Biology subjects

Mukhopadhyay, A. K.

Publications and source records attributed to Mukhopadhyay, A. K..

8 recordsLinked to original sources

bioRxiv2025

Exposure to third generation cephalosporin induces L-form transition in Shigella sonnei, potentially acting as a bacterial reservoir for persistent infection

Shigellosis remains a major global health burden, and the increasing prevalence of multidrug-resistant (MDR) Shigella strains is complicating effective antibiotic therapy. Bacteria may survive antibiotics by transitioning into cell wall-deficient L-forms, which are intrinsically resistant to {beta}-lactams and can revert to a virulent state, potentially causing relapsing infections. Here we characterized a clinical MDR isolate Shigella sonnei HK8, a.k.a. PD552A, whose genome contains key resistance (gyrA, PBP3) and virulence (icsA) genes. Exposure to ceftriaxone induced a transition into a viable L-form state that was hyper-adhesive to macrophages in vitro. However, this survival adaptation was linked to a profound loss of pathogenicity. Using murine and guinea pig models, the L-form variant was shown to be profoundly attenuated, failing to cause the keratoconjunctivitis, diarrheal disease, or significant histopathology characteristic of the wild-type strain. These findings reveal a critical virulence-survival trade-off, positioning the L-form as a "stealth" phenotype that enables bacterial persistence at the expense of acute virulence. This offers a potential mechanism for asymptomatic carriage and recurrent infections, highlighting a previously underappreciated mechanism by which antibiotic treatment may resolve acute symptoms while permitting the persistence of a cryptic bacterial reservoir capable of driving recurrent infection. ImportanceOur research provides critical insight into the challenge of antibiotic treatment failure in shigellosis. By integrating experimental validation of reversible L-form transitions with mathematical modelling, we uncover a crucial virulence-survival trade-off. We show that MDR S. sonnei survives ceftriaxone by adopting a "stealth" phenotype, quantified by a high Stealth Index - maintaining bacterial burden while evading host inflammatory detection. These findings imply that standard antibiotics may resolve acute symptoms while inadvertently selecting for a cryptic, persistent reservoir poised for relapse. This work challenges the conventional view of therapeutic success and highlights an urgent need to develop novel diagnostic and therapeutic strategies capable of identifying and eliminating these resilient, "stealth" L-form persisters to achieve true bacterial clearance and prevent chronic infections.

microbiology↗

bioRxiv2025

In vivo efficacy of L-ascorbic acid in restricting cholera pathogenesis

In the last three centuries, our world has experienced seven cholera pandemics. Cholera is a deadly diarrheal disease caused by Vibrio cholerae, an important member of the gamma-proteobacteria. Despite conventional treatments like antibiotics, vaccines, and ORS, the 7th cholera pandemic is still a major threat to developing nations. L-ascorbic acid was recently shown to effectively kill the Vibrio cholerae cells in vitro under various growth conditions mimicking the in vivo host conditions, including growth in the presence of bile salts, growth of acid-adapted V. cholerae, and growth in the presence of various ORS components. In the current study, we extend this work and test the efficacy of L-ascorbic acid in animal models (rabbit ileal loop and the removable intestinal tie adult rabbit diarrhea). We show that L-ascorbic acid can effectively reduce the bacterial load in both the rabbit models as well as fast-track the recovery from the diarrheal symptoms. ImportanceCholera is a severe human diarrheal disease that affects millions each year. Cholera treatment is primarily oral rehydration to balance the voluminous fluid loss. Previous studies have found that L-ascorbic acid (L-AA) is effective at inhibiting growth of Vibrio cholerae, the causative agent of cholera. Thus L-AA has potential as am inexpensive cholera therapeutic. Here, L-AA is tested in rabbit models to determine whether it can inhibit the effects of V. cholerae infection. Results suggest that L-AA treatment inhibits bacterial proliferation and leads to shortened recovery from disease.

microbiology↗

bioRxiv2023

Potential use of Sodium Butyrate (SB) as an anti-virulence agent against Vibrio cholerae targeting ToxT virulence protein.

