Rotavirus non-structural protein 4 usurps host cellular RIPK1-RIPK3 complex to induce MLKL-dependent necroptotic cell death.

The dynamic interface between invading viral pathogens and programmed cell death (PCD) of the host is a finely regulated process. Host cellular demise at the end of the viral life cycle ensures the release of progeny virions to initiate new infection cycles. Rotavirus (RV), a diarrheagenic virus with double-stranded RNA genome, has been reported to trigger different types of PCD such as apoptosis and pyroptosis in a highly regulated way to successfully disseminate progeny virions. Recently our lab also showed that induction of MLKL-driven programmed necroptosis by RV. However, the host cellular machinery involved in RV-induced necroptosis and the upstream viral trigger responsible for it remained unaddressed. In the present study, the signalling upstream of MLKL-driven necroptosis has been delineated where the involvement of Receptor interacting serine/threonine kinase 3 (RIPK3) and 1 (RIPK1) from the host side and RV non-structural protein 4 (NSP4) as the viral trigger for necroptosis has been shown. Interestingly, RV-NSP4 was found to be an integral component of the necrosome complex by interacting with RIPK1, thereby bypassing the requirement of RIPK1 kinase activity. Subsequently, NSP4-driven elevated cytosolic Ca2+ concentration and Ca2+-binding to NSP4 lead further to RHIM domain-dependent RIPK1-RIPK3 interaction, RIPK3-dependent MLKL phosphorylation, and eventual necroptosis. Overall, this study presents the interplay between RV-NSP4 and the host cellular necrosome complex to induce necroptotic death of host cells.

Establishment of an intragastric surgical model using C57BL/6 mice to study the vaccine efficacy of OMV-based immunogens against Helicobacter pylori.

Chronic gastritis is one of the major symptoms of gastro-duodenal disorders typically induced by Helicobacter pylori (H. pylori). To date, no suitable model is available to study pathophysiology and therapeutic measures accurately. Here, we have presented a successful surgical infection model of H. pylori-induced gastritis in C57BL/6 mice that resembles features similar to human infection. The proposed model does not require any preparatory treatment other than surgical intervention. C57BL/6 mice were injected with wild-type SS1 (Sydney strain 1, reference strain) directly into the stomach. Seven days post infection, infected animals showed alterations in cytokine responses along with inflammatory cell infiltration in the lamina propria, depicting a prominent inflammatory response due to infection. To understand the immunogenicity and protective efficacy, the mice were immunized with outer membrane vesicles (OMVs) isolated from an indigenous strain with putative virulence factors of H. pylori [A61C (1), cag+/vacA s1m1]. In contrast to the non-immunized cohort, the OMV-immunized cohort showed a gradual increase in serum immunoglobulin(s) levels on the 35th day after the first immunization. This conferred protective immunity against subsequent challenge with the reference strain (SS1). Direct inoculation of H. pylori into the stomach influenced infection in a short time and, more importantly, in a dose-dependent manner, indicating the usefulness of the developed model for pathophysiology, therapeutic and prophylactic studies.

Controlling the bacterial load of Salmonella Typhi in an experimental mouse model by a lytic Salmonella phage STWB21: a phage therapy approach.

BACKGROUND: Salmonella enterica serotype Typhi is one of the major pathogens causing typhoid fever and a public health burden worldwide. Recently, the increasing number of multidrug-resistant strains of Salmonella spp. has made this utmost necessary to consider bacteriophages as a potential alternative to antibiotics for S. Typhi infection treatment. Salmonella phage STWB21, isolated from environmental water, has earlier been reported to be effective as a safe biocontrol agent by our group. In this study, we evaluated the efficacy of phage STWB21 in reducing the burden of salmonellosis in a mammalian host by inhibiting Salmonella Typhi invasion into the liver and spleen tissue. RESULTS: Phage treatment significantly improved the survival percentage of infected mice. This study also demonstrated that oral administration of phage treatment could be beneficial in both preventive and therapeutic treatment of salmonellosis caused by S. Typhi. Altogether the result showed that the phage treatment could control tissue inflammation in mice before and after Salmonella infection. CONCLUSIONS: To the best of our knowledge, this is the first report of phage therapy in a mouse model against a clinically isolated Salmonella Typhi strain that includes direct visualization of histopathology and ultrathin section microscopy images from the liver and spleen sections.

