19 or E. coli.sup.pApyr were orally gavaged in the morning. At the end of cefoperazone treatment E. coli.sup.pHND19 or E. coli.sup.pApyr administration was continued for 3 additional days.

[0418] At the end of the experiment, the body weight of the animals was assessed. Results are shown in FIG. 29. The data reveal that cefoperazone-induced dysbiosis results in significantly decreased body weight (control mice compared to mice treated with cefoperazone alone or in association with E. coli.sup.pHND19). In contrast, mice treated with cefoperazone and bacteria expressing apyrase (E. Coli.sup.PAPYr) showed body weight gains that were not significantly different from untreated animals, while they were significantly higher with respect to mice treated with cefoperazone alone or in association with E. coli.sup.pHND19. These data indicate that E. coli.sup.pApyr administration attenuates body weight loss induced by cefoperazone mediated dysbiosis.

[0419] Mice were sacrificed at the end of the experiment and white perigonadal adipose tissue was collected and quantified. Results are shown in FIG. 30. Quantification of white adipose tissue (WAT) as percentage of total body weight revealed a significant reduction of WAT in cefoperazone-induced dysbiosis (control mice compared to mice treated with cefoperazone alone or in association with E. Coli.sup.pHND19), while no significant variation was observed between control mice and mice treated with cefoperazone and bacteria expressing apyrase (E. Coli.sup.PAPYr). Accordingly, E. coli.sup.pApyr treatment attenuates WAT reduction induced by cefoperazone mediated dysbiosis.

Example 9: Effects of Apyrase Administration in a Mouse Model of Antibiotics-Induced Dysbiosis

[0420] Next, the effects of administration of apyrase were investigated in a mouse model of antibiotics-induced dysbiosis. To this end, dysbiosis was induced by daily oral gavage of ABX for 4 consecutive days. Together with the antibiotic treatment, mice were orally gavaged with PBS or 40 μg of purified recombinant apyrase every 12 h.

[0421] At the end of the experiment, blood glucose levels were analyzed. Results are shown in FIG. 32. The analysis of blood glucose in mice orally gavaged with ABX and apyrase after four days of antibiotic treatment revealed values comparable to untreated mice and significantly higher blood glucose levels than mice treated with ABX without apyrase. Accordingly, the administration of the apyrase protein is sufficient to mitigate this metabolic modification in antibiotics-induced dysbiosis and apyrase attenuates the induction of hypoglycemia caused by antibiotic mediated dysbiosis.

[0422] Mice were sacrificed at the end of the experiment and white perigonadal adipose tissue was collected and quantified. Results are shown in FIG. 33. Quantification of the WAT weight showed a significant decrease due to antibiotics-induced dysbiosis, while this effect was no longer observed in animals treated with apyrase, which show significantly higher WAT weight than animals suffering from antibiotics-induced dysbiosis without apyrase treatment. Accordingly, Apyrase treatment attenuates WAT reduction induced by antibiotics-induced dysbiosis.

Example 10: Apyrase Attenuates Caecum Enlargement and Bacterial Translocation to the Mesenteric Lymph Node (MLN) Caused by Antibiotics-Mediated Dysbiosis

[0423] Intestinal dysbiosis caused by antibiotic treatment is characterized by a reduction in bacterial load and diversity, altered microbiota composition and impaired intestinal barrier integrity. Host features indicative of reduced bacterial load after antibiotic treatment could be examined by assessing cecum size, length and weight, which are reported to be grossly enlarged in germ-free animals (Devkota, S., Wang, Y., Musch, M. W., Leone, V., Fehlner-Peach, H., Nadimpalli, A., Antonopoulos, D. A., Jabri, B., and Chang, E. B. (2012). Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10−/− mice. Nature 487, 104-108; Poteres, E., Hubert, N., Poludasu, S., Brigando, G., Moore, J., Keeler, K., Isabelli, A., Ibay, I. C. V., Alt, L., Pytynia, M., et al. (2020). Selective Regional Alteration of the Gut Microbiota by Diet and Antibiotics. Front Physiol 11, 797). To investigate the effect of apyrase on caecum enlargement caused by antibiotics-mediated dysbiosis, bacteria expressing apyrase were administered as described in Example 2 (experimental schedule as shown in FIG. 4). Caecum weight was analysed after 4 days of antibiotic treatment and 4 days of recovery (see FIG. 4). Results are shown in FIG. 46. Dysbiosis induced by antibiotics resulted in a pronounced increase in caecum weight. However, mice treated with E. coli.sup.pApyr showed significantly reduced caecum weight as compared to mice treated with ABX or ABX and E. coli.sup.pHN19. These data indicate that E. coli.sup.pApyr administration mitigates antibiotics-induced caecum enlargement.

[0424] Antibiotic treatment induces impairment of gut barrier integrity and translocation of live commensal bacteria to the mesenteric lymph node (MLN) boosting an inflammatory response (Knoop K A, McDonald K G, Kulkarni D H, Newberry R D. Antibiotics promote inflammation through the translocation of native commensal colonic bacteria. (2016) Gut 65, 1100-9. doi: 10.1136/gutjnl-2014-309059). In order to investigate the effect of apyrase on antibiotics mediated bacterial translocation to the MLN, mice were sacrificed at the end of the experiment (see FIG. 4), MLN were harvested aseptically into RPMI and mechanically homogenized. Dilutions of homogenates were plated onto Schaedler agar (BD Biosciences). Plates were grown under aerobic or anaerobic culture conditions at 37° C. for 24-72 hours before enumeration of colonies. Results are shown in FIG. 47 and FIG. 48. Quantification of CFU from the MLN, both in aerobic and anaerobic conditions, revealed a significant increase in mice treated with ABX or ABX and E. coli.sup.pHN19 compared to untreated animals. However, mice treated with the combination of ABX and E. Coli.sup.pApyr showed a number of CFU in the MLN that was not significantly different from untreated animals, while significantly lower with respect to mice treated with ABX alone or in association with E. coli.sup.pHN19. These data indicate that E. coli.sup.pApyr administration attenuates intestinal bacterial translocation induced by antibiotic-mediated dysbiosis.

