Biology subjects

Kudva, I. T.

Publications and source records attributed to Kudva, I. T..

5 recordsLinked to original sources

bioRxiv2026

Varied influence of curli on Shiga toxin-producing Escherichia coli O157, O26, O111 biofilm formation and animal cell adherence.

Shiga toxin-producing Escherichia coli (STEC) persist in cattle and can colonize the bovine recto-anal junction, making it an important reservoir for transmission. Curli are extracellular amyloid fibers associated with biofilm formation and environmental persistence, but their contribution to STEC adherence to bovine epithelial cells is unclear. This study examined the relationship between curli production, biofilm formation, and adherence of O157, O26, and O111 STEC isolates to bovine recto-anal junction (RAJ) squamous epithelial (RSE) cells. Thirty isolates were evaluated for RSE adherence, curli-associated phenotypes, and biofilm formation under environmental and host-associated growth conditions. O157 and O111 isolates predominantly exhibited strong aggregative adherence to RSE cells, whereas O26 isolates showed more heterogeneous, primarily diffuse adherence. O111 isolates produced the strongest biofilms under environmental conditions and displayed relatively stable curli phenotypes, while O157 and O26 isolates showed greater variation. However, environmental biofilm formation did not consistently correlate with RSE adherence. Biofilm formation was also markedly reduced under host-simulated conditions in DMEM-LG at 26, 37, and 39{degrees}C, including among isolates that produced strong environmental biofilms. Differences in Shiga toxin genotype and expression likewise did not account for the major adherence patterns, as O111 isolates lacking stx2 retained strong aggregative adherence. These findings indicate that curli-associated biofilm formation is primarily influenced by environmental conditions and does not directly predict STEC adherence to bovine RSE cells. The results suggest that environmental persistence and host-cell attachment represent distinct phenotypes and that STEC adherence to bovine epithelial cells is likely mediated by additional strain- and serotype-specific factors.

microbiology↗

bioRxiv2025

The Carbon Starvation-Inducible Lipoprotein (Slp) Influences Differential Adherence of Escherichia coli O157:H7 at the Bovine Rectoanal Junction.

Shiga toxin-producing Escherichia coli O157:H7 (O157), a foodborne human pathogen, persists at the rectoanal junction (RAJ) of the bovine intestinal tract, in asymptomatic cattle reservoirs. Identifying mechanisms used by O157 for initial adherence before persistence at the RAJ could help develop effective O157 control modalities. We recently established the role of carbon starvation-inducible lipoprotein (Slp) in initial adherence of O157 to Caco-2 cells, with the human polymeric immunoglobulin receptor (pIgR) protein as the Slp-receptor. Here, we evaluated the role of Slp in O157 adherence to the bovine RAJ using the RAJ squamous epithelial (RSE) cell- and RAJ-In vitro Organ Culture (IVOC)- adherence assays. The wild-type O157 strain EDL932 (EDL932-WT), its isogenic slp deletion mutant (EDL932 {Delta}slp), and the slp complemented mutant (EDL932 {Delta}slp-p:slp), were tested with no bacteria controls. Adherence was verified by culture and immunofluorescence (IF) staining of O157. Tissue integrity was determined using nuclear/cell staining dyes and histopathological examination. All test strains adhered in a diffuse-moderate pattern on RSE cells. However, differential adherence was observed on the RAJ-IVOC with the strains preferentially adhering to the columnar cells. Additionally, EDL932- WT and EDL932 {Delta}slp-p:slp strains adhered in slightly greater numbers than the EDL932 {Delta}slp strain to the RAJ-IVOC, causing disruptions primarily in the columnar region of otherwise intact RAJ-IVOC tissues. Interestingly, pIgR was also predominantly detected by IF microscopy and RNAscope in situ hybridization at the columnar region of the RAJ-IVOC tissue. In silico modeling demonstrated the possibility of a bovine pIgR- bacterial Slp interaction. Hence, our observations support the role for Slp in the initial adherence of O157 to the columnar cells at the bovine RAJ, unlike the squamous cells where the loss of slp did not impact attachment. In addition, a possible mucosal immune-interference resulting from the bovine pIgR-Slp interaction could contribute towards long-term O157 colonization of cattle. AUTHOR SUMMARYEscherichia coli O157:H7 (O157) is a foodborne pathogen that causes disease in humans with symptoms ranging from watery/bloody diarrhea to kidney failure. Cattle are the main reservoirs of this human pathogen, where O157 tends to persist at the rectoanal junction (RAJ) of the bovine gastrointestinal tract. However, the exact mechanisms by which O157 adheres and persists at the bovine RAJ is not fully understood. Here, we identified that the O157 carbon starvation-inducible lipoprotein (Slp) allows O157 to attach to cells at the RAJ using polymeric immunoglobulin receptor (pIgR) protein as the Slp-receptor. Not only does this attachment of Slp to the cattle receptor allow O157 to colonize cattle, but the interaction could potentially interfere with intestinal immune responses, further promoting long-term O157 colonization of cattle. This makes Slp an attractive target for therapeutic interventions, such as vaccines that could interfere with O157 attachment to cattle intestinal cells.

