Biology subjects

Bhuiyan, T. R.

Publications and source records attributed to Bhuiyan, T. R..

10 recordsLinked to original sources

bioRxiv2024

Vibrio cholerae lineage and pangenome diversity varies geographically across Bangladesh over one year

Cholera is a diarrhoeal disease caused by Vibrio cholerae. It remains a major public health challenge in the endemic region around the Bay of Bengal. Over decadal time scales, one lineage typically dominates the others and spreads in global pandemic waves. However, it remains unclear to what extent diverse lineages co-circulate during a single outbreak season. Defining the pool of diversity during finer time scales is important because the selective pressures that impact V. cholerae - namely antibiotics and phages - are dynamic on these time scales. To study the nationwide diversity of V. cholerae, we long-read sequenced 273 V. cholerae genomes from seven hospitals over one year (2018) in Bangladesh. Four major V. cholerae lineages were identified: known lineages BD-1, BD-2a, and BD-2b, and a novel lineage that we call BD-3. In 2022, BD-1 caused a large cholera outbreak in Dhaka, apparently outcompeting BD-2 lineages. We show that, in 2018, BD-1 was already dominant in the five northern regions, including Dhaka, consistent with an origin from India in the north. By contrast, we observed a higher diversity of lineages in the two southern regions near the coast. The four lineages differed in pangenome content, including integrative and conjugative elements (ICEs) and genes involved in resistance to bacteriophages and antibiotics. Notably, BD-2a lacked an ICE and is predicted to be more sensitive to phages and antibiotics, but nevertheless persisted throughout the year-long sampling period. Genes associated with antibiotic resistance in V. cholerae from Bangladesh in 2006 were entirely absent from all lineages in 2018-19, suggesting shifting costs and benefits of encoding these genes. Together, our results highlight the dynamic nature of the V. cholerae pangenome and the geographic structure of its lineage diversity.

microbiology↗

bioRxiv2024

Limited O-specific polysaccharide (OSP)-specific functional antibody responses in young children with Shigella infection in Bangladesh

Shigellosis is the second leading cause of diarrheal death in children younger than five years of age globally. At present, there is no broadly licensed vaccine against shigella infection. Previous vaccine candidates have failed at providing protection for young children in endemic settings. Improved understanding of correlates of protection against Shigella infection and severe shigellosis in young children living in endemic settings is needed. Here, we applied a functional antibody profiling approach to define Shigella-specific antibody responses in young children versus older individuals with culture-confirmed shigellosis in Bangladesh, a Shigella endemic area. We analyzed Shigella-specific antibody isotypes, FcR binding and antibody-mediated innate immune cell activation in longitudinal serum samples collected at clinical presentation and up to 1 year later. We found that higher initial Shigella O-specific polysaccharide (OSP)-specific and protein-specific IgG and Fc{gamma}R binding levels correlated with less severe disease regardless of patient age, but that individuals under 5 years of age developed a less prominent class switched, FcR-binding, functional and durable antibody response against both OSP and protein Shigella antigens than older individuals. Focusing on the largest cohort, we found that functional S. flexneri 2a OSP-specific responses were significantly induced only in individuals over age 5 years, and that these responses promoted monocyte phagocytosis and activation. Our findings suggest that in a Shigella endemic region, young children with shigellosis harbor a functional antibody response that fails to maximally activate monocytes; such a response may be important in facilitating subsequent innate cell clearance of Shigella, especially via recruitment and activation of polymorphonuclear cells capable of directly killing Shigella.

immunology↗

bioRxiv2024

Assembly and performance of a cholera RDT prototype that detects both Vibrio cholerae and associated bacteriophage as a proxy for pathogen detection