ABSTRACTCholera, a diarrhoeal disease caused by gram-negative bacterium Vibrio cholerae remains a global health threat in developing countries owing to its high transmissibility and increase in antibiotic resistance. The current issue is to overcome the problem of resistance by antimicrobial therapy. There is a need for alternative strategies with an emphasis on anti-virulent approaches to alter the outcome of bacterial infections. Vibrio cholerae causes cholera by secreting virulence factors in the intestinal epithelial cells. Virulence factors help in cholera toxin production and colonisation during infection. Here, we show that sodium butyrate (SB), a small molecule, had no effect on bacterial viability but was effective in suppressing the virulence attributes of V. cholerae. The production of cholera toxin (CT) was downregulated in a standard V. cholerae El Tor strain and two clinical isolates when grown in presence of sodium butyrate. Analysis of mRNA and protein levels further demonstrated that sodium butyrate reduced the expression of the ToxT-dependent virulence genes like tcpA and ctxAB. DNA-protein interaction assays conducted at cellular (ChIP) and in in vitro conditions (EMSA) indicated that sodium butyrate weakens the binding between ToxT and its downstream promoter DNA, likely by blocking DNA binding. Furthermore, the efficacy of sodium butyrate was confirmed by showing its anti-virulence activity and tissue damage recovery in animal models. Collectively, these findings suggest that sodium butyrate (SB) has the potential to be developed as an anti-virulence agent against V. cholerae in place of conventional antibiotics or as an adjunctive therapy to combat cholera. IMPORTANCEThe world has been facing an upsurge in cholera cases since 2021 with a similar trend continuing into 2022 with over 29 countries reporting cholera outbreaks (World Health Organization 16 December 2022 Disease Outbreak News; Cholera - Global situation). Treatment of cholera involves oral rehydration therapy coupled with antibiotics to reduce the duration of the illness. However, over the last few years, there has been indiscriminate use of antibiotics that contributed largely to the reservoir of antibiotic-resistant strains. In this study, we have addressed the problem of antibiotic resistance by targeting virulence factors. The screening of several compounds led to the identification of a small molecule, sodium butyrate that inhibits the virulence cascade in V. cholerae. We demonstrated that (i) sodium butyrate intervened with ToxT protein-DNA binding and subsequently affected the expression of ToxT-regulated virulence genes (ctxAB and tcpA) (ii) Sodium Butyrate is a potential therapeutic candidate for development of novel antimicrobial agents.

microbiology↗

bioRxiv2023

Comparative metagenomic analysis of diarrheal and non-diarrheal gut microbiome delineating the prospective development of prognostic markers and probiotics to protect from diarrhea

A cross-sectional gut microbiome analysis of 23 non-diarrheal and 5 diarrheal fecal samples was conducted by employing 16s rRNA amplicon sequencing and subsequent analysis for taxonomic profiling of OTUs and abundance interpretation of reads. Significant differences in the structural composition of the two groups were observed. In both Firmicutes was the most abundant phylum in majority of the samples. B/F ratio was consistently <1 in all diarrheal samples. Significant difference in mean B/F ratio of the two groups was found. Proteobacteria was significantly more abundant in the diarrheal group. Prevotellaceae was the most abundant family in non-diarrheal samples and was suppressed significantly in diarrheal samples. Streptococcaceae was the most abundant family in 60% diarrheal samples and where Streptococcaceae was suppressed, Bacteroideaceae and Nocardeaceae were the most abundant. In non-diarrheal samples where Streptococcaceae was almost completely suppressed Bifidobacteriaceae was the most abundant and suppressed other families significantly. A negative correlation was observed between Prevotellaceae and Bacteroideaceae in the non-diarrheal group. Prevotella copri was the most abundant species in 70% non-diarrheal samples and was significantly suppressed in diarrheal samples. Proteus mirabilis was identified in all the non-diarrheal samples while they were absent in diarrheal samples. The OTUs associated with diarrheal dysbiosis can serve as prognostic markers. This is the first report on the comparative analysis of diarrheal and non-diarrheal microbiome, to our knowledge, and distinctly addressing the gut microbiome dysbiosis from the context that can lead to the development of prognostic markers and probiotics for protecting the endemic population from diarrhea.

microbiology↗

bioRxiv2023

The gut microbiome of the healthy population in Kolkata, India, is a reservoir of antimicrobial resistance genes emphasizing the need of enforcing antimicrobial stewardship

Antimicrobial resistance (AMR) alleviation warrants antimicrobial stewardship (AS) entailing indispensability of epidemiological surveillance. We undertook a small-scale surveillance in Kolkata to detect the presence of antimicrobial resistance genes (ARGs) in the healthy gut microbiome. We found that it was a reservoir of ARGs against common antibiotics. A targeted PCR and sequencing-based ARGs detection against tetracyclines, macrolides, trimethoprim, sulfamethoxazole, aminoglycosides, amphenicol and mobile genetic element (MGE) markers was deployed in twenty-five fecal samples. Relative abundance and frequency of ARGs was calculated. We detected markers against all these classes of antibiotics. 100% samples carried aminoglycoside resistance marker and int1U. A comparison with our previously published diarrheal resistome from the same spatial and temporal frame revealed that a higher diversity of ARGs were detected in the community and a higher rate of isolation of tetC, msrA, tmp and sul-2 was found. The presence of common markers in the two cohorts proves that the gut microbiome has been contaminated with ARGs and which are being disseminated among different ecosystems. This is an issue of discerning concern for public health. The study raises an alarming picture of the AMR crisis in low-middle and emergent economies. It emphasizes the strict enforcement of AS in the community.

microbiology↗

bioRxiv2022

Glycyrrhizin, an inhibitor of HMGB1 induces autolysosomal degradation function and inhibits H. pylori infection

