VK Viswanathan

Interests

Teaching

Bacterial Pathogenesis, Host-pathogen interactions; Senior undergraduate & Graduate levels

Research

Host-pathogen interactions, Bacterial pathogenesis, intestinal pathogens, EPEC, EHEC, Clostridium difficile

Courses

Scholarly Contributions

Journals/Publications

• Calton, C. M., Carothers, K., Ramamurthy, S., Jagadish, N., Phanindra, B., Garcia, A., Viswanathan, V. K., & Halpern, M. D. (2024). Clostridium scindens exacerbates experimental necrotizing enterocolitis via upregulation of the apical sodium-dependent bile acid transporter. American journal of physiology. Gastrointestinal and liver physiology, 326(1), G25-G37.
  More info

  Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Evidence indicates that bile acid homeostasis is disrupted during NEC: ileal bile acid levels are elevated in animals with experimental NEC, as is expression of the apical sodium-dependent bile acid transporter (Asbt). In addition, bile acids, which are synthesized in the liver, are extensively modified by the gut microbiome, including via the conversion of primary bile acids to more cytotoxic secondary forms. We hypothesized that the addition of bile acid-modifying bacteria would increase susceptibility to NEC in a neonatal rat model of the disease. The secondary bile acid-producing species exacerbated both incidence and severity of NEC. upregulated the bile acid transporter Asbt and increased levels of intraenterocyte bile acids. Treatment with also altered bile acid profiles and increased hydrophobicity of the ileal intracellular bile acid pool. The ability of to enhance NEC requires bile acids, as pharmacological sequestration of ileal bile acids protects animals from developing disease. These findings indicate that bile acid-modifying bacteria can contribute to NEC pathology and provide additional evidence for the role of bile acids in the pathophysiology of experimental NEC. Necrotizing enterocolitis (NEC), a life-threatening gastrointestinal emergency in premature infants, is characterized by dysregulation of bile acid homeostasis. We demonstrate that administering the secondary bile acid-producing bacterium enhances NEC in a neonatal rat model of the disease. -enhanced NEC is dependent on bile acids and driven by upregulation of the ileal bile acid transporter Asbt. This is the first report of bile acid-modifying bacteria exacerbating experimental NEC pathology.
• Anwar, F., Clark, M., Lindsey, J., Claus-Walker, R., Mansoor, A., Nguyen, E., Billy, J., Lainhart, W., Shehab, K., Viswanathan, V. K., & Vedantam, G. (2023). Prevalence of diagnostically-discrepant clinical specimens: insights from longitudinal surveillance. Frontiers in medicine, 10, 1238159.
  More info

  Infection (CDI) is a healthcare-associated diarrheal disease prevalent worldwide. A common diagnostic algorithm relies on a two-step protocol that employs stool enzyme immunoassays (EIAs) to detect the pathogen, and its toxins, respectively. Active CDI is deemed less likely when the Toxin EIA result is negative, even if the pathogen-specific EIA is positive for We recently reported, however, that low-toxin-producing strains recovered from Toxin-negative ('discrepant') clinical stool specimens can be fully pathogenic, and cause lethality in a rodent CDI model. To document frequency of discrepant CDI specimens, and evaluate strain diversity, we performed longitudinal surveillance at a Southern Arizona tertiary-care hospital.
• Harishankar, A., & Viswanathan, V. K. (2023). Attaching and effacing pathogens modulate host mitochondrial structure and function. International review of cell and molecular biology, 377, 65-86.
  More info

  Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are human enteric pathogens that contribute significantly to morbidity and mortality worldwide. These extracellular pathogens attach intimately to intestinal epithelial cells and cause signature lesions by effacing the brush border microvilli, a property they share with other "attaching and effacing" (A/E) bacteria, including the murine pathogen Citrobacter rodentium. A/E pathogens use a specialized apparatus called a type III secretion system (T3SS) to deliver specific proteins directly into the host cytosol and modify host cell behavior. The T3SS is essential for colonization and pathogenesis, and mutants lacking this apparatus fail to cause disease. Thus, deciphering effector-induced host cell modifications is critical for understanding A/E bacterial pathogenesis. Several of the ∼20-45 effector proteins delivered into the host cell modify disparate mitochondrial properties, some via direct interactions with the mitochondria and/or mitochondrial proteins. In vitro studies have uncovered the mechanistic basis for the actions of some of these effectors, including their mitochondrial targeting, interaction partners, and consequent impacts on mitochondrial morphology, oxidative phosphorylation and ROS production, disruption of membrane potential, and intrinsic apoptosis. In vivo studies, mostly relying on the C. rodentium/mouse model, have been used to validate a subset of the in vitro observations; additionally, animal studies reveal broad changes to intestinal physiology that are likely accompanied by mitochondrial alterations, but the mechanistic underpinnings remain undefined. This chapter provides an overview of A/E pathogen-induced host alterations and pathogenesis, specifically focusing on mitochondria-targeted effects.
• Anwar, F., Roxas, B. A., Shehab, K. W., Ampel, N. M., Viswanathan, V. K., & Vedantam, G. (2022). Low-toxin RT027 strains exhibit robust virulence. Emerging microbes & infections, 11(1), 1982-1993.
  More info