[0425] In order to understand whether secretory IgA were involved in apyrase mediated intestinal adaptation to dysbiosis by antibiotics, the same experiment as shown in FIG. 4 was performed using Igh-J.sup.−/− mice. Caecum weight was analysed after 4 days of antibiotic treatment and 4 days of recovery. Results are shown in FIG. 49. Dysbiosis induced by antibiotics resulted in a pronounced increase in caecum weight in Igh-J.sup.−/− mice treated with ABX, ABX+E. coli.sup.pHN19 and ABX+E. coli.sup.pApyr compared to untreated animals. Quantification of CFU in the MLN of Igh-J.sup.−/− mice, both in aerobic and anaerobic conditions, revealed the prominent translocation of bacteria in all the three different groups compared to the untreated group (FIG. 50 and FIG. 51). Strikingly, no amelioration of this feature was observed in animals treated with ABX in combination with E. coli.sup.pApyr as compared to Igh-J.sup.−/− mice treated with ABX or ABX+E. coli.sup.pHN19. These data show the importance of secretory IgA elicited by Apyrase in controlling gut barrier impairment mediated by ABX treatment.

Example 11: Generation of Recombinant Bacteria Heterologously Expressing Apyrase, which Carry the Apyrase Gene Integrated in their Genome (EcN::Phon2)

[0426] The apyrase expressing bacteria designed and produced as described in Example 1 above were obtained by transforming bacteria with plasmids encoding apyrase. Such plasmids may contain antibacterial resistance for the selection of the transformants. Such bacterial transformants typically bear multiple copies of the apyrase-encoding plasmid (and may be selected for antibiotic resistance). To investigate whether similar effects can be obtained in recombinant bacteria encoding apyrase in a heterologous manner in a single copy in their genome instead of multiple copies of extrachromosomal plasmids, bacteria having a single copy of the (heterologous) apyrase (phoN2) gene in the bacterial chromosome (non-transmissible) (without antibiotic resistance) were created.

[0427] To this end, the chromosomal integration of the Shigella flexneri phoN2 apyrase-encoding gene in the EcN genome (GenBank accession number CP007799.1) was performed by the λ Red recombineering approach (Datsenko K. A. and Wanner B. L. 2000 One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 97, 6640).

[0428] FIG. 52 schematically shows the DNA fragment used for the recombineering, including: [0429] A portion of the EcN malP gene, coding for the maltodextrin phosphorylase enzyme; [0430] The E. coli cat gene, which codes for the chloramphenicol acetyltransferase enzyme conferring resistance to the chloramphenicol antibiotic, flanked by the Flippase Recognition Target (FRT) sequences; [0431] The Shigella flexneri phoN2 apyrase-encoding gene fused upstream with the P.sub.proD synthetic promoter (Davis J. H., Rubin A. J. and Sauer R. T. 2011 Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 9, 1131) and the BBa_BB0032 Ribosome Binding Site (RBS; iGEM Parts Registry), and downstream with the phoN2 transcriptional terminator; [0432] A portion of the EcN malT gene, coding for the transcriptional activator of the maltose and maltodextrins operon.

[0433] FIGS. 53 and 54 show the nucleotide sequences of EcN malP and malTgene portions, respectively (SEQ ID NOs 6 and 7, respectively). FIG. 55 shows the nucleotide sequence of the DNA fragment, including the P.sub.proD promoter, the BBa_BB0032 RBS, the S. flexneri phoN2 gene and the phoN2 transcriptional terminator (SEQ ID NO: 8). FIG. 56 shows the nucleotide sequence of the DNA fragment, including the E. coli cat gene flanked by the FRT sequences (SEQ ID NO: 9).

[0434] To perform the recombineering in the EcN genome, the insertion DNA fragment was transformed in an EcN strain carrying the pKD46 plasmid, which expresses the phage λ Red recombinase. The λ Red-mediated homology recombination at the malP and malT sites promoted the integration of the insertion DNA fragment in the malP-malT intergenic region of EcN. After pKD46 removal, the EcN clones carrying the insertion DNA fragment in the genome were selected for chloramphenicol resistance and checked by PCR for the correct integration in the genome. The EcN clones selected for the correct integration of the insertion DNA fragment were transformed with the pCP20 plasmid, which expresses the yeast Flp recombinase (Flippase), to excise the chloramphenicol resistance cassette from the genome. After pCP20 removal, the EcN recombinant clones not carrying the chloramphenicol cassette in the genome were selected for chloramphenicol sensitivity and checked by PCR for the correct excision of the cassette from the genome. The resulting EcN recombinant clones carrying the S. flexneri phoN2 gene in the malP-malT intergenic region were named EcN::phoN2. FIG. 57 schematically shows the malP-phoN2-malT recombinant genomic region of the obtained EcN::phoN2 clones. FIG. 58 shows the expression of apyrase in one selected EcN::phoN2 clone (cl 1) in a Western-Blot of periplasmic extracts. In addition, the activity of the enzyme in EcN::phoN2 cl 1 was verified. FIG. 59 shows the dose-dependent degradation of ATP by EcN::phoN2 cl 1 periplasmic extract in an in vitro ATP-degradation assay. In both assays, the EcN wild type strain (EcN) was used as negative control. The EcN wild type and EcN::phoN2 bacterial strains were grown in LB medium.