Pentavalent outer membrane vesicles immunized mice sera confers passive protection against five prevalent pathotypes of diarrhoeagenic Escherichia coli in neonatal mice.

Diarrhoeagenic Escherichia coli (DEC) pathotypes are one of the major causative agents of diarrhoea induced childhood morbidity and mortality in developing countries. Licensed vaccines providing broad spectrum protection against DEC mediated infections are not available. Outer membrane vesicles (OMVs) are microvesicles released by gram-negative bacteria during the growth phase and contain multiple immunogenic proteins. Based on prevalence of infections, we have formulated a pentavalent outer-membrane vesicles (POMVs) based immunogen targeting five main pathotypes of DEC responsible for diarrhoeal diseases. Following isolation, OMVs from five DEC pathotypes were mixed in equal proportions to formulate POMVs and 10 µg of the immunogen was intraperitoneally administered to adult BALB/c mice. Three doses of POMVs induced significant humoral immune response against whole cell lysates (WCLs), outer membrane proteins (OMPs) and lipopolysaccharides (LPS) isolated from DEC pathotypes along with significant induction of cellular immune response in adult mice. Passive transfer of POMVs immunized adult mice sera protected neonatal mice significantly against DEC infections. Overall, this study finds POMVs to be immunogenic in conferring broad-spectrum passive protection to neonatal mice against five main DEC pathotypes. Altogether, these findings suggest that POMVs can be used as a potent vaccine candidate to ameliorate the DEC-mediated health burden.

Bacterial ghost cell based bivalent candidate vaccine against Salmonella Typhi and Salmonella Paratyphi A: A prophylactic study in BALB/c mice.

Typhoid and emerging paratyphoid fever are a severe enteric disease worldwide with high morbidity and mortality. Licensed typhoid vaccines are in the market, but no paratyphoid vaccine is currently available. In the present study we developed a bivalent vaccine against Salmonella Typhi and Paratyphi A using a bacterial ghost platform. Bacterial ghost cells (BGs) are bacteria-derived cell membranes without cytoplasmic contents that retain their cellular morphology, including all cell surface features. Furthermore, BGs have inherent adjuvant properties that promote an enhanced humoral and cellular immune reaction to the target antigen. Sodium hydroxide was used to prepare ghost cells of Salmonella Typhi and Paratyphi A. The bacterial ghost cells were characterised using electron microscopy. Then BALB/c mice were immunized three times (0th, 14th and 28th day) with the bivalent typhoidal bacterial ghost cells. Haematological study of adult mice throughout immunization period reflected that the immunogen was safe to administer and does not affect the animals' health. After complete immunization, we found significant serum antibody titter against whole cell lysate, outer membrane protein and lipopolysaccharide of both bacteria, and cell-mediated immunity was observed in an ex-vivo experiment. CD4+, CD8a+ and CD19+ splenic cell populations were increased in immunized animals. Bivalent Typhoidal ghost cell immunized mice showed better survival, less bacterial colonization in systemic organs, and less inflammation and/or destruction of tissue in histopathological analysis than non-immunized control mice.Serum antibodies of immunized animals can significantly inhibit bacterial motility and mucin penetration ability with better killing properties against Salmonella Typhi and Paratyphi A. Furthermore, significant passive protection was observed through the adoptive transfer of serum antibody and lymphocytes of immunized animals to naïve animals after bacterial infection. In summary, Bivalent Typhoidal Bacterial Ghost cells (BTBGs) enhances immunogenic properties and serves as a safe and effective prevention strategy against Salmonella Typhi and Paratyphi A.

Cloning, improved expression and purification of invasion plasmid antigen D (IpaD): an effector protein of enteroinvasive Escherichia coli (EIEC).