IntroductionCholera rapid diagnostic tests (RDTs) are vulnerable to virulent bacteriophage predation. We hypothesized that an enhanced cholera RDT that detects the common virulent bacteriophage ICP1 might serve as a proxy for pathogen detection. We previously developed a monoclonal antibody (mAb) to the ICP1 major capsid protein. Our objective herein was to design and assemble a first-of-its-kind RDT that detects both a bacterial pathogen (Vibrio cholerae) and associated virulent bacteriophage (ICP1). MethodCandidate mAbs were expanded to increase design options and evaluated by immunological assays (ELISA; western blot). A subset of mAbs were selected for gold conjugation and printing on the RDT. The limit of detection (LOD) of prototype RDTs were determined in diarrheal stools with the addition of ICP1. ResultsThree mAb candidates were developed and evaluated for the capsid decoration protein (ORF123) and tail fiber protein (ORF93), and the prior mAb for the major capsid protein (ORF122). A single mAb sandwich RDT prototype for ORF122 was able to detect ICP1; RDTs with mAbs to ORF123 and ORF93 failed to detect ICP1 in single or dual sandwich configurations. Biologically meaningful LODs for ICP1 were achieved only after boiling the stool with ICP1; analysis by electron microscopy suggested increased epitope availability after boiling. ConclusionIn this study, we demonstrate a proof of concept for a functional RDT that can detect both the primary pathogen and a common virulent bacteriophage as a proxy for pathogen detection. Further optimization is required before scaled production and implementation.

immunology↗

bioRxiv2024

Host-derived CEACAM-laden vesicles engage enterotoxigenic E. coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections. Significance statementEnterotoxigenic E. coli, characterized by the production of heat-labile (LT) and heat-stable (ST) toxins, are a very common cause of diarrhea in low-income regions responsible for hundreds of millions of infections each year, and the major cause of diarrhea in travelers to endemic areas. Although these infections may be severe and cholera-like, they are typically self-limited. These studies demonstrate that extracellular vesicles produced by host intestinal cells can capture the bacteria and its secreted toxins at a distance from the cell surface, potentially acting as molecular decoys to neutralize the enterotoxins and extinguish the infection.

microbiology↗

bioRxiv2024

Prevalent chromosome fusion in Vibrio cholerae O1

Two circular chromosomes are a defining feature of the family Vibrionaceae, including the pathogen Vibrio cholerae, with rare reports of isolates with a single, fused chromosome. Here we report chromosome fusions in clinical V. cholerae O1 isolates, including several independent fusion events stable enough to be transmitted between patients within a household. Fusion occurs in a 12 kilobase-pair homologous sequence shared between the two chromosomes, which may lead to reversible chromosomal fusion.

microbiology↗

bioRxiv2023

Shigella O-specific polysaccharide functional IgA responses mediate protection against shigella infection in an endemic high-burden setting

Shigella is the second leading cause of diarrheal disease-related death in young children in low and middle income countries. The mechanism of protection against shigella infection and disease in endemic areas is uncertain. While historically LPS-specific IgG titers have been associated with protection in endemic settings, emerging deeper immune approaches have recently elucidated a protective role for IpaB-specific antibody responses in a controlled human challenge model in North American volunteers. To deeply interrogate potential correlates of immunity in areas endemic for shigellosis, here we applied a systems approach to analyze the serological response to shigella across endemic and non-endemic populations. Additionally, we analyzed shigella-specific antibody responses over time in the context of endemic resistance or breakthrough infections in a high shigella burden location. Individuals with endemic exposure to shigella possessed broad and functional antibody responses across both glycolipid and protein antigens compared to individuals from non-endemic regions. In high shigella burden settings, elevated levels of OSP-specific FcR binding antibodies were associated with resistance to shigellosis. OSP-specific FcR binding IgA found in resistant individuals activated bactericidal neutrophil functions including phagocytosis, degranulation and reactive oxygen species production. Moreover, IgA depletion from resistant serum significantly reduced binding of OSP-specific antibodies to FcR and antibody mediated activation of neutrophils and monocytes. Overall, our findings suggest that OSP-specific functional IgA responses contribute to protective immunity against shigella infection in high-burden settings. These findings will assist in the development and evaluation of shigella vaccines.

immunology↗

bioRxiv2021

A Co-abundant Gene Analysis for Identification of Gut Microbial Strains Associated with Immune Responses to Oral Cholera Vaccine

The degree of protection conferred after receiving an oral cholera vaccine (OCV) varies based on age, prior exposure to Vibrio cholerae, and unknown factors. Recent evidence suggests that the microbiota may mediate some of the unexplained differences in oral vaccine responses. We used metagenomic sequencing of the microbiota at the time of vaccination, and then related microbial features to immune responses after OCV using a reference-independent gene-level analysis. We found that the presence of sphingolipid-producing bacteria was associated with the development of protective immune responses after OCV. We experimentally tested these results by stimulating human macrophages with Bacteroides xylanisolvens metabolites and found that sphingolipid-containing extracts increased innate immune responses to OCV antigens. Our findings demonstrate a new analytic method for translating metagenomic sequencing data into strain-specific results associated with a biological outcome, and in validating this tool, we identified that microbe-derived sphingolipids impact in vitro immune responses to OCV antigens.