Helicobacter pylori a key agent for causing gastric complications is linked with peptic ulcer, gastritis, and in severe cases gastric cancer. In response to infection, host cells stimulate autophagy to maintain cellular homeostasis. However, H. pylori have evolved the ability to usurp the hosts autophagic machinery. High mobility group box1 (HMGB1), an alarmin molecule is a regulator of autophagy and its expression is augmented in gastric cancer and many other cancers. Therefore, this study aims to explore the role of glycyrrhizin (a known inhibitor of HMGB1) in autophagy during H. pylori infection. Human gastric cancer (AGS) cells were infected with H. pylori SS1 strain and further treatment was done with glycyrrhizin. Western blot was used to examine the expression levels of autophagy proteins. Autophagy and lysosomal activity were monitored by immunofluorescence. We have performed knockdown of HMGB1 to verify the effect of glycyrrhizin by siRNA transfection method. H. pylori-infection in vivo C56BL/6 mice model was established and the effect of glycyrrhizin treatment was studied. We found that the autophagy-lysosomal pathway was impaired due to a significant increase in lysosomal membrane permeabilization during H. pylori infection in AGS cells. Subsequently, glycyrrhizin treatment restored the lysosomal membrane integrity, accompanied by an increase in cathepsin B activity and reduction of ROS and inflammatory cytokine IL-8. The recovered lysosomal function enhanced autolysosome formation and concomitantly attenuated the intracellular H. pylori growth by eliminating the pathogenic niche from gastric cells. Additionally, glycyrrhizin treatment inhibited inflammation and improved gastric tissue damages in mice.

microbiology↗

bioRxiv2021

Elucidating the correlation between the number of TTTTGAT heptamer repeats and cholera toxin promoter activity in Vibrio cholerae O1 pandemic strains

A complex regulatory cascade controls expression of the cholera toxin genes (ctxAB) in Vibrio cholerae; which eventually leads to choleragen (CT) production and secretion, resulting in rice watery diarrhoea. The cholera toxin promoter (PctxAB) contains a series of heptad repeats (5-TTTTGAT-3); which have been previously shown to play crucial role in ctxAB transcriptional regulation by recruiting the transcriptional activators ToxT, ToxR, and the nucleoid-associated protein H-NS along the ctx promoter. The numbers of these repeats vary between the two biotypes of V. cholerae O1 strains, and even among strains of the same biotype. In this study, we examined PctxAB activation of V. cholerae O1 pandemic strains to understand the significance of the distal heptad repeats in regulating ctx expression. Interestingly, we found that ctx activation may depend on the number of TTTTGAT heptad repeats within PctxAB, and we posit that the occupation of the distal repeats by H-NS could further prevent transcriptional activation of ctx genes in V. cholerae. We hypothesize that ToxT-dependent transcriptional activation may not require entire displacement of H-NS and propose a revision in the currently accepted model of ToxT dependent PctxAB transcriptional activation. IMPORTANCECT production by pathogenic V. cholerae O1 strains is regulated through the transcriptional silencing of CTX promoter by H-NS and counter repression by ToxT. The highly AT rich PctxAB is composed of the tandem repeats 5{square} TTTTGAT 3{square}; the numbers of which differ among the classical and El Tor biotypes. However, it is still not very clear whether the numbers of these repeats could be correlated with promoter activation of the ctx operon. Here we report the role of the distal repeats in ctxAB expression levels. We demonstrate that PctxAB activation changes with the number of the heptad repats within the core promoter element, thereby suggesting a model for ctx regulation in the toxigenic strains of V. cholerae.

microbiology↗

bioRxiv2017

Presence of WH2 like domain in VgrG-1 toxin of Vibrio cholerae reveals the molecular mechanism of actin cross-linking

Type VI secretion systems (T6SS) plays a crucial role in Vibrio cholerae mediated pathogenicity and predation. Tip of T6SS is homologous to gp27/gp5 complex or tail spike of T4 bacteriophage. VgrG-1 of V. cholerae T6SS is unusual among other VgrG because its effector domain is trans-located into the cytosol of eukaryotic cells with an additional actin cross-linking domain (ACD) at its C terminal end. ACD of VgrG-1 (VgrG-1-ACD) causes T6SS dependent host cell cytotoxicity through actin cytoskeleton disruption to prevent bacterial engulfment by macrophages. ACD mediated actin cross-linking promotes survival of the bacteria in the small intestine of humans, along with other virulence factors; establishes successful infection with the onset of diarrhoea in humans. Our studies demonstrated VgrG-1-ACD can bind to actin besides actin cross-linking activity. Computational analysis of ACD revealed the presence of WH2 domain through which it binds actin. Mutations in WH2 domain lead to loss of actin binding in vitro. VgrG-1-ACD having the mutated WH2 domain cannot cross-link actin efficiently in vitro and manifests less actin cytoskeleton disruption when transfected in HeLa cells.\n\nSummary statementActin cross-linking (ACD) domain of VgrG-1 toxin of Type VI secretion in Vibrio cholera has WASP Homology domain 2 (WH2) domain. ACD interact with actin through WH2 domain, WH2 is essential for ACD mediated cross-linking and disruption of actin cytoskeleton in the host cell.

biochemistry↗