The widespread increase in broad-spectrum antimicrobial resistance is making it more difficult to treat gastrointestinal infections. Enteroinvasive Escherichia coli is a prominent etiological agent of bacillary dysentery, invading via the fecal-oral route and exerting virulence on the host via the type III secretion system. IpaD, a surface-exposed protein on the T3SS tip that is conserved among EIEC and Shigella, may serve as a broad immunogen for bacillary dysentery protection. For the first time, we present an effective framework for improving the expression level and yield of IpaD in the soluble fraction for easy recovery, as well as ideal storage conditions, which may aid in the development of new protein therapies for gastrointestinal infections in the future. To achieve this, uncharacterized full length IpaD gene from EIEC was cloned into pHis-TEV vector and induction parameters were optimized for enhanced expression in the soluble fraction. After affinity-chromatography based purification, 61% pure protein with a yield of 0.33 mg per litre of culture was obtained. The purified IpaD was retained its secondary structure with a prominent α-helical structure as well as functional activity during storage, at 4°C, -20°C and -80°C using 5% sucrose as cryoprotectants, which is a critical criterion for protein-based treatments.

Genomic insights into extensively drug-resistant Pseudomonas aeruginosa isolated from a diarrhea case in Kolkata, India.

Aim: To characterize extensively drug-resistant Pseudomonas aeruginosa from a patient with diarrhea. Materials & methods: Antimicrobial susceptibility was tested by the disk diffusion method. The P. aeruginosa genome was sequenced to identify virulence, antibiotic resistance and prophages encoding genes. Results: P. aeruginosa had a wide spectrum of resistance to antibiotics. Genomic analysis of P. aeruginosa revealed 76 genes associated with antimicrobial resistance, xenobiotic degradation and the type three secretion system. Conclusion: This is the first report on diarrhea associated with P. aeruginosa. Since no other organism was identified, the authors assume that the patient had dysbiosis due to antibiotic exposure, leading to antibiotic-associated diarrhea. The in vivo toxicity expressed by the pathogen may be associated with T3SS.

Facile synthesis of multi-faceted, biomimetic and cross-protective nanoparticle-based vaccines for drug-resistant Shigella: a flexible platform technology.

BACKGROUND: No commercial vaccines are available against drug-resistant Shigella due to serotype-specific/narrow-range of protection. Nanoparticle-based biomimetic vaccines involving stable, conserved, immunogenic proteins fabricated using facile chemistries can help formulate a translatable cross-protective Shigella vaccine. Such systems can also negate cold-chain transportation/storage thus overcoming challenges prevalent in various settings. METHODS: We explored facile development of biomimetic poly (lactide-co-glycolide)/PLGA 50:50 based nanovaccines (NVs), encapsulating conserved stabilized antigen(s)/immunostimulant of S. dysenteriae 1 origin surface-modified using simple chemistries. All encapsulants (IpaC/IpaB/LPS) and nanoparticles (NPs)-bare and modified (NV), were thoroughly characterized. Effect of IpaC on cellular uptake of NPs was assessed in-vitro. Immunogenicity of the NVs was assessed in-vivo in BALB/c mice by intranasal immunization. Cross-protective efficacy was assessed by intraperitoneally challenging the immunized groups with a high dose of heterologous S. flexneri 2a and observing for visible diarrhea, weight loss and survival. Passive-protective ability of the simplest NV was assessed in the 5-day old progeny of vaccinated mice. RESULTS: All the antigens and immunostimulant to be encapsulated were successfully purified and found to be stable both before and after encapsulation into NPs. The ~ 300 nm sized NPs with a zeta potential of ~ - 25 mV released ~ 60% antigen by 14th day suggesting an appropriate delivery kinetics. The NPs could be successfully surface-modified with IpaC and/or CpG DNA. In vitro experiments revealed that the presence of IpaC can significantly increase cellular uptake of NPs. All NVs were found to be cytocompatible and highly immunogenic. Antibodies in sera of NV-immunized mice could recognize heterologous Shigella. Immunized sera also showed high antibody and cytokine response. The immunized groups were protected from diarrhea and weight loss with ~ 70-80% survival upon heterologous Shigella challenge. The simplest NV showed ~ 88% survival in neonates. CONCLUSIONS: Facile formulation of biomimetic NVs can result in significant cross-protection. Further, passive protection in neonates suggest that parental immunization could protect infants, the most vulnerable group in context of Shigella infection. Non-invasive route of vaccination can also lead to greater patient compliance making it amenable for mass-immunization. Overall, our work contributes towards a yet to be reported platform technology for facile development of cross-protective Shigella vaccines.