microbiology↗

bioRxiv2024

Growth dynamics and protein-expression of Escherichia coli serotypes O26:H11, O111:H8 and O145:NM in the bovine rumen.

To adapt to the ruminal environment, Shiga toxin-producing Escherichia coli (STEC) O157:H7 (O157) expresses proteins involved in survival rather than virulence. Additionally, STEC O157 strains exhibit distinct in vitro but shared in vivo survival patterns in rumen fluids that sets them apart from non-STEC, commensal E. coli. To determine if similar responses would be observed with other STEC, we evaluated three non-O157 serotypes, O26:H11, O111:H8 and O145:NM, along with a non-STEC E. coli, under the growth conditions used for STEC O157: (i) anaerobically, in vitro, in rumen fluid from cattle on a lactation (low fiber, high protein) or maintenance (high fiber, low protein) diet, at 39{degrees}C for 48 hr and (ii) in vivo for 48 hr within the rumen of cattle on the same diets using a non-terminal, rumen-fistulated animal model that allows for introduction of bacteria without ruminal contamination. On the lactation diet, the ruminal pH was acidic ranging from 5.2 - 6.0 and the total volatile fatty acids (VFA) concentration, 141 - 230 M/ml. On the maintenance diet, the ruminal fluid pH was close to neutral ranging from 6.0 - 7.0, with a total VFA of 87 - 190 M/ml. Unlike STEC O157, the three non-O157 serotypes demonstrated survival patterns similar to each other and the control non-STEC E. coli. A greater reduction in viable STEC counts was observed in vitro in rumen fluid from cattle fed the lactation diet than in vivo, corroborating previous reports that in vitro conditions cannot mimic those observed in vivo. Like STEC O157, the non-O157 serotypes mainly expressed proteins supporting ruminal adaptation although not all proteins matched those expressed by STEC O157, and included the virulence protein, intimin. Explorative studies such as this could provide insights into common conditions/ targets that may have application in broader STEC control in cattle. Importance of this studyThis study demonstrates that non-O157 serotypes O26:H11, O111:H8, O145:NM have similar survival and protein expression patterns in rumen fluid with variations being influenced primarily by rumen fluid composition associated with diet. Unlike STEC O157, under in vivo conditions, the growth dynamics of the non-O157 serotypes were comparable to that of non-STEC, commensal E. coli. Hence, exploring bacterial protein expression within the host is critical in discerning therapeutic targets, unique to/shared between STEC, for broad control strategies. In addition, this study further validates the value of using a non-terminal animal model for rumen studies that reduces number of animals used for an experiment.

microbiology↗

bioRxiv2022

Phenotypic and genomic comparison of human outbreak and cattle-associated Shiga toxin-producing Escherichia coli O157:H7