  is a leading cause of healthcare-associated infections worldwide. Currently, there is a lack of consensus for an optimal diagnostic method for infection (CDI). Multi-step diagnostic algorithms use enzyme immunosorbent analysis (EIA)-based detection of toxins TcdA/TcdB in stool, premised on the rationale that EIA toxin-negative (Tox) patients have less severe disease and shorter diarrhoea duration. The aim of this study was to characterize toxigenic (i.e. -positive) strains isolated from diarrheic patient stool with an EIA Tox (i.e. "discrepant") CDI diagnostic test result. Recovered strains were DNA fingerprinted (ribotyped), subjected to multiple toxin, genome and proteome evaluations, and assessed for virulence. Overall, of 1243 -positive patient stool specimens from Southern Arizona hospitals, 31% were discrepant. For RT027 (the most prevalent ribotype)-containing specimens, 34% were discrepant; the corresponding RT027 isolates were cytotoxic to cultured fibroblasts, but their total toxin levels were comparable to, or lower than, the historic low-toxin-producing strain CD630. Nevertheless, these low-toxin RT027 strains (LT-027) exhibited similar lethality to a clade-matched high-toxin RT027 strain in Golden Syrian hamsters, and heightened colonization and persistence in mice. Genomics and proteomics analyses of LT-027 strains identified unique genes and altered protein abundances, respectively, relative to high-toxin RT027 strains. Collectively, our data highlight the robust virulence of LT-027 , provide a strong argument for reconsidering the clinical significance of a Tox EIA result, and underscore the potential limitations of current diagnostic protocols.
• Clark, A. E., Adamson, C. C., Carothers, K. E., Roxas, B. A., Viswanathan, V. K., & Vedantam, G. (2022). The Alternative Sigma Factor SigL Influences Toxin Production, Sporulation, and Cell Surface Properties. Frontiers in microbiology, 13, 871152.
  More info

  The alternative sigma factor SigL (Sigma-54) facilitates bacterial adaptation to the extracellular environment by modulating the expression of defined gene subsets. A homolog of the gene encoding SigL is conserved in the diarrheagenic pathogen . To explore the contribution of SigL to biology, we generated -disruption mutants () in strains belonging to two phylogenetically distinct lineages-the human-relevant Ribotype 027 (strain BI-1) and the veterinary-relevant Ribotype 078 (strain CDC1). Comparative proteomics analyses of mutants and isogenic parental strains revealed lineage-specific SigL regulons. Concomitantly, loss of SigL resulted in pleiotropic and distinct phenotypic alterations in the two strains. Sporulation kinetics, biofilm formation, and cell surface-associated phenotypes were altered in CDC1 relative to the isogenic parent strain but remained unchanged in BI-1 . In contrast, secreted toxin levels were significantly elevated only in the BI-1 mutant relative to its isogenic parent. We also engineered SigL overexpressing strains and observed enhanced biofilm formation in the CDC1 background, and reduced spore titers as well as dampened sporulation kinetics in both strains. Thus, we contend that SigL is a key, pleiotropic regulator that dynamically influences 's virulence factor landscape, and thereby, its interactions with host tissues and co-resident microbes.
• Roxas, J. L., Ramamurthy, S., Cocchi, K., Rutins, I., Harishankar, A., Agellon, A., Wilbur, J. S., Sylejmani, G., Vedantam, G., & Viswanathan, V. K. (2022). Enteropathogenic Escherichia coli regulates host-cell mitochondrial morphology. Gut microbes, 14(1), 2143224.
  More info