A tetravalent Shigella outer membrane vesicles based candidate vaccine offered cross-protection against all the serogroups of Shigella in adult mice.

In today's world and mostly in low and middle income countries, Shigella flexneri and Shigella sonnei remains the major causative agent of clinical bacillary dysentery. Based on contemporary epidemiology, a tetravalent Outer Membrane Vesicle (OMVs) based immunogen was formulated using the most commonly circulating Shigella strains, namely, S. flexneri 2a, S. flexneri 3a, S. flexneri 6 and S. sonnei I, in a 1:1:1:1 ratio. Adult BALB/c mice were orally immunized in a prime-boost-boost manner. Tetravalent Shigella OMVs immunogen induced significant and persistent serum and mucosal antibodies against OMVs, Outer Membrane Proteins (OMPs) and lipopolysaccharides (LPS). Tetravalent OMVs also primed cell mediated immune response effectively. Protective efficacy against six heterologous Shigella strains was checked in an intra-peritoneal mouse model. Immunized mice survived lethal infection better than the non-immunized mice cohort with fewer replicating bacteria isolated from their gut. This study establishes the possibilities of tetravalent OMVs immunogen to become a potent vaccine candidate against human shigellosis, overcoming the limitations of sero-specific cross-protection of Shigella species.

Suppression of classical nuclear import pathway by importazole and ivermectin inhibits rotavirus replication.

BACKGROUND: Rotavirus is the foremost cause of acute gastroenteritis among infants in resource-poor countries, causing severe morbidity and mortality. The currently available rotavirus vaccines are effective in reducing severity of the disease but not the infection rates, thus antivirals as an adjunct therapy are needed to reduce the morbidity in children. Viruses rely on host cellular machinery for nearly every step of the replication cycle. Therefore, targeting host factors that are indispensable for virus replication could be a promising strategy. OBJECTIVES: To assess the therapeutic potential of ivermectin and importazole against rotaviruses. METHODS: Antirotaviral activity of importazole and ivermectin was measured against various rotavirus strains (RV-SA11, RV-Wa, RV-A5-13, RV-EW) in vitro and in vivo by quantifying viral protein expression by western blot, analysing viroplasm formation by confocal microscopy, and measuring virus yield by plaque assay. RESULTS: Importin-ß1 and Ran were found to be induced during rotavirus infection. Knocking down importin-ß1 severely impaired rotavirus replication, suggesting a critical role for importin-ß1 in the rotavirus life cycle. In vitro studies revealed that treatment of ivermectin and importazole resulted in reduced synthesis of viral proteins, diminished production of infectious virus particles, and decrease in viroplasm-positive cells. Mechanistic study proved that both drugs perform antirotavirus activity by inhibiting the function of importin-ß1. In vivo investigations in mice also confirmed the antirotavirus potential of importazole and ivermectin at non-toxic doses. Treatments of rotavirus-infected mice with either drug resulted in diminished shedding of viral particles in the stool sample, reduced expression of viral protein in the small intestine and restoration of damaged intestinal villi comapared to untreated infected mice. CONCLUSIONS: The study highlights the potential of importazole and ivermectin as antirotavirus therapeutics.

Protective efficacy of fish oil nanoemulsion against non-typhoidal Salmonella mediated mucosal inflammation and loss of barrier function.