Example 12: Recombinant Bacteria Encoding Apyrase in their Genome for Heterologous Expression Improve Dysbiosis-Induced Hypoglycemia and WAT Weight Loss

[0435] To investigate whether administration of E. coli Nissle 1917 (EcN) probiotic bacteria with phoN2 gene integrated in the genome (obtained as described above, Example 11) were effective in ameliorating hypoglycemia due to antibiotics-mediated dysbiosis, antibiotics (Vancomycin 1.25 mg, ampicillin 2.5 mg and metronidazole 1.25 mg in 200 μl sterile water per mouse) were administered to C57BL/6 mice that were subsequently gavaged with EcN or EcN::phoN2 strain (experimental schedule as shown in FIG. 60). Blood glucose was analysed after 4 days of antibiotic treatment and 4 days of recovery (day −4 and 3, respectively, in FIG. 60). As shown in FIG. 61, antibiotic treatment resulted in a significant decrease in blood glucose (hypoglycemia). Notably, mice treated with EcN::phoN2 showed serum glucose levels similar to untreated mice and significantly higher as compared to ABX and ABX+EcN treated mice. These data indicate that EcN::phoN2 administration restores blood glucose levels compromised by antibiotic treatments.

[0436] In order to evaluate the effect of EcN::phoN2 on antibiotics-mediated loss of WAT, white adipose tissue was collected and quantified. As shown in FIG. 62, WAT weight was significantly decreased due to antibiotics-induced dysbiosis, while this effect was attenuated in animals treated with EcN::phoN2, which showed significantly increased WAT weight than animals treated with ABX or ABX+EcN. These data indicate that EcN::phoN2 administration mitigates the antibiotics-induced WAT loss.

Example 13: Recombinant Bacteria Encoding Apyrase in their Genome for Heterologous Expression Attenuate Caecum Enlargement and Bacterial Translocation to the Mesenteric Lymph Node (MLN) Caused by Dysbiosis

[0437] In order to investigate a possible beneficial effect of EcN::phoN2 in the recovery from dysbiosis, a mouse model of antibiotics-induced dysbiosis was used. The treatment schedule is shown in FIG. 60. 8-week old C57BL/6 male mice were randomly assigned to 4 different experimental groups: not treated (control), treated with antibiotics (ABX: Vancomycin 1.25 mg, ampicillin 2.5 mg and metronidazole 1.25 mg in 200 μl sterile water per mouse), treated with ABX and 10.sup.10 CFU of EcN and treated with ABX and 10.sup.10 CFU of EcN::phoN2. At the end of the experiment, mice were sacrificed by CO.sub.2 inhalation and caecum and mesenteric lymph node (MLN) were harvested and analysed.

[0438] Caecum weight was analysed after 4 days of antibiotic treatment and 4 days of recovery (see FIG. 60). Results are shown in FIG. 63. Dysbiosis induced by antibiotics treatment resulted in a pronounced increased in caecum weight. However, mice treated with Ecn::phoN2 showed significantly lower caecum weight as compared to mice treated with ABX or ABX+EcN. These data indicate that EcN::phoN2 administration mitigates the antibiotic-mediated induction of caecum enlargement.

[0439] In order to investigate the effect of EcN::phoN2 in controlling bacterial translocation, MLN were harvested aseptically into RPMI and mechanically homogenized. Dilutions were plated onto Schaedler agar (BD Biosciences). Plates were grown under aerobic or anaerobic culture conditions at 37° C. for 24-72 h before enumeration of colonies. Results are shown in FIG. 64 and FIG. 65. Quantification of CFU from the MLN, both in aerobic and anaerobic conditions, revealed a significant increase in ABX and ABX+EcN treated mice compared to control animals. However, mice treated with EcN::phoN2 showed significantly reduced CFU both in aerobic and anaerobic conditions, compared to ABX and ABX+EcN treated mice. These data indicate that EcN::phoN2 administration attenuates bacterial translocation induced by antibiotics-mediated dysbiosis.

Example 14: Design and Production of Apyrase Expressing Lactococcus lactis

[0440] To further expand our platform of apyrase expressing biotherapeutics, we selected Lactococcus lactis, as a Gram-positive strain. Due to its noninvasive and nonpathogenic characteristics, L. lactis has been demonstrated to be a promising candidate for the intestinal delivery of functional proteins (Varma, N. R., Toosa, H., Foo, H. L., Alitheen, N. B., Nor Shamsudin, M., Arbab, A. S., Yusoff, K., and Abdul Rahim, R. (2013). Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71. Biotechnol Res Int 2013, 431315). Genetically engineered L. lactis expressing interleukin-10 (IL-10) was used for the treatment of inflammatory bowel diseases (IBD) (Braat, H., Rottiers, P., Hommes, D. W., Huyghebaert, N., Remaut, E., Remon, J. P., van Deventer, S. J., Neirynck, S., Peppelenbosch, M. P., and Steidler, L. (2006). A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol 4, 754-759). Moreover, a recombinant L. lactis strain expressing the Pancreatitis-associated Protein (PAP) has been shown to efficiently preserve gut homeostasis in chemotherapy induced mucositis (Carvalho, R., Vaz, A., Pereira, F. L., Dorella, F., Aguiar, E., Chatel, J. M., Bermudez, L., Langella, P., Fernandes, G., Figueiredo, H., et al. (2018). Gut microbiome modulation during treatment of mucositis with the dairy bacterium Lactococcus lactis and recombinant strain secreting human antimicrobial PAP. Sci Rep 8, 15072).