  The diarrheagenic pathogen enteropathogenic is responsible for significant childhood mortality and morbidity. EPEC and related attaching-and-effacing (A/E) pathogens use a type III secretion system to hierarchically deliver effector proteins into host cells and manipulate epithelial structure and function. Subversion of host mitochondrial biology is a key aspect of A/E pathogen virulence strategy, but the mechanisms remain poorly defined. We demonstrate that the early-secreted effector EspZ and the late-secreted effector EspH have contrasting effects on host mitochondrial structure and function. EspZ interacts with FIS1, a protein that induces mitochondrial fragmentation and mitophagy. Infection of epithelial cells with either wildtype EPEC or an isogenic deletion mutant (Δ) robustly upregulated FIS1 abundance, but a marked increase in mitochondrial fragmentation and mitophagy was seen only in Δinfected cells. FIS1-depleted cells were protected against Δinduced fission, and EspZ-expressing transfected epithelial cells were protected against pharmacologically induced mitochondrial fission and membrane potential disruption. Thus, EspZ interacts with FIS1 and blocks mitochondrial fragmentation and mitophagy. In contrast to WT EPEC, Δinfected epithelial cells had minimal FIS1 upregulation and exhibited hyperfused mitochondria. Consistent with the contrasting impacts on organelle shape, mitochondrial membrane potential was preserved in Δinfected cells, but profoundly disrupted in Δinfected cells. Collectively, our studies reveal hitherto unappreciated roles for two essential EPEC virulence factors in the temporal and dynamic regulation of host mitochondrial biology.
• Roxas, B. A., Roxas, J. L., Claus-Walker, R., Harishankar, A., Mansoor, A., Anwar, F., Jillella, S., Williams, A., Lindsey, J., Elliott, S. P., Shehab, K. W., Viswanathan, V. K., & Vedantam, G. (2020). Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes. Scientific reports, 10(1), 22135.
  More info

  Clostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.
• Maseda, D., Zackular, J. P., Trindade, B., Kirk, L., Roxas, J. L., Rogers, L. M., Washington, M. K., Du, L., Koyama, T., Viswanathan, V. K., Vedantam, G., Schloss, P. D., Crofford, L. J., Skaar, E. P., & Aronoff, D. M. (2019). Nonsteroidal Anti-inflammatory Drugs Alter the Microbiota and Exacerbate Colitis while Dysregulating the Inflammatory Response. mBio, 10(1).
  More info

  infection (CDI) is a major public health threat worldwide. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with enhanced susceptibility to and severity of CDI; however, the mechanisms driving this phenomenon have not been elucidated. NSAIDs alter prostaglandin (PG) metabolism by inhibiting cyclooxygenase (COX) enzymes. Here, we found that treatment with the NSAID indomethacin prior to infection altered the microbiota and dramatically increased mortality and the intestinal pathology associated with CDI in mice. We demonstrated that in -infected animals, indomethacin treatment led to PG deregulation, an altered proinflammatory transcriptional and protein profile, and perturbed epithelial cell junctions. These effects were paralleled by increased recruitment of intestinal neutrophils and CD4 cells and also by a perturbation of the gut microbiota. Together, these data implicate NSAIDs in the disruption of protective COX-mediated PG production during CDI, resulting in altered epithelial integrity and associated immune responses. infection (CDI) is a spore-forming anaerobic bacterium and leading cause of antibiotic-associated colitis. Epidemiological data suggest that use of nonsteroidal anti-inflammatory drugs (NSAIDs) increases the risk for CDI in humans, a potentially important observation given the widespread use of NSAIDs. Prior studies in rodent models of CDI found that NSAID exposure following infection increases the severity of CDI, but mechanisms to explain this are lacking. Here we present new data from a mouse model of antibiotic-associated CDI suggesting that brief NSAID exposure prior to CDI increases the severity of the infectious colitis. These data shed new light on potential mechanisms linking NSAID use to worsened CDI, including drug-induced disturbances to the gut microbiome and colonic epithelial integrity. Studies were limited to a single NSAID (indomethacin), so future studies are needed to assess the generalizability of our findings and to establish a direct link to the human condition.
• Roxas, J. L., & Viswanathan, V. K. (2018). Modulation of Intestinal Paracellular Transport by Bacterial Pathogens. Comprehensive Physiology, 8(2), 823-842.
  More info

  The passive and regulated movement of ions, solutes, and water via spaces between cells of the epithelial monolayer plays a critical role in the normal intestinal functioning. This paracellular pathway displays a high level of structural and functional specialization, with the membrane-spanning complexes of the tight junctions, adherens junctions, and desmosomes ensuring its integrity. Tight junction proteins, like occludin, tricellulin, and the claudin family isoforms, play prominent roles as barriers to unrestricted paracellular transport. The past decade has witnessed major advances in our understanding of the architecture and function of epithelial tight junctions. While it has been long appreciated that microbes, notably bacterial and viral pathogens, target and disrupt junctional complexes and alter paracellular permeability, the precise mechanisms remain to be defined. Notably, renewed efforts will be required to interpret the available data on pathogen-mediated barrier disruption in the context of the most recent findings on tight junction structure and function. While much of the focus has been on pathogen-induced dysregulation of junctional complexes, commensal microbiota and their products may influence paracellular permeability and contribute to the normal physiology of the gut. Finally, microbes and their products have become important tools in exploring host systems, including the junctional properties of epithelial cells. © 2018 American Physiological Society. Compr Physiol 8:823-842, 2018.
• Roxas, J. L., Monasky, R. C., Roxas, B. A., Agellon, A. B., Mansoor, A., Kaper, J. B., Vedantam, G., & Viswanathan, V. K. (2018). Enteropathogenic EspH-Mediated Rho GTPase Inhibition Results in Desmosomal Perturbations. Cellular and molecular gastroenterology and hepatology, 6(2), 163-180.
  More info