Non-typhoidal Salmonella serotypes are well adapted to utilize the inflammation for colonization in the mammalian gut mucosa and cause loss of the integrity of the epithelial barrier in the mammalian intestine. The present study assessed the protective efficacy of fish oil-in-water nanoemulsion, compared to the conventional emulsion, towards the intestinal epithelial barrier against invasive infection of Salmonella enterica serovar Typhimurium strain SL1344 in an in vivo streptomycin-treated mouse model. Non-typhoidal Salmonella enterica serovar Typhimurium strain SL1344 expresses its invasiveness by creating extreme inflammatory assault in the mammalian host lumen via its repertoire of secretory or membrane-bound proteins. Prophylactic treatment of ω-3 polyunsaturated fatty acid-rich fish oil nanoemulsion not only reduced the inflammatory markers by 4-5 fold against the established infection but also retained the gut barrier efficiency as shown by FITC-dextran permeability assay. Though the conventional emulsion also showed similar trends, the efficacy was significantly better with nanoemulsion treatment but neither the nanoemulsion nor conventional emulsion caused any significant change in the microbial colonization of the murine gut mucosa. Mechanistic assessment of the nanoemulsion against inflammation and invasion across the Caco-2 cell monolayer revealed that nanoemulsion treatment protected the expression of Zona occludens-1 along the tight junction, almost by 3-fold as compared to the infected cell monolayer. Such protection was evinced by the trans-epithelial electrical resistance value and the FITC-dextran permeability analysis as well. Fish oil nanoemulsion treatment has also shown significant reduction in pro-inflammatory cytokine expression by the Salmonella strain SL1344 infected Caco-2 cell monolayer. Conventional emulsion also showed distinct protection, but the nanoemulsion offered better protection at the same dosage of fish oil, probably due to its better bioavailability. The results proved that fish oil-loaded nanoemulsion can be efficacious towards maintaining the barrier function and protecting against systemic bacteremia during invasive intestinal infection.

Stable Recombinant Invasion Plasmid Antigen C (IpaC)-Based Single Dose Nanovaccine for Shigellosis.

Shigellosis, caused by the bacteria Shigella, is the leading cause of bacterial diarrhea and the second leading cause of diarrheal death among children under the age of five. Unfortunately, Shigella strains have acquired resistance to antibiotics, and a commercial vaccine is yet to be available. We have previously demonstrated that Shigella dysenteriae serotype 1 (Sd1)-based recombinant, stabilized, "invasion plasmid antigen C" (IpaC; 42 kDa) protein can induce robust immune responses in BALB/c mice against a challenge of a high dose of heterologous Shigella when immunized via three intranasal doses of IpaC without an adjuvant. In this work, in order to reduce the frequency of dosing and increase possible patient compliance, based on our previous screening, the minimum protective dose of stabilized IpaC (20 µg) was encapsulated in biodegradable polymeric poly(lactide-co-glycolide) nanoparticles (∼370 nm) and intranasally administered in BALB/c mice in a single dose. Interestingly, a single intranasal dose of the developed vaccine particles encapsulating only 20 µg of Sd1 IpaC led to a temporal increase in the antibody production with an improved cytokine response compared to free IpaC administered three times as described in our previous report. Upon intraperitoneal challenge with a high dose of heterologous Shigella flexneri 2a (common in circulation), the immunized animals were protected from diarrhea, lethargy, and weight loss with ∼67% survival, while all the control animals died by 36 h of the challenge. Overall, the developed nanovaccine could be explored as a potential noninvasive, cross-protective, single-dose, single-antigen Shigella vaccine amenable for scale-up and eventual mass immunization.

Quercetin, a flavonoid, combats rotavirus infection by deactivating rotavirus-induced pro-survival NF-κB pathway.