[0441] For the expression of Shigella flexneri apyrase in the Lactococcus lactis NZ900 strain, the apyrase encoding gene phoN2 was PCR amplified from the S. flexneri genome and cloned into the pNZ8123 plasmid, generating the pNZ-Apyr plasmid (FIG. 66). Apyrase expression in the pNZ-Apyr plasmid is controlled by the P.sub.nisA promoter, which is inducible by the nisin anti-microbial peptide. The phoN2 gene was in-frame cloned with the signal sequence of the L. lactis major secreted protein Usp45 to allow apyrase secretion. L. lactis.sup.pNZ and L. lactis.sup.pNZ-Apyr strains were grown in M17 medium supplemented with glucose (0.5% w/v) and nisin (4 ng/ml).

Example 15: Administration of L. lactis.SUP.pNZ-Apyr .Counteracts Intestinal Barrier Disruption and Bacterial Translocation to MLN Caused by Diet Induced Dysbiosis in Adult Mice

[0442] The intestinal barrier defines the morpho-functional unit responsible for the defence of the intestinal mucosa and consists of the intestinal microbiota, intestinal epithelial cells (IECs) and mucosal immunity tightly linked through a complex network of cytokines, antimicrobial peptides (AMPs), metabolic products, and numerous regulatory molecules (Meng, M., Klingensmith, N. J., and Coopersmith, C. M. (2017). New insights into the gut as the driver of critical illness and organ failure. Curr Opin Crit Care 23, 143-148). The intestinal mucosa is the largest body surface at risk of infectious threats, the anatomic and functional homeostasis of the intestinal barrier is a key step in the anti-infectious defence of the human organism. The intestinal microbiota represents the first line of defence of the intestinal barrier. The microbiota entails millions of microorganisms colonizing the gastrointestinal tract most of which are bacteria. This large number of microorganisms withstands the unfavourable intestinal habitat thanks to the symbiotic relationships with the human organism. These symbiotic host-commensal relationships develop after birth and enable the metabolic, immune and anti-infectious processes through which the microbiota contributes to gut homeostasis (O'Hara, A. M., and Shanahan, F. (2006). The gut flora as a forgotten organ. EMBO Rep 7, 688-693). The structural and functional stability of commensal populations is regulated through numerous signaling molecules (quorum sensing) and cellular regulators (miRNAs) as well as through other physiologic and pathologic factors. Qualitative or quantitative alterations of this microbial community broadly defined as dysbiosis impair the relationships between the host and commensal species, modify the balance between commensals and pathogens, decrease the intestinal barrier protection and favour infectious pathogens (McDonald, D., Ackermann, G., Khailova, L., Baird, C., Heyland, D., Kozar, R., Lemieux, M., Derenski, K., King, J., Vis-Kampen, C., et al. (2016). Extreme Dysbiosis of the Microbiome in Critical Illness. mSphere 1: e00199-16. doi: 10.1128/mSphere.00199-16). Diet is a major element affecting the intestinal microbiota. Natural variations in food intake cause transient changes in microbial composition, although predominant components such as meat, fish, and fibers have durable effects on the microbiota and leave typical signatures characterized by shifts in specific bacterial groups (Scott, K. P., Gratz, S. W., Sheridan, P. O., Flint, H. J., and Duncan, S. H. (2013). The influence of diet on the gut microbiota. Pharmacol Res 69, 52-60). Changing food composition as well as food shortage or oversupply affect the gut microbiota. The absence of nutrients in the gut occurring in parenteral feeding increases the levels of Proteobacteria, which promote inflammation at the mucosal wall and eventually cause a breakdown of the epithelial barrier (Demehri, F. R., Barrett, M., and Teitelbaum, D. H. (2015). Changes to the Intestinal Microbiome With Parenteral Nutrition: Review of a Murine Model and Potential Clinical Implications. Nutr Clin Pract 30, 798-806). The influence of diet on the composition of the microbiota has been shown during the initial colonization phase: breast fed infants have higher levels of Bifidobacteria spp. while formula fed infants have higher levels of Bacteroides spp., as well as increased Clostridium coccoides and Lactobacillus spp. (Fallani, M., Young, D., Scott, J., Norin, E., Amarri, S., Adam, R., Aguilera, M., Khanna, S., Gil, A., Edwards, C. A., et al. (2010). Intestinal microbiota of 6-week-old infants across Europe: geographic influence beyond delivery mode, breast-feeding, and antibiotics. J Pediatr Gastroenterol Nutr 51, 77-84). Beyond the postnatal period, the microbiota was suspected to be relatively stable throughout life. However, several recent studies have shown that dietary factors alter the microbial community resulting in biological changes to the host. In fact, the composition of the gut microbiota strongly correlates with diet as demonstrated by a study assessing the relative contributions of host genetics and diet in shaping the gut microbiota and modulating metabolic syndrome phenotypes in mice (Zhang, C., Zhang, M., Wang, S., Han, R., Cao, Y., Hua, W., Mao, Y., Zhang, X., Pang, X., Wei, C., et al. (2010). Interactions between gut microbiota, host genetics and diet relevant to development of metabolic syndromes in mice. ISME J 4, 232-241).

[0443] Food is not only a source of nutrients but may also modulate some physiological functions of the body. This is especially true for the intestinal tract because of the continuous interaction of the intestine with dietary antigens (Ulluwishewa, D., Anderson, R. C., McNabb, W. C., Moughan, P. J., Wells, J. M., and Roy, N.C. (2011). Regulation of tight junction permeability by intestinal bacteria and dietary components. J Nutr 141, 769-776). Recent studies demonstrated the effects of the interaction between food and IECs. In fact, dietary antigens are able to modulate transporter activity, tight junction permeability, metabolic enzyme expression, immune functions, and microbiota (Shimizu, M. (2010). Interaction between food substances and the intestinal epithelium. Biosci Biotechnol Biochem 74, 232-241). Food entering the gastrointestinal tract provides nutrition to the organism. In addition, there are many metabolites produced by enzymatic conversion of nutrients, either by the host enzymes or by the gut microbiota, or by stimulating release of non-enzymatic molecules that influence diverse functions including alterations in the intestinal barrier.