  The diarrheagenic pathogen, enteropathogenic (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss.
• Roxas, J. L., Vedantam, G., & Viswanathan, V. K. (2018). Epithelial maturity influences EPEC-induced desmosomal alterations. Gut microbes, 1-5.
  More info

  Desmosomes are junctional protein complexes that confer strong adhesive capacity to adjacent host cells. In a recent study, we showed that enteropathogenic Escherichia coli (EPEC) disrupts desmosomes, weakens cell-cell adhesion and perturbs barrier function of intestinal epithelial (C2) cells. Desmosomal damage was dependent on the EPEC effector protein EspH and its inhibitory effect on Rho GTPases. EspH-mediated Rho inactivation resulted in retraction of keratin intermediate filaments and degradation of desmosomal cadherins. Immunofluorescence studies of EPEC-infected C2 cells revealed keratin retraction towards the nucleus coincident with significant cytoplasmic redistribution of the desmosomal cadherin desmoglein-2 (DSG2). In this addendum, we expand on how EPEC-induced keratin retraction leads to loss of DSG2 anchoring at the junctions, and show that maturity of the epithelial cell monolayer impacts the fate of desmosomes during infection.
• Vedantam, G., Kochanowsky, J., Lindsey, J., Mallozzi, M., Roxas, J. L., Adamson, C., Anwar, F., Clark, A., Claus-Walker, R., Mansoor, A., McQuade, R., Monasky, R. C., Ramamurthy, S., Roxas, B., & Viswanathan, V. K. (2018). An Engineered Synthetic Biologic Protects Against Infection. Frontiers in microbiology, 9, 2080.
  More info

  Morbidity and mortality attributed to infection (CDI) have increased over the past 20 years. Currently, antibiotics are the only US FDA-approved treatment for primary infection, and these are, ironically, associated with disease relapse and the threat of burgeoning drug resistance. We previously showed that non-toxin virulence factors play key roles in CDI, and that colonization factors are critical for disease. Specifically, a adhesin, Surface Layer Protein A (SlpA) is a major contributor to host cell attachment. In this work, we engineered Syn-LAB 2.0 and Syn-LAB 2.1, two synthetic biologic agents derived from lactic acid bacteria, to stably and constitutively express a host-cell binding fragment of the adhesin SlpA on their cell-surface. Both agents harbor conditional suicide plasmids expressing a codon-optimized chimera of the lactic acid bacterium's cell-wall anchoring surface-protein domain, fused to the conserved, highly adherent, host-cell-binding domain of SlpA. Both agents also incorporate engineered biocontrol, obviating the need for any antibiotic selection. Syn-LAB 2.0 and Syn-LAB 2.1 possess positive biophysical and properties compared with their parental antecedents in that they robustly and constitutively display the SlpA chimera on their cell surface, potentiate human intestinal epithelial barrier function , are safe, tolerable and palatable to Golden Syrian hamsters and neonatal piglets at high daily doses, and are detectable in animal feces within 24 h of dosing, confirming robust colonization. In combination, the engineered strains also delay (in fixed doses) or prevent (when continuously administered) death of infected hamsters upon challenge with high doses of virulent . Finally, fixed-dose Syn-LAB ameliorates diarrhea in a non-lethal model of neonatal piglet enteritis. Taken together, our findings suggest that the two synthetic biologics may be effectively employed as non-antibiotic interventions for CDI.
• Chu, M., Mallozzi, M. J., Roxas, B. P., Bertolo, L., Monteiro, M. A., Agellon, A., Viswanathan, V. K., & Vedantam, G. (2016). A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence. PLoS pathogens, 12(10), e1005946.
  More info

  Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence.
• Wilbur, J. S., Byrd, W., Ramamurthy, S., Ledvina, H., Khirfan, K., Riggs, M., Boedeker, E., Vedantam, G., & Viswanathan, V. K. (2015). The secreted effector protein EspZ is essential for virulence of rabbit enteropathogenic Escherichia coli. Infection and immunity.
  More info

  Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (ΔespZ), and corresponding cis-complemented derivatives, of rabbit enteropathogenic Escherichia coli, and compared their ability to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs and intestinal tissue alterations. Consistent with earlier reports, espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, compared to the parent and complemented strains, ΔespZ induced greater host cell death. In the rabbit infections, fecal ΔespZ levels were ten-fold lower than the parent strain one day post-infection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZ fecal carriage progressively decreased on subsequent days. ΔespZ-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. TUNEL staining of intestinal sections revealed increased epithelial cell apoptosis on Day 1 post-infection with ΔespZ, as compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host-cell cytoprotection likely prevents epithelial cell death and sloughing and, thereby, promotes bacterial colonization.
• Wilbur, J. S., Wilbur, J. S., Byrd, I. W., Byrd, I. W., Ramamurthy, S., Ramamurthy, S., Ledvina, H., Ledvina, H., Khirfan, K., Khirfan, K., Riggs, M. W., Riggs, M. W., Boediker, E., Boediker, E., Vedantam, G., Vedantam, G., Viswanathan, V., & Viswanathan, V. (2015). The secreted effector protein EspZ is Essential for virulence of rabbit enteropathogenic Escherichia coli. Infection and Immunity, 83(3), 1139-49. doi:10.1128
  More info

  Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (ΔespZ), and corresponding cis-complemented derivatives, of rabbit enteropathogenic Escherichia coli, and compared their ability to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs and intestinal tissue alterations. Consistent with earlier reports, espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, compared to the parent and complemented strains, ΔespZ induced greater host cell death. In the rabbit infections, fecal ΔespZ levels were ten-fold lower than the parent strain one day post-infection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZ fecal carriage progressively decreased on subsequent days. ΔespZ-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. TUNEL staining of intestinal sections revealed increased epithelial cell apoptosis on Day 1 post-infection with ΔespZ, as compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host-cell cytoprotection likely prevents epithelial cell death and sloughing and, thereby, promotes bacterial colonization.
• Roxas, J. L., Ryan, K., Vedantam, G., & Viswanathan, V. K. (2014). Enteropathogenic Escherichia coli dynamically regulates EGFR signaling in intestinal epithelial cells. American journal of physiology. Gastrointestinal and liver physiology, 307(3), G374-80.
  More info

  The diarrheagenic pathogen enteropathogenic Escherichia coli (EPEC) dynamically modulates the survival of infected host intestinal epithelial cells. In the initial stages of infection, several prosurvival signaling events are activated in host cells. These include the phosphorylation of epidermal growth factor receptor (EGFR) and the consequent activation of the phosphatidylinositol-3 kinase/Akt pathway. While studying this pathway in infected epithelial cells, we observed EGFR depletion at later stages of infection, followed subsequently by a decrease in phospho-EGFR. EGFR loss was not dependent on receptor phosphorylation, or on canonical proteasome- and lysosome-dependent processes. Although a type III secretion mutant (ΔescN) stimulated EGFR phosphorylation, it failed to induce receptor degradation. To identify the specific EPEC effector molecule(s) that influenced EGFR stability, epithelial cells infected with isogenic mutant EPEC strains were examined. An EPEC ΔespF strain failed to induce EGFR degradation, whereas EPEC ΔespZ accentuated receptor loss in infected cells. Given the known and contrasting effects of EspF and EspZ on caspase activation, and the known role of proteases in cleaving EGFR, we explored the effect of caspase inhibitors on infection-dependent EGFR loss. The pan-caspase inhibitor Q-VD-OPh blocked EPEC-induced EGFR cleavage in a dose-dependent manner. Taken together, our data suggest that EPEC EspF stimulates caspase-dependent EGFR cleavage and loss, whereas EspZ inhibits this process. Whereas EGFR phosphorylation contributes to the survival of host cells early in infection, EspF-driven caspase activation and consequent EGFR loss likely induce a precipitous increase in host cell death later in the infectious process.
• Viswanathan, V. K. (2014). Memories of a virulent past. Gut Microbes, 5(2), 143-145.
• Viswanathan, V. K. (2014). Memories of a virulent past. Gut microbes, 5(2), 143-5.
  More info