Rotavirus (RV) is the leading cause of acute gastroenteritis and watery diarrhea in children under 5 years accounting for high morbidity and mortality in countries with poor socioeconomic status. Although vaccination against RV has been implemented in more than 100 countries, the efficacy of vaccine has been challenged in low-income settings. The lack of any FDA-approved drug against RV is an additional concern regarding the treatment associated with rotavirus-induced infantile death. With the purpose for the discovery of anti-RV therapeutics, we assessed anti-rotaviral potential of quercetin, a well-characterized antioxidant flavonoid. In vitro study revealed that quercetin treatment resulted in diminished production of RV-SA11 (simian strain) viral particles in a concentration-dependent manner as estimated by the plaque assay. Consistent with this result, Western blot analysis also revealed reduced synthesis of viral protein in quercetin-treated RV-SA11-infected MA104 cells compared to vehicle (DMSO) treated controls. Not surprisingly, infection of other RV strains A5-13 (bovine strain) and Wa (Human strain) was also found to be abridged in the presence of quercetin compared to DMSO. The IC50 of quercetin against three RV strains ranges between 2.79 and 4.36 Mm, and S.I. index is greater than 45. Concurrent to the in vitro results, in vivo study in mice model also demonstrated reduced expression of viral proteins and viral titer in the small intestine of quercetin-treated infected mice compared to vehicle-treated infected mice. Furthermore, the result suggested anti-rotaviral activity of quercetin to be interferon-independent. Mechanistic study revealed that the antiviral action of quercetin is co-related with the inhibition of RV-induced early activation of NF-κB pathway. Overall, this study delineates the strong anti-RV potential of quercetin and also proposes it as future therapeutics against rotaviral diarrhea.

An Experimental Adult Zebrafish Model for Shigella Pathogenesis, Transmission, and Vaccine Efficacy Studies.

Shigellosis has been a menace to society for ages. The absence of an effective vaccine against Shigella, improper sanitation, and unhygienic use of food and water allow the disease to flourish. Shigella can also be transmitted via natural water bodies. In the absence of a good animal model, the actual nature of pathogenesis and transmission remains unclear. Zebrafish larvae have previously been described as a model for Shigella pathogenesis. However, larval fish lack a mature intestinal microbiota and immune system. Here, the adult zebrafish was assessed as a potential model for Shigella pathogenesis. Their well-developed innate and adaptive immune responses mimic the mammalian immune system. Shigella showed a clear dose-, time-, and temperature-dependent colonization of the adult zebrafish gut. Efficacy of a three-dose immunization regime was tested using bath immunization with heat-killed trivalent Shigella immunogen. The present study demonstrates the efficacy of an adult zebrafish model for pathogenesis, transmission, and vaccine efficacy studies. IMPORTANCE Shigellosis is a diarrheal disease that is prevalent in developing countries and especially dangerous in young children. Currently, animal models for shigellosis are unable to model some aspects of the infectious cycle. Here, we describe a new shigellosis model in adult zebrafish, an increasingly common model organism for studying bacterial pathogens. The zebrafish model can be used to study Shigella colonization, transmission, and immune responses, as well as test vaccine efficacy.

Development of a novel trivalent invasive non-typhoidal Salmonella outer membrane vesicles based vaccine against salmonellosis and fowl typhoid in chickens.

Poultry animals act as natural reservoirs of invasive non-typhoidal Salmonella [iNTS] serovars and consumption of iNTS contaminated poultry meat and eggs is one of the major sources of iNTS infection in developed and developing countries. Irrational use of antibiotics in the poultry industry gives rise to the global emergence of multi drug resistant iNTS strains. Among different strategies to control iNTS infection in poultry farms, vaccination is now being widely used. There are several licensed vaccines available in the market for poultry animals to ameliorate iNTS infection but none of them have broad spectrum protective efficacy. In this study we have formulated a single novel trivalent iNTS outer membrane vesicles [OMVs] based immunogen which can confer long term broad spectrum protection against most prevalent iNTS serovars. We have isolated OMVs from Salmonella Typhimurium [ST], Salmonella Enteritidis [SE], and Salmonella Gallinarum [SG] and formulated the trivalent immunogen by mixing OMVs in a 1:1:1 ratio. One day old chicks were immunized thrice via oral route at two week intervals. Vaccination significantly induced serovar specific antibodies detected up to 180 days post immunization. Post challenge with both homologous and heterologous [S. Infantis] serovars, immunized birds showed reduced level of fecal shedding and organ invasion. A long term efficacy study also showed reduced levels of tissue invasion up to one year post immunization. These results demonstrate that our novel formulation of immunogen could be a broad spectrum potential vaccine for both layer and broiler breeds against iNTS mediated salmonellosis and fowl typhoid.