[0444] The metabolites produced in the lumen may enter the bloodstream and reach sufficient concentrations to affect the functions of body organs (Dodd, D., Spitzer, M. H., Van Treuren, W., Merrill, B. D., Hryckowian, A. J., Higginbottom, S. K., Le, A., Cowan, T. M., Nolan, G. P., Fischbach, M. A., et al. (2017). A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature 551, 648-652). These interactions between diet, digestion, microbiota, and the intestinal barrier may impact gut homeostasis (Farre, R., Fiorani, M., Abdu Rahiman, S., and Matteoli, G. (2020). Intestinal Permeability, Inflammation and the Role of Nutrients. Nutrients 12:1185. doi: 10.3390/nu12041185).

[0445] Diet induced dysbiosis trigger also mechanisms that unbalance the intestinal homeostasis and cause inflammation. The translocation of bacteria across the gut epithelium increases in dysbiosis (Sato, J., Kanazawa, A., Ikeda, F., Yoshihara, T., Goto, H., Abe, H., Komiya, K., Kawaguchi, M., Shimizu, T., Ogihara, T., et al. (2014). Gut dysbiosis and detection of “live gut bacteria” in blood of Japanese patients with type 2 diabetes. Diabetes Care 37, 2343-2350). Small numbers of translocated commensal bacteria, as they occur in a healthy human gut, are removed by the action of Th1 and Th17 cells that are particularly induced by polysaccharides of Bacteroides spp. (Mazmanian, S. K., and Kasper, D. L. (2006). The love-hate relationship between bacterial polysaccharides and the host immune system. Nat Rev Immunol 6, 849-858) and mucosa-adherent segmented filamentous bacteria (SFB) (Ivanov, I I, Atarashi, K., Manel, N., Brodie, E. L., Shima, T., Karaoz, U., Wei, D., Goldfarb, K. C., Santee, C. A., Lynch, S. V., et al. (2009). Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485-498). Conversely, high numbers of invading bacteria activate TLRs and elicit an overexpression of pro-inflammatory cytokines, which damage the gut epithelium and lead to chronic intestinal inflammation (Karczewski, J., Poniedzialek, B., Adamski, Z., and Rzymski, P. (2014). The effects of the microbiota on the host immune system. Autoimmunity 47, 494-504). Disruption of the intestinal barrier, increased intestinal permeability and translocation of bacterial antigens towards metabolically active tissue would result in a chronic inflammatory state and impaired metabolic functions such as insulin resistance, hepatic fat deposition, excessive adipose tissue development (Tehrani, A. B., Nezami, B. G., Gewirtz, A., and Srinivasan, S. (2012). Obesity and its associated disease: a role for microbiota? Neurogastroenterol Motil 24, 305-311; Camilleri, M. (2019). Leaky gut: mechanisms, measurement and clinical implications in humans. Gut 68, 1516-1526), rheumatoid arthritis (Guerreiro, C. S., Calado, A., Sousa, J., and Fonseca, J. E. (2018). Diet, Microbiota, and Gut Permeability—The Unknown Triad in Rheumatoid Arthritis. Front Med (Lausanne) 5, 349) and ulcerative colitis (Den Hond, E., Hiele, M., Evenepoel, P., Peeters, M., Ghoos, Y., and Rutgeerts, P. (1998). In vivo butyrate metabolism and colonic permeability in extensive ulcerative colitis. Gastroenterology 115, 584-590).

[0446] A mouse model of diet induced dysbiosis (DID) was created to investigate possible beneficial effects of apyrase on this condition. Dysbiosis was induced by feeding mice with a diet characterized by 7% protein, 5% fat and 88% carbohydrates. Normal diet (ND) characterized by 20% protein, 15% fat and 65% carbohydrate was used as control. A schematic of the components of each diet is shown in FIG. 67. Experimental layout showed in FIG. 68 shows the DID model in 5 weeks old mice. At 5 weeks of age, female C67BL/6 mice were randomized into receiving either ND or DID diet. During this period, DID diet fed mice were orally gavaged every day with 10.sup.10 of L. lactis.sup.pNZ or L. lactis.sup.pNZ-Apyr. After 8 weeks, mice were sacrificed and analyzed in order to evaluate the effects of the different diets.

[0447] DID is characterized by a modification of gut microbiota that can impair the gut barrier functions of the intestinal mucosa, leading to enhanced mucosa permeability and subsequent translocation of commensal bacteria and or bacterial products into blood circulation (Fukui, H. (2019). Role of Gut Dysbiosis in Liver Diseases: What Have We Learned So Far? Diseases 7:58 doi: 10.3390/diseases7040058).

[0448] In order to investigate if L. lactis.sup.pNZ-Apyr could preserve intestinal integrity, dysbiosis was induced and bacteria expressing apyrase were administered as described above (experimental schedule as shown in FIG. 68). After 8 weeks of ND or DID diet, mice were orally gavaged with fluorescein isothiocyanate (FITC)-labeled dextran and subsequently FITC levels were measured in the serum. Results are shown in FIG. 69. DID mice either treated or not with L. lactis.sup.pNZ were characterized by significantly higher concentrations of FITC in serum compared to ND mice. In contrast, mice fed with DID diet and treated with L. lactis.sup.pApyr showed levels of FITC in serum that were comparable to ND animals and significantly lower with respect to mice fed with stand-alone DID diet or in association with L. lactis.sup.pNZ. These data indicate that L. lactis.sup.pNZ-Apyr administration attenuates gut barrier disruption caused by DID. Moreover, in order to investigate the effect of apyrase in the mitigation of bacterial translocation to the MLN, mice were sacrificed at the end of the experiment (see FIG. 68), MLN were harvested aseptically into RPMI and mechanically homogenized. Dilutions were plated onto Schaedler agar (BD Biosciences). Plates were grown under aerobic or anaerobic culture conditions at 37° C. for 24-72 h before enumeration of colonies. Results are shown in FIG. 70 and FIG. 71. Quantification of CFU from the MLN, both in aerobic and anaerobic conditions, revealed a significant increase of MLN CFU in DID diet or DID diet+L. lactis.sup.pNZ treated mice compared to ND animals. In contrast, DID mice treated with L. lactis.sup.pNZ-Apyr showed a number of CFU in the MLN that was not significantly different from ND animals and significantly lower with respect to DID diet and DID diet+L. lactis.sup.pNZ mice. These data indicate that L. lactis.sup.pNZ-Apyr administration attenuates bacterial translocation induced by DID.