  Studies on bacterial virulence and host-pathogen interactions usually rely on type strains isolated from human or veterinary populations. For instance, the El Tor strain N16961 has been extensively used to characterize Vibrio cholerae virulence, while E2348/69 is a default choice for studies on enteropathogenic E. coli interactions with host cells. Subsequent to isolation, such strains are passaged under laboratory conditions in rich medium, and often genetically manipulated to induce specific mutations or other alterations. While the cumulative knowledge gained by focused studies on a limited number of bacterial isolates allows for rapid scientific progress, strain diversity resulting from prolonged propagation and manipulation in different laboratories may lead to a distorted perspective and, at times, confound attempts to replicate specific experiments. Exploring such aberrations is an inevitable and necessary, if not always welcome, part of scientific progress.
• Viswanathan, V. K. (2014). Muramyl dipeptide. Gut Microbes, 5(3), 275-276.
• Viswanathan, V. K. (2014). Muramyl dipeptide: Not just another brick in the wall. Gut microbes, 5(3), 275-6.
  More info

  The robust expression of microbial pattern recognition receptors such as TLR4 and Nod2 in intestinal stem cells reflects an active communication dynamic between the host and the gut microbiota. A new study reveals that muramyl dipeptide, the bacterial cell wall peptidoglycan motif, activates Nod2 within crypt base columnar Lgr5-positive stem cells and promotes their survival. Apart from the immediate relevance to the growth of organoids for in vitro experiments, the study raises new questions about the molecular mechanisms whereby gut microbes influence intestinal physiology.
• Viswanathan, V. K. (2014). Off-label abuse of antibiotics by bacteria. Gut Microbes, 5(1), 3-4.
• Viswanathan, V. K. (2014). Off-label abuse of antibiotics by bacteria. Gut microbes, 5(1), 3-4.
  More info

  Antibiotics and antibiotic resistance made news on several fronts in the past year. Many public health organizations, including the CDC, used terms such as "crisis", "catastrophic consequences", and "nightmare scenario" to highlight the rapid emergence and spread of antibiotic resistance. A report from the Pew Commission on Industrial Farm Animal Production, on the fifth anniversary of the publication of its landmark 2008 report, noted that state and federal legislative efforts to limit non-therapeutic use of antibiotics in animal production were thwarted by drug and food animal industries. In its lobbying disclosures, the Farm Bureau stated that such efforts to limit use of animal antibiotics were "based on emotion and no credible peer reviewed science." Meanwhile, there have been inexorable advances in our understanding of the molecular mechanisms by which antibiotics induce diversity and resistance in bacteria. This article reviews one study that probed the role of the bacterial general stress response in sub-inhibitory antibiotic-induced mutagenesis and antibiotic resistance.
• Viswanathan, V. K. (2014). The trendy microbes. Gut Microbes, 5(4), 439-440.
• Viswanathan, V. K. (2014). The trendy microbes. Gut microbes, 5(4), 439-40.
  More info

  After dominating the earth in numbers and biomass for a good 3.5 billion years, microbes are now finally in the public consciousness. The vigorous surge in the study of microbes promises to yield novel approaches to treating diseases and maintaining health. In the middle of the deluge of scientific publications and lay articles, we pause to reflect on this trend.
• Merrigan, M. M., Venugopal, A., Roxas, J. L., Anwar, F., Mallozzi, M. J., Roxas, B. A., Gerding, D. N., Viswanathan, V. K., & Vedantam, G. (2013). Surface-layer protein A (SlpA) is a major contributor to host-cell adherence of Clostridium difficile. PloS one, 8(11).
  More info

  Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.
• Viswanathan, V. K. (2013). Oh what tangled webs we weave.... Gut Microbes, 4(3).
  More info

  PMID: 23519061;PMCID: PMC3669162;Abstract: A wide range of molecules in plants and animals have the capacity to form net-like structures to trap pathogens. In concert with antimicrobial compounds, these aggregates can become efficient killing machines. In other instances, as with the intracellular septins, such structures may constrain pathogenic organisms and direct them to an autophagic tryst with deadly lysosomes. Most curiously, a meshwork of proteins in the mosquito gut appears to protect luminal bacteria from immune cells. Several recent studies delve into the intricacies of net formation and its role in microbial containment. © 2013 Landes Bioscience.
• Viswanathan, V. K. (2013). Sensing bacteria, without bitterness?. Gut Microbes, 4(2), 91-93.
  More info

  PMID: 23380647;PMCID: PMC3595082;Abstract: In addition to their recognized roles in intra- and inter-species signaling, bacterial quorum-sensing molecules have been implicated in inter-kingdom signaling. A new study in Pseudomonas aeruginosa suggests that mammalian bitter taste receptors may recognize bacterial quorum sensing molecules, and widens the scope of such inter-kingdom communication. Intestinal cells also harbor these receptors, but whether they eavesdrop on bacterial conversations remains an open question. © 2013 Landes Bioscience.
• Viswanathan, V. K. (2013). Shigella takes the temperature. Gut Microbes, 4(4), 267-268.
  More info