Development of a Self-Adjuvanting, Cross-Protective, Stable Intranasal Recombinant Vaccine for Shigellosis.

With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.

Virulence of Cholera Toxin Gene-Positive Vibrio cholerae Non-O1/non-O139 Strains Isolated From Environmental Water in Kolkata, India.

Cholera toxin (CT)-producing Vibrio cholerae O1 and O139 cause acute diarrheal disease and are proven etiological agents of cholera epidemics and pandemics. On the other hand, V. cholerae non-O1/non-O139 are designated as non-agglutinable (NAG) vibrios and are not associated with epidemic cholera. The majority of NAG vibrios do not possess the gene for CT (ctx). In this study, we isolated three NAG strains (strains No. 1, 2, and 3) with ctx from pond water in Kolkata, India, and examined their pathogenic properties. The enterotoxicity of the three NAG strains in vivo was examined using the rabbit ileal intestinal loop test. Strain No. 1 induced the accumulation of fluid in the loop, and the volume of fluid was reduced by simultaneous administration of anti-CT antiserum into the loop. The volume of fluid in the loop caused by strains No. 2 and 3 was small and undetectable, respectively. Then, we cultured these three strains in liquid medium in vitro at two temperatures, 25°C and 37°C, and examined the amount of CT accumulated in the culture supernatant. CT was accumulated in the culture supernatant of strain No.1 when the strain was cultured at 25°C, but that was low when cultured at 37°C. The CT amount accumulated in the culture supernatants of the No. 2 and No. 3 strains was extremely low at both temperature under culture conditions examined. In order to clarify the virulence properties of these strains, genome sequences of the three strains were analyzed. The analysis showed that there was no noticeable difference among three isolates both in the genes for virulence factors and regulatory genes of ctx. However, vibrio seventh pandemic island-II (VSP-II) was retained in strain No. 1, but not in strains No. 2 or 3. Furthermore, it was revealed that the genotype of the B subunit of CT in strain No. 1 was type 1 and those of strains No. 2 and 3 were type 8. Histopathological examination showed the disappearance of villi in intestinal tissue exposed to strain No. 1. In addition, fluid accumulated in the loop due to the action of strain No. 1 had hemolytic activity. This indicated that strain No. 1 may possesses virulence factors to induce severe syndrome when the strain infects humans, and that some strains of NAG vibrio inhabiting pond water in Kolkata have already acquired virulence, which can cause illness in humans. There is a possibility that these virulent NAG vibrios, which have acquired genes encoding factors involved in virulence of V. cholerae O1, may emerge in various parts of the world and cause epidemics in the future.

Bivalent non-typhoidal Salmonella outer membrane vesicles immunized mice sera confer passive protection against gastroenteritis in a suckling mice model.

Invasive non-typhoidal Salmonella (iNTS) serovars, especially Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE), cause gastroenteritis worldwide. Due to the emergence of multi-drug resistance in iNTS, a broad-spectrum vaccine is urgently needed for the prevention of iNTS infection. Currently, there is no effective licensed vaccine against iNTS available in the market. We have formulated an outer membrane vesicles (OMVs) based bivalent immunogen as a vaccine candidate to generate broad-spectrum protective immunity against both recently circulating prevalent ST and SE. We have isolated OMVs from ST and SE and formulated the immunogen by mixing both OMVs (1:1 ratio). Three doses of bivalent immunogen significantly induced humoral immune responses against lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) as well as a cell-mediated immune response in adult mice. We also observed that proteins of OMVs act as an adjuvant for generation of high levels of anti-LPS antibodies through T cell activation. We then characterized the one-day old suckling mice model for both ST and SE mediated gastroenteritis and used the model for a passive protection study. In the passive protection study, we found the passive transfer of bivalent OMVs immunized sera significantly reduced ST and SE mediated colonization and gastroenteritis symptoms in the colon of suckling mice compared to non-immunized sera recipients. The overall study demonstrated that OMVs based bivalent vaccine could generate broad-spectrum immunity against prevalent iNTS mediated gastroenteritis. This study also established the suckling mice model as a suitable animal model for vaccine study against iNTS mediated gastroenteritis.