[0449] Concomitantly, DID and DID+L. lactis.sup.pNZ mice developed mild signs of intestinal inflammation quantified as increased levels of lipocalin-2 (LCN-2) in the stools compared to ND mice. Strikingly, mice fed with DID diet and treated with L. lactis.sup.pNZ-Apyr showed levels of fecal LCN-2 that were not significantly different from ND animals and significantly lower with respect to mice fed with stand-alone DID diet or in association with L. lactis.sup.pNZ. These data indicate that L. lactis.sup.pNZ-Apyr administration attenuates intestinal inflammation caused by DID (results are shown in FIG. 72).

Example 16: Administration of L. lactis.SUP.pNZ-Apyr .Counteracts Intestinal Barrier Disruption and Bacterial Translocation to MLN Caused by Diet Induced Dysbiosis in Neonatal Mice

[0450] Transmission of metabolic diseases from mother to child is multifactorial and includes genetic, epigenetic and environmental influences. Evidence in rodents, humans and non-human primates support the scientific premise that exposure to diet induced dysbiosis during pregnancy creates a long-lasting metabolic signature on the infant immune system and juvenile microbiota, which predisposes the offspring to obesity and metabolic diseases. In neonates, gastrointestinal microbes introduced through the mother are noted for their ability to serve as direct inducers/regulators of the infant immune system. Neonates have a limited capacity to initiate an immune response. Thus, disruption of microbial colonization during the early neonatal period results in disrupted postnatal immune responses. Although the mechanisms are poorly understood, increasing evidence suggests that diet induced dysbiosis during pregnancy influences the development and modulation of the infant microbiota composition, liver and other organs through direct communication via the portal system, metabolite production, alterations in gut barrier integrity and the hematopoietic immune cell axis (Collado, M. C., Isolauri, E., Laitinen, K., and Salminen, S. (2010). Effect of mother's weight on infant's microbiota acquisition, composition, and activity during early infancy: a prospective follow-up study initiated in early pregnancy. Am J Clin Nutr 92, 1023-1030; Collado, M. C., Laitinen, K., Salminen, S., and Isolauri, E. (2012). Maternal weight and excessive weight gain during pregnancy modify the immunomodulatory potential of breast milk. Pediatr Res 72, 77-85).

[0451] A neonatal model of diet-induced dysbiosis (DID) was created to investigate possible beneficial effect of apyrase on DID in neonates. Dysbiosis was induced starting from the mothers that were fed with a diet characterized by 7% protein, 5% fat and 88% carbohydrates. A normal diet (ND) characterized by 20% protein, 15% fat and 65% carbohydrate was used as control. A schematic of the components of each diet is shown in FIG. 67. Experimental layout in FIG. 73 shows the neonatal model of DID. At eight weeks of age, female C57BL/6 mice were randomized into receiving either a ND or a DID diet. After 15 days, ND and DID female C57BL/6 mice were mated with a ND male mice. Starting immediately after birth, DID pups were orally gavaged with 10.sup.8 of L. lactis.sup.pNZ or L. lactis.sup.pNZ-Apyr two times a week until 21 days after birth. Pups were daily monitored for body weight, tail length and behavior until 21 days after birth.

[0452] In order to investigate if L. lactis.sup.pNZ-Apyr could preserve intestinal integrity, 21 days old DID or ND mice were orally gavaged with fluorescein isothiocyanate (FITC)-labeled dextran and subsequently FITC levels were measured in the serum. Results are shown in FIG. 74. DID and DID+L. lactis.sup.pNZ mice were characterized by higher concentration of FITC in serum compared to ND mice. In contrast, mice fed with DID diet and treated with L. lactis.sup.pNZ-Apyr showed levels of FITC in serum that were not significantly different from ND animals, while they were significantly lower with respect to mice fed with stand-alone DID diet or in association with L. lactis.sup.pNZ. These data indicate that L. lactis.sup.pNZ-Apyr administration attenuates gut barrier disruption caused by diet induced dysbiosis.

[0453] Moreover, in order to investigate the effect of apyrase in the mitigation of bacterial translocation to the MLN, mice were sacrificed at the end of the experiment (see FIG. 73), MLN were harvested aseptically into RPMI and mechanically homogenized. Dilutions were plated onto Schaedler agar (BD Biosciences). Plates were grown under aerobic or anaerobic culture conditions at 37° C. for 24-72 h before enumeration of colonies. Results are shown in FIG. 75 and FIG. 76. Quantification of CFU in the MLN, both in aerobic and anaerobic conditions, revealed a significant increase of CFU in DID or DID+L. lactis.sup.pNZ mice compared to the ND animals. In contrast, DID mice treated with L. lactis.sup.pNZ-Apyr showed a number of CFU in the MLN that was at all similar to control animals and significantly lower with respect to DID and DID+L. lactis.sup.pNZ mice. These data indicate that L. lactis.sup.pNZ-Apyr administration counteracts bacterial translocation induced by diet induced dysbiosis.