  PMID: 23851363;PMCID: PMC3744510;Abstract: For bacteria, temperature is a fairly reliable cue for entry into a vertebrate host. Many pathogenic bacteria sense elevated temperatures (37°C) and turn on virulence and metabolic genes appropriate for survival and growth within the host. A recent study demonstrated that the expression of a heme uptake protein in Shigella dysenteriae and pathogenic Escherichia coli was dependent on an RNA thermometer. © 2013 Landes Bioscience.
• Viswanathan, V. K. (2013). The meddling microbes midst our medicines. Gut Microbes, 4(5).
  More info

  Abstract: It is not surprising that the complex metabolic machinery of the gut microbiome has accidental, or directed, ability to alter our medicines and influence their efficacy. What is not known is the extent to which this has contributed to drug failures or contraindications, or to the derailment of clinical trials. Some studies are unraveling the mechanisms by which the microbiota alter specific drugs, such as digoxin, and contribute to variations in efficacies between patient populations. Microbiome profiling, therefore, may well become an inevitable arm of all clinical trials in the future. © 2013 Landes Bioscience.
• Chatfield, C., Mulhern, B., Viswanathan, V., & Cianciotto, N. (2012). The Major Facilitator Superfamily-Type Protein LbtC Promotes the Utilization of the Legiobactin Siderophore by Legionella pneumophila. Microbiology, 158, 721-735.
• Roxas, J., Wilbur, J. S., Zhang, X., Martinez, G., Vedantam, G., & Viswanathan, V. (2012). The enteropathogenic Escherichia coli secreted protein EspZ inhibits host cell apoptosis. Infection & Immunit, 80(11), 3850-7.
• Viswanathan, V. -. (2012). Clostridium difficile clinical isolates exhibit variable susceptibility and proteome alterations upon exposure to mammalian cationic antimicrobial peptides. Anaerobe, 18(6), 614-20.
• Viswanathan, V. K. (2012). Sizing up microbes. Gut Microbes, 3(6), 483-484.
  More info

  PMID: 23117415;PMCID: PMC3495784;Abstract: The size range of life forms is dictated by basic principles of physics. Large microorganisms, with sizes approaching a millimeter, have compensating features that address the immutable laws of physics. For pathogens, size may impact a range of functions, such as adherence and immune evasion. We review several recent studies on factors impacting, and impacted by, the size of microorganisms. © 2012 Landes Bioscience.
• Viswanathan, V. (2011). A more perfect union. Gut Microbes, 2(3), 125-6.
  More info

  Regular Column "Inside Passage" for the Journal
• Viswanathan, V. (2011). Bacteriophages: the ultimate agents of change. Gut Microbes, 2(4), 209-10.
  More info

  Regular Column "Inside Passage" for the Journal
• Viswanathan, V. (2011). Mind-bending infections. Gut Microbes, 2(1), 1-2.
  More info

  Regular Column "Inside Passage" for the Journal
• Viswanathan, V. -. (2011). Of Cabbages & Kings. Gut Microbes, 2(2), 67-8.
  More info

  Regular Column "Inside Passage" for the Journal
• Mallozzi, M., Viswanathan, V., & Vedantam, G. (2010). Spore-forming Bacilli and Clostridia in human disease. Future Microbiol, 5(7), 1109-23.
• Merrigan, M., Venugopal, A., Mallozzi, M., Roxas, B., Viswanathan, V., Johnson, S., Gerding, D., & Vedantam, G. (2010). Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production. J Bacteriol, 192(19), 4904-11.
• Royan, S. V., Jones, R. M., Koutsouris, A., Roxas, J. L., Falzari, K., Weflen, A. W., Kim, A., Bellmeyer, A., Turner, J. R., Neish, A. S., Rhee, K., Viswanathan, V. K., & Hecht, G. A. (2010). Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-κB activation. Molecular Microbiology, 78(5), 1232-1245.
  More info

