In aquatic environments, Vibrio cholerae colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of chiA2 is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of V. cholerae chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for V. cholerae survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that V. cholerae could utilize mucin as a nutrient source. In comparison to the wild type strain, ΔchiA2 mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also ∼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the ΔchiA2 mutant caused ∼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the ΔchiA2 mutant and wild type V. cholerae was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by V. cholerae in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by V. cholerae for growth and survival in the host intestine.

The Vibrio cholerae O1 serogroup is responsible for pandemic cholera and is divided into the classical and El Tor biotypes. Classical V. cholerae produces acid when using glucose as a carbon source, whereas El Tor V. cholerae produces the neutral product acetoin when using glucose as a carbon source. An earlier study demonstrated that Escherichia coli strains that metabolize glucose to acidic byproducts drastically reduced the survival of V. cholerae strains in vitro. In the present study, zebrafish were fed 1% glucose and either inoculated with single V. cholerae or E. coli strains or coinfected with both V. cholerae and E. coli. A significant decrease in classical biotype colonization was observed after glucose feeding due to acid production in the zebrafish intestine. El Tor colonization was unaffected by glucose alone. However, the El Tor strain exhibited significantly lower colonization of the zebrafish when either of the acid-producing E. coli strains was coinoculated in the presence of glucose. An E. coli sugar transport mutant had no effect on V. cholerae colonization even in presence of glucose. Glucose and E. coli produced a prophylactic effect on El Tor colonization in zebrafish when E. coli was inoculated before V. cholerae infection. Thus, the probiotic feeding of E. coli inhibits V. cholerae colonization in a natural host. This suggests that a similar inhibitory effect could be seen in cholera patients, especially if a glucose-based oral rehydration solution (ORS) is administered in combination with probiotic E. coli during cholera treatment. FIG 2 Effect of glucose feeding on classical and El Tor V. cholerae colonization of zebrafish intestine. Zebrafish were fed 1% glucose for 12 h, and ϳ5 ϫ 10 6 CFU/ml of either V. cholerae O395 or N16961 was inoculated in zebrafish. V. cholerae levels were determined by plating of serial dilutions of the intestinal homogenates. *, P ϭ 0.005. Probiotic Inhibition of V. cholerae Colonization Infection and Immunity December 2018 Volume 86 Issue 12 e00486-18 iai.asm.org 3 on July 5, 2020 by guest http://iai.asm.org/ Downloaded from FIG 3 Effect of E. coli strains plus glucose on V. cholerae colonization of zebrafish intestine. Zebrafish were fed 1% glucose for 12 h, and ϳ5 ϫ 10 6 CFU/ml of E. coli 40 and N was coinoculated with ϳ5 ϫ 10 6 CFU/ml of either V. cholerae O395 or N16961. (A) Coinfection of E. coli with classical V. cholerae strain O395. (B) Coinfection of E. coli with El Tor V. cholerae strain N16961. V. cholerae levels were determined by plating of serial dilutions of the intestinal homogenates. Horizontal bars indicate the mean colonization level for each group, and individual symbols indicate the results for individual fish. ***, P Ͻ 0.0001; **, P Ͻ 0.001; NS, nonsignificant differences.Probiotic Inhibition of V. cholerae Colonization Infection and Immunity December 2018 Volume 86 Issue 12 e00486-18 iai.asm.org 5 on July 5, 2020 by guest http://iai.asm.org/ Downloaded from FIG 5 Effect of E. coli feeding plus glucose before V. cholerae infection on N16...