microbiology↗

bioRxiv2020

A combination of metagenomic and cultivation approaches reveals hypermutator phenotypes within Vibrio cholerae infected patients

Vibrio cholerae can cause a range of symptoms, ranging from severe diarrhea to asymptomatic infection. Previous studies using whole genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous in V. cholerae DNA repair genes in five out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome, and in one of three patients with isolate genomes sequenced. Second, mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, non-mutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients. IMPORTANCEPathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease, or asymptomatic infection. Here we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

microbiology↗

bioRxiv2020

Humans surviving cholera develop antibodies against Vibrio cholerae O-specific polysaccharide that inhibit pathogen motility

The mechanism of protection against cholera afforded by previous illness or vaccination is currently unknown. We have recently shown that antibodies targeting O-specific polysaccharide (OSP) of Vibrio cholerae correlate highly with protection against cholera. V. cholerae is highly motile and possesses a flagellum sheathed in O-specific polysaccharide (OSP), and motility of V. cholerae correlates with virulence. Using high speed video microscopy, and building upon previous animal-related work, we demonstrate that sera, polyclonal antibody fractions, and OSP-specific monoclonal antibodies recovered from humans surviving cholera block V. cholerae motility at both subagglutinating and agglutinating concentrations. This anti-motility effect is reversed by pre-adsorbing sera and polyclonal antibody fractions with purified OSP; and is associated with OSP-specific but not flagellin-specific monoclonal antibodies. F[ab] fragments of OSP-specific polyclonal antibodies do not inhibit motility, suggesting a requirement for antibody-mediated crosslinking in motility inhibition. We show that OSP-specific antibodies do not directly affect V. cholerae viability, but that OSP-specific monoclonal antibody highly protects against death in the murine cholera model. We used in vivo competitive index studies to demonstrate that OSP-specific antibodies impede colonization and survival of V. cholerae in intestinal tissues, and that this impact is motility-dependent. Our findings suggest that the impedance of motility by antibodies targeting V. cholerae OSP contributes to protection against cholera. IMPORTANCECholera is a severe dehydrating illness of humans caused by Vibrio cholerae. V. cholerae is a highly motile bacterium that has a single flagellum covered in lipopolysaccharide (LPS) displaying O-specific polysaccharide (OSP), and V. cholerae motility correlates with its ability to cause disease. The mechanisms of protection against cholera are not well understood; however, since V. cholerae is a non-invasive intestinal pathogen, it is likely that antibodies that bind the pathogen or its products in the intestinal lumen contribute to protection from infection. Here, we demonstrate that OSP-specific antibodies isolated from humans surviving cholera in Bangladesh inhibit V. cholerae motility and are associated with protection against challenge in a motility-dependent manner.

microbiology↗

bioRxiv2018

Cognate T and B cell interaction and association of Follicular helper T cells with B cell responses in Vibrio cholerae O1 infected Bangladeshi adults

Vibrio cholerae O1 can cause life threatening diarrheal disease if left untreated. A long lasting immune response, producing 3-5 years of protection from subsequent, symptomatic disease following natural infection, is mediated by B cell mediated humoral immunity. T cells can play critical roles in inducing such immunity. However, the mechanism of T cell dependent B cell maturation and whether a key sub-population of T cells are involved is not well established in cholera. We hypothesized that a specific population of T cells, follicular helper T (Tfh) cells, are involved in B cell maturation following cholera; we used flow cytometry, culture and colorimetric assays to address this question. We found that V. cholerae infection induces significant increase in circulating Tfh cells expressing B cell maturation associated protein CD40L early in disease. The increased Tfh cells expressing CD40L recognize cholera toxin most prominently, with lessened responses to two antigens tested, V. cholerae membrane preparation (MP) and Vibrio cholerae cytolysin (VCC). We further showed that early induction of Tfh cells and CD40L was associated with later memory B cell responses to same antigens. Lastly, we demonstrated in vitro that Tfh cells isolated after cholera can stimulate class switching of cocultured, isolated B cells from patients with cholera, leading to production of the more durable IgG antibody isotype. These studies were conducted on circulating Tfh cells; future studies will be directed at examining role of Tfh cells during cholera directly in the gut mucosa of biopsied samples, at the single cell level if feasible.

immunology↗