  PMID: 21091507;PMCID: PMC3325542;Abstract: Summary: Enteric bacterial pathogens have evolved sophisticated strategies to evade host immune defences. Some pathogens deliver anti-inflammatory effector molecules into the host cell cytoplasm via a type III secretion system (T3SS). Enteropathogenic Escherichia coli (EPEC) inhibits inflammation by an undefined, T3SS-dependent mechanism. Two proteins encoded outside of the EPEC locus of enterocyte effacement (LEE) pathogenicity island, non-LEE-encoded effector H1 (NleH1) and H2 (NleH2), display sequence similarity to Shigella flexneri OspG, which inhibits activation of the pro-inflammatory transcription factor NF-κB. We hypothesized that the anti-inflammatory effects of EPEC were mediated by NleH1 and NleH2. In this study, we examined the effect of NleH1/H2 on the NF-κB pathway. We show that NleH1/H2 are secreted via the T3SS and that transfection of cells with plasmids harbouring nleH1 or nleH2 decreased IKK-β-induced NF-κB activity and attenuated TNF-α-induced degradation of phospho-IκBα by preventing ubiquitination. Serum KC levels were higher in mice infected with δnleH1H2 than those infected with WT EPEC, indicating that NleH1/H2 dampen pro-inflammatory cytokine expression. δnleH1H2 was cleared more rapidly than WT EPEC while complementation of δnleH1H2 with either NleH1 or NleH2 prolonged colonization. Together, these data show that NleH1 and NleH2 function to dampen host inflammation and facilitate EPEC colonization during pathogenesis. © 2010 Blackwell Publishing Ltd.
• Viswanathan, V. (2010). Self portraits. Gut microbiology.. Gut microbes, 1(6), 357-358.
  More info

  PMID: 21468215;PMCID: PMC3056098;
• Viswanathan, V. K. (2010). Fascinating science, compelling cause. Gut Microbes, 1(1), 3-.
• Viswanathan, V. K. (2010). Humor me. Gut Microbes, 1(2), 75-76.
• Viswanathan, V. K. (2010). Tossed salads. Gut Microbes, 1(5).
• Viswanathan, V. K. (2010). What is black and white and a puzzle all over?. Gut microbes, 1(3), 129-130.
  More info

  PMID: 21327019;PMCID: PMC3023592;
• Weflen, A. W., Alto, N. M., Viswanathan, V. K., & Hecht, G. (2010). E. coli secreted protein F promotes EPEC invasion of intestinal epithelial cells via an SNX9-dependent mechanism. Cellular Microbiology, 12(7), 919-929.
  More info

  PMID: 20088948;PMCID: PMC2896985;Abstract: Enteropathogenic Escherichia coli (EPEC) infection requires the injection of effector proteins into intestinal epithelial cells (IECs) via type 3 secretion. Type 3-secreted effectors can interfere with IEC signalling pathways via specific protein-protein interactions. For example, E. coli secreted protein F (EspF) binds sorting nexin 9 (SNX9), an endocytic regulator, resulting in tubulation of the plasma membrane. Our aim was to determine the mechanism of EspF/SNX9-induced membrane tubulation. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. Although characterized as non-invasive, EPEC are known to invade IECs in vitro and in vivo. Indeed, we found significant invasion of Caco-2 cells by EPEC, which, like tubulation, was blocked by pharmacological inhibition of CCPs. Interestingly, however, inhibition of dynamin activity did not prevent tubulation or EPEC invasion, which is in contrast to Salmonella invasion, which requires dynamin activity. Our data also indicate that EPEC invasion is dependent on EspF and its interaction with SNX9. Together, these findings suggest that EspF promotes EPEC invasion of IECs by harnessing the membrane-deforming activity of SNX9. © 2010 Blackwell Publishing Ltd.
• Bellmeyer, A., Cotton, C., Kanteti, R., Koutsouris, A., Viswanathan, V. K., & Hecht, G. (2009). Enterohemorrhagic Escherichia coli suppresses inflammatory response to cytokines and its own toxin. American Journal of Physiology - Gastrointestinal and Liver Physiology, 297(3), G576-G581.
  More info

  PMID: 19556613;PMCID: PMC2739818;Abstract: Infection with the enteric pathogen enterohemorrhagic Escherichia coli (EHEC) causes a variety of symptoms ranging from nonbloody diarrhea to more severe sequelae including hemorrhagic colitis, altered sensorium and seizures, and even life-threatening complications, such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. The more severe consequences of EHEC infection are attributable to the production of Shiga toxin (Stx) and its subsequent effects on the vasculature, which expresses high levels of the Stx receptor, Gb3. Interestingly, the intestinal epithelium does not express Gb3. Despite the lack of Gb3 receptor expression, intestinal epithelial cells translocate Stx. The effect of Stx on intestinal epithelial cells is controversial with some studies demonstrating induction of inflammation and others not. This may be difficult to resolve because EHEC expresses both proinflammatory molecules, such as flagellin, and factor(s) that dampen the inflammatory response of epithelial cells. The goal of our study was to define the effect of Stx on the inflammatory response of intestinal epithelial cells and to determine whether infection by EHEC modulates this response. Here we show that Stx is a potent inducer of the inflammatory response in intestinal epithelial cells and confirm that EHEC attenuates the induction of IL-8 by host-derived proinflammatory cytokines. More importantly, however, we show that infection with EHEC attenuates the inflammatory response by intestinal epithelial