Example 17: Administration of L. lactis.SUP.pNZ-Apyr .Improves Growth Parameters Affected by Diet Induced Dysbiosis in Neonatal Mice

[0454] Maternal protein deficiency causes severe dysbiosis that results in fetal growth retardation and predisposition to diseases in adult life (Rees, W. D., Hay, S. M., Buchan, V., Antipatis, C., and Palmer, R. M. (1999). The effects of maternal protein restriction on the growth of the rat fetus and its amino acid supply. Br J Nutr 81, 243-250.).

[0455] In order to understand if apyrase could have an impact on growth parameters in the offspring, neonates born from ND and DID dams were orally gavaged with 10.sup.8 of L. lactis.sup.pNZ or L. lactis.sup.pNZ-Apyr two times a week until 21 days after birth (see FIG. 73). At day 21 after birth, body weight, tail length, small intestine and colon length were evaluated. Diet induced dysbiosis in the mothers significantly affected body weight variation (FIG. 77), tail length (FIG. 78), small intestine (FIG. 79) and colon length (FIG. 80) in both DID and DID+L. lactis.sup.pNZ neonates. Notably, DID neonates treated with L. lactis.sup.pNZ-Apyr showed an amelioration of all the different growth parameters compared to the others DID groups. Therefore, L. lactis.sup.pNZ-Apyr administration improves the growth retardation caused by DID.