Escherichia coli O157:H7 (O157)-adulterated food products, including beef and produce, are associated with disease outbreaks in humans. Although cattle feces are a source for the contamination, it is unclear if diverse O157 human-associated outbreak isolates expressing a specific virulence phenotype can colonize and shed in the feces of cattle at a quantitatively similar levels to non-outbreak isolates. It is also unclear if other phenotypes, such as biofilm, cell attachment, and toxin production, differentiate environmental O157 isolates from O157 isolates associated with human illness. Genomic profiling of O157 isolates acquired through routine surveillance can inform if the isolates encode virulence genes associated with human disease, but many genotype-phenotype relationships remain unclear for O157. Therefore, the objective of this study was to compare a diverse set of O157 isolates, with the intent of identifying potential genotypic differences that could inform phenotypes such as cattle colonization and fecal shedding, in vitro cell attachment, biofilm production, and Shiga toxin production. In addition, the relationship between phenotypes and potential for foodborne illness as it relates to genomic virulence traits was explored. No significant differences in cattle colonization and fecal shedding were detected for the tested isolates, despite broad genomic differences. In addition, the in vitro phenotypic differences noted in biofilm and cell attachment did not associate with one LSPA-6 lineage compared to another. Overall, no differences in cattle shedding were observed, yet variations in genotype and phenotype were identified indicating further work is warranted to better understand the relationship between O157 genome and virulence. ImportanceFoodborne illness has a major impact on the health and wellbeing of the global population, besides creating substantial financial hardships for industry. While many bacteria and viruses are implicated in foodborne illness, Escherichia coli serotype O157:H7 (O157) is a common food adulterant that can cause human disease and food recalls. Cattle feces are a significant source of food-adulterating O157. A greater understanding of O157 genetics and its relation to phenotype is needed to develop mitigation strategies to limit spread of O157 into the food chain. The goal of the research was to identify O157 genomic and phenotypic attributes of O157 associated with cattle colonization and fecal shedding along with other factors involved in environmental persistence and illness in humans. It was observed that variations in biofilm formation and in vitro cellular adherence did not associate with enhanced cattle colonization or fecal shedding, indicating that the processes involved in cattle colonization are complex and not well understood.

microbiology↗

bioRxiv2018

The Escherichia coli O157:H7 Carbon Starvation-Inducible Lipoprotein Slp Contributes to Initial Adherence In Vitro via the Human Polymeric Immunoglobulin Receptor

Escherichia coli O157:H7 is the most well-studied serotype of enterohemorrhagic E. coli (EHEC) class of E. coli intestinal pathogens, and is responsible for many outbreaks of serious food-borne illness worldwide each year. Adherence mechanisms are a critical component of its pathogenesis, persistence in natural reservoirs, and environmental contamination. E. coli O157:H7 has a highly effective virulence operon, the Locus of Enterocyte Effacement (LEE), and its encoded intimate adherence mechanism is well characterized. However, factors involved in the preceding initial attachment are not well understood. In this study, we propose a mechanism of initial adherence used by E. coli O157:H7 in vitro. We describe a bacterial protein not previously reported to be involved in adherence, Slp, and its interactions with the human host protein polymeric immunoglobulin receptor (pIgR). The human pIgR has previously been shown to act as an adherence receptor for some mucosal pathogens, and is highly expressed in the intestine. Following observation of significant colocalization between E. coli O157:H7 and pIgR location on Caco-2 cells, a co-immunoprecipitation (Co-IP) assay using a human recombinant Fc-tagged pIgR protein led to the identification of this protein. Disruption of Slp expression in E. coli O157:H7, through deletion of its encoding gene slp, produced a significant adherence deficiency to Caco-2 cells at early time points associated with initial adherence. Plasmid complementation of slp fully restored the wild-type phenotype. Furthermore, immunofluorescence microscopy revealed evidence that this interaction is specific to the pathogenic strains of E. coli tested, and not the nonpathogenic strain E. coli K12. Additionally, deletion of slp resulted in the absence of the corresponding protein band in further Co-IP assays, while the plasmid-encoded slp complementation of the deletion mutant strain restored the wild-type pattern. These data support the proposal that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor.\n\nAuthor summaryEscherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) are responsible for tens of thousands of cases of food-borne illness in the United States each year. E. coli O157:H7 has a particularly effective intimate adherence mechanism. However, the mechanisms of initial adherence, which facilitate attachment and virulence prior to the engagement of intimate adherence, are not well understood. In this study, we describe an initial adherence interaction between the E. coli O157:H7 Slp and the human polymeric immunoglobulin receptor (pIgR) expressed by the human colonic epithelial cell line Caco-2. The relationship was first demonstrated as a significant colocalization between the locations of E. coli O157:H7 bacterial cells and pIgR protein using immunofluorescence microscopy. The E. coli O157:H7 Slp protein was identified, and disruption of the slp gene resulted in a severe adherence deficiency to Caco-2 cells during initial adherence. This effect was reversed upon complementation of the {Delta}slp strain with a plasmid-encoded slp gene, and the constitutive over-expression of slp resulted in hyper-adherence exceeding that of the wild-type E. coli O157:H7. These data support the proposition that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor.

microbiology↗