Multifunctional transcription factor CytR of Vibrio cholerae is important for pathogenesis.

Vibrio cholerae, the Gram-negative facultative pathogen, resides in the aquatic environment and infects humans and causes diarrhoeagenic cholera. Although the environment differs drastically, V. cholerae thrives in both of these conditions aptly and chitinases play a vital role in their persistence and nutrient acquisition. Chitinases also play a role in V. cholerae pathogenesis. Chitinases and its downstream chitin utilization genes are regulated by sensor histidine kinase ChiS, which also plays a significant role in pathogenesis. Recent exploration suggests that CytR, a transcription factor of the LacI family in V. cholerae, also regulates chitinase secretion in environmental conditions. Since chitinases and chitinase regulator ChiS is involved in pathogenesis, CytR might also play a significant role in pathogenicity. However, the role of CytR in pathogenesis is yet to be known. This study explores the regulation of CytR on the activation of ChiS in the presence of mucin and its role in pathogenesis. Therefore, we created a CytR isogenic mutant strain of V. cholerae (CytR¯) and found considerably less ß-hexosaminidase enzyme production, which is an indicator of ChiS activity. The CytR¯ strain greatly reduced the expression of chitinases chiA1 and chiA2 in mucin-supplemented media. Electron microscopy showed that the CytR¯ strain was aflagellate. The expression of flagellar-synthesis regulatory genes flrB, flrC and class III flagellar-synthesis genes were reduced in the CytR¯ strain. The isogenic CytR mutant showed less growth compared to the wild-type in mucin-supplemented media as well as demonstrated highly retarded motility and reduced mucin-layer penetration. The CytR mutant revealed decreased adherence to the HT-29 cell line. In animal models, reduced fluid accumulation and colonization were observed during infection with the CytR¯ strain due to reduced expression of ctxB, toxT and tcpA. Collectively these data suggest that CytR plays an important role in V. cholerae pathogenesis.

Molecular insights into the genome dynamics and interactions between core and acquired genomes of Vibrio cholerae.

Bacterial species are hosts to horizontally acquired mobile genetic elements (MGEs), which encode virulence, toxin, antimicrobial resistance, and other metabolic functions. The bipartite genome of Vibrio cholerae harbors sporadic and conserved MGEs that contribute in the disease development and survival of the pathogens. For a comprehensive understanding of dynamics of MGEs in the bacterial genome, we engineered the genome of V. cholerae and examined in vitro and in vivo stability of genomic islands (GIs), integrative conjugative elements (ICEs), and prophages. Recombinant vectors carrying the integration module of these GIs, ICE and CTXΦ, helped us to understand the efficiency of integrations of MGEs in the V. cholerae chromosome. We have deleted more than 250 acquired genes from 6 different loci in the V. cholerae chromosome and showed contribution of CTX prophage in the essentiality of SOS response master regulator LexA, which is otherwise not essential for viability in other bacteria, including Escherichia coli In addition, we observed that the core genome-encoded RecA helps CTXΦ to bypass V. cholerae immunity and allow it to replicate in the host bacterium in the presence of similar prophage in the chromosome. Finally, our proteomics analysis reveals the importance of MGEs in modulating the levels of cellular proteome. This study engineered the genome of V. cholerae to remove all of the GIs, ICEs, and prophages and revealed important interactions between core and acquired genomes.