TABLE-US-00002 TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NO Sequence Remarks SEQ ID NO: 1 MKTKNFLLFCIATNMIFIPSANALKAEGFLTQQTSPDSLSI Apyrase LPPPPAEDSVVFLADKAHYEFGRSLRDANRVRLASEDAY YENFGLAFSDAYGMDISRENTPILYQLLTQVLQDSHDYA VRNAKEYYKRVRPFVIYKDATCTPDKDEKMAITGSYPSG HASFGWAVALILAEINPQRKAEILRRGYEFGESRVICGAH WQSDVEAGRLMGASVVAVLHNTPEFTKSLSEAKKEFEEL NTPTNELTP SEQ ID NO: 2 MKTKNFLLFCIATNMIFIPSANALKAEGFLTQQTSPDSLSI Loss-of- LPPPPAEDSVVFLADKAHYEFGRSLRDANRVRLASEDAY function YENFGLAFSDAYGMDISRENTPILYQLLTQVLQDSHDYA isoform  VRNAKEYYKRVRPFVIYKDATCTPDKDEKMAITGSYPSG of apyrase HASFGWAVALILAEINPQRKAEILRRGYEFGESPVICGAH WQSDVEAGRLMGASVVAVLHNTPEFTKSLSEAKKEFEEL NTPTNELTP SEQ ID NO: 3 ATGAAAACCAAAAACTTTCTTCTTTTTTGTATTGCTACA phoN2 gene  AATATGATTTTTATCCCCTCAGCAAATGCTCTGAAGGC encoding AGAAGGTTTTCTCACTCAACAAACTTCACCAGACAGTT apyrase TGTCAATACTTCCGCCGCCTCCGGCAGAGGATTCAGT AGTATTTCTGGCTGACAAAGCTCATTATGAATTCGGCC GCTCGCTCCGGGATGCTAATCGTGTACGTCTCGCTAG CGAAGATGCATACTACGAGAATTTTGGTCTTGCATTTT CAGATGCTTATGGCATGGATATTTCAAGGGAAAATAC CCCAATCTTATATCAGTTGTTAACACAAGTACTACAGG ATAGCCATGATTACGCCGTGCGTAACGCCAAAGAATA TTATAAAAGAGTTCGTCCATTCGTTATTTATAAAGACG CAACCTGTACACCTGATAAAGATGAGAAAATGGCTAT CACTGGCTCTTATCCCTCTGGTCATGCATCCTTTGGTT GGGCAGTAGCACTGATACTTGCGGAGATTAATCCTCA ACGTAAAGCGGAAATACTTCGACGTGGATATGAGTTT GGAGAAAGTCGGGTCATCTGCGGTGCGCATTGGCAA AGCGATGTAGAGGCTGGGCGTTTAATGGGAGCATCG GTTGTTGCAGTACTTCATAATACACCTGAATTTACCAA AAGCCTTAGCGAAGCCAAAAAAGAGTTTGAAGAATTA AATACTCCTACCAATGAACTGACCCCATAA SEQ ID NO: 4 CCTACGGGNGGCWGCAG forward primer SEQ ID NO: 5 GACTACHVGGGTATCTAATCC reverse primer SEQ ID NO: 6 CGAGCAGGCACACTGGAAGTATTGCTGCATCAGGCG EcN malP gene  CAGCTTTTTACCGGCAGTATGGTTGTCGTTTGGATAGA portion GAACTTTGGTCAGTTTTTCCGCGTTGATGCCCTGCTGT TCGGCACGCAGGAAATCACCGTCGTTAAATTTAGTCA GATCAAACGGATGCGCATGCGTCGCCTGCCACAGACG CAGTGGCTGCGCCACGCCATTACGATAGCCGACAACG GGGAGATCCCACGCTTGACCGGTAATGGTAAACTCCG GCTCCCAGCGTCCATCTTTCGTCACTTTACCGCCAATC CCTACCTGCACATCCAGTGCTTCGTTGTGGCGGAACC ACGGGTAGTTACCGCGATGCCAGTCATCCGGCGCTTC AACCTGTTTGCCATCGACAAATGACTGGCGGAACAAG CCATATTGATAATTAAGGCCGTAGCCAGTAGCTGACT GCCCGACAGTTGCCATTGAGTCGAGGAAGCACGCCG CCAGACGTCCCAGACCACCGTTCCCCAGCGCCGGGTC GATCTCTTCTTCCAACAGGTCAGTCAGGTTGATGTCAT AAGCCTTCAACGAATCCTGTACATCCTGATACCAGCCG AGATTCAACAGGTTGTTGCCCGTCAGGCGACCAATCA AAAACTCCATTGAGATGTAGTTAACATGTCGCTGATTC GCCACTGGCTTGGCGAATGGCTGAGCACGCAGCATTT CGGCCAGTGCTTCGCTCACTGCCAGCCACCACTGGCG AGGAGTCATTTCAGCCGCAGAATTTAAGCCATAACGC TGCCACTGACGTGAAAGCGCTTCCTGAAATTGCTTATC GTTAAAAATAGGTTGTGACAT SEQ ID NO: 7 ATGCTGATTCCGTCAAAATTAAGTCGTCCGGTTCGACT EcN malT gene  CGACCATACCGTGGTTCGTGAGCGCCTGCTGGCTAAA portion CTTTCCGGCGCGAACAACTTCCGGCTGGCGCTGATCA CAAGTCCTGCGGGCTACGGAAAGACCACGCTCATTTC CCAGTGGGCGGCAGGCAAAAACGATATCGGCTGGTA CTCGCTGGATGAAGGTGATAACCAGCAAGAGCGTTTC GCCAGCTATCTCATTGCCGCCGTGCAACAGGCAACCA ACGGTCACTGCGCGATATGTGAGACGATGGCGCAAA AACGGCAATATGCCAGCCTGACGTCACTCTTCGCCCA GCTTTTCATTGAGCTGGCGGAATGGCATAGCCCACTTT ATCTGGTCATCGATGACTATCATCTGATCACTAATCCT GTGATCCACGAGTCAATGCGCTTCTTTATTCGCCATCA ACCAGAAAATCTCACCCTTGTGGTGTTGTCACGCAACC TTCCGCAACTGGGCATTGCCAATCTGCGTGTTCGTCCA GCTAGCGAATTCGCTGGAAATTGGCAGTCAGCAACTG GCATTTACCCATCAGGAAGCGAAGCAGTTTTTTGATT GCCGTCTGTCATCGCCGATTGAAGCTGCAGAAAGCAG TCGGATTTGTGATGATGTTTCCGGTTGGGCGACGGCA CTGCAGCTAATCGCCCTCTCCGCCCGGCAGAATACTCA CTCAGCCCATAAGTCGGCACGCCGCCTGGCGGGAATC AATGCCAGCCATCTTTCGGATTATCTGGTCGATGAGG TTTTGGATAACGTCGATCTCGCAACGCGCCA SEQ ID NO: 8 CAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGT DNA fragment CTATGAGTGGTTGCTGGATAACTTTACGGGCATGCAT including  AAGGCTCGTATAATATATTCAGGGAGACCACAACGGT the P.sub.proD TTCCCTCTACAAATAATTTTGTTTAACTTTTACTAGAGT promoter,  CACACAGGAAAGTACTAGATGAAAACCAAAAACTTTC the BBa_BB0032  TTCTTTTTTGTATTGCTACAAATATGATTTTTATCCCCTC RBS, the AGCAAATGCTCTGAAGGCAGAAGGTTTTCTCACTCAA S. flexneri  CAAACTTCACCAGACAGTTTGTCAATACTTCCGCCGCC phoN2 gene TCCGGCAGAGGATTCAGTAGTATTTCTGGCTGACAAA and the phoN2 GCTCATTATGAATTCGGCCGCTCGCTCCGGGATGCTA transcriptional ATCGTGTACGTCTCGCTAGCGAAGATGCATACTACGA terminator GAATTTTGGTCTTGCATTTTCAGATGCTTATGGCATGG ATATTTCAAGGGAAAATACCCCAATCTTATATCAGTTG TTAACACAAGTACTACAGGATAGCCATGATTACGCCG TGCGTAACGCCAAAGAATATTATAAAAGAGTTCGTCC ATTCGTTATTTATAAAGACGCAACCTGTACACCTGATA AAGATGAGAAAATGGCTATCACTGGCTCTTATCCCTCT GGTCATGCATCCTTTGGTTGGGCAGTAGCACTGATAC TTGCGGAGATTAATCCTCAACGTAAAGCGGAAATACT TCGACGTGGATATGAGTTTGGAGAAAGTCGGGTCATC TGCGGTGCGCATTGGCAAAGCGATGTAGAGGCTGGG CGTTTAATGGGAGCATCGGTTGTTGCAGTACTTCATA ATACACCTGAATTTACCAAAAGCCTTAGCGAAGCCAA AAAAGAGTTTGAAGAATTAAATACTCCTACCAATGAA CTGACCCCATAAAGCTGGACAGCCTGT SEQ ID NO: 9 GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGGC DNA fragment GCGCCTACCTGTGACGGAAGATCACTTCGCAGAATAA including the  ATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCT E. coli cat    GGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCA gene flanked  CGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGA by the TCACTACCGGGCGTATTTTTTGAGTTGTCGAGATTTTC FRT sequences AGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCAC TGGATATACCACCGTTGATATATCCCAATGGCATCGTA AAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGT ACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTT TTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTAT CCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGC TCATCCGGAATTACGTATGGCAATGAAAGACGGTGAG CTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGT TTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGA GTGAATACCACGACGATTTCCGGCAGTTTCTACACATA TATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGG CCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTC GTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGA TTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCG TTTTCACCATGGGCAAATATTATACGCAAGGCGACAA GGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCC GTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATG AATTACAACAGTACTGCGATGAGTGGCAGGGCGGGG CGTAAGGCGCGCCATTTAAATGAAGTTCCTATTCCGA AGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAAGCA GCTCCAGCCTACACAATGAATTC