Purpose: The radiosensitivity of the normal intestinal epithelium is the major limiting factor for definitive radiotherapy against abdominal malignancies. Radiosensitizers, which can be used without augmenting radiation toxicity to normal tissue, are still an unmet need. Inhibition of proteosomal degradation is being developed as a major therapeutic strategy for anticancer therapy as cancer cells are more susceptible to proteasomal inhibition-induced cytotoxicity compared with normal cells. Auranofin, a goldcontaining antirheumatoid drug, blocks proteosomal degradation by inhibiting deubiquitinase inhibitors. In this study, we have examined whether auranofin selectively radiosensitizes colon tumors without promoting radiation toxicity in normal intestine.Experimental Design: The effect of auranofin (10 mg/kg i.p.) on the radiation response of subcutaneous CT26 colon tumors and the normal gastrointestinal epithelium was deter-mined using a mouse model of abdominal radiation. The effect of auranofin was also examined in a paired human colonic organoid system using malignant and nonmalignant tissues from the same patient.Results: Both in the mouse model of intestinal injury and in the human nonmalignant colon organoid culture, auranofin pretreatment prevented radiation toxicity and improved survival with the activation of p53/p21-mediated reversible cellcycle arrest. However, in a mouse model of abdominal tumor and in human malignant colonic organoids, auranofin inhibited malignant tissue growth with inhibition of proteosomal degradation, induction of endoplasmic reticulum stress/ unfolded protein response, and apoptosis.Conclusions: Our data suggest that auranofin is a potential candidate to be considered as a combination therapy with radiation to improve therapeutic efficacy against abdominal malignancies.

Like most other Gram‐negative bacteria, Shigella releases outer membrane vesicles (OMVs) into the surrounding environment during growth. In this study, we have exploited OMVs of Shigella as a protective immunogen in a mice model against Shigellosis. Distinctive vesicle secretion was noticed from different Shigella strains. Among them, Shigella boydii type 4 (BCH612) was secreting relatively higher amounts. We immunized female adult mice orally with 32 μg of purified Shigella boydii type 4 (BCH612) OMVs four times at 1‐week intervals. Antibodies against these vesicles were detected in immunized sera until 120 days, indicating a persistent immune response. To observe whether the passive immunity had been transferred to the neonates, the immunized female mice were mated and the offspring were challenged orally, with wild‐type homologous and heterologous Shigella strains. All offspring of immunized mothers survived the challenge with homologous strain BCH612 and up to 81% protective efficacy was noted against heterologous strains Shigella dysenteriae 1, Shigella flexneri 2a, Shigella flexneri 3a, Shigella flexneri 6 and Shigella sonnei. Our results exhibited for the first time that oral immunization of adult female mice with purified OMVs of Shigella, without any adjuvant, conferred passive protection to their offspring against shigellosis. These findings will contribute to the future development of a potential non‐living vaccine candidate against shigellosis.

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Background Vibrio cholerae non-O1/ non-O139 serogroups have been reported to cause sporadic diarrhoea in humans. Cholera toxins have been mostly implicated for hypersecretion of ions and water into the small intestine. Though most of the V. cholerae non-O1/ non-O139 strains lack these cholera toxins, several other innate virulence factors contribute towards their pathogenicity. The environmental isolates may thus act as reservoirs for potential spreading of these virulence genes in the natural environment which may cause the emergence of epidemic-causing organisms.ResultsThe environmental isolates of vibrios were obtained from water samples, zooplanktons and phytoplanktons, from a village pond in Gandhinagar, Gujarat, India. They were confirmed as Vibrio cholerae non-O1/ non-O139 using standard biochemical and serotyping tests. PCR experiments revealed that the isolates lacked ctxA, ctxB, tcpA, zot and ace genes whereas other pathogenicity genes like toxR, rtxC, hlyA, hapA and prtV were detected in these isolates. Compared with epidemic strain V. cholerae O1 El Tor N16961, culture supernatants from most of these isolates caused higher cytotoxicity to HT29 cells and higher hemolytic, hemagglutinin and protease activities. In rabbit ileal loop assays, the environmental isolates showed only 2-4 folds lesser fluid accumulation in comparison to N16961 and a V. cholerae clinical isolate IDH02365 of 2009. Pulsed Field Gel electrophoresis and Random amplification of Polymorphic DNA indicated that these isolates showed considerable diversity and did not share the same clonal lineage even though they were derived from the same water source. All the isolates showed resistance to one or more antibiotics.ConclusionThough these environmental isolates lacked the cholera toxins, they seem to have adopted other survival strategies by optimally utilising a diverse array of several other toxins. The current findings indicate the possibility that these isolates could cause some gastroenteric inflammation when ingested and may serve as progenitors for overt disease-causing organisms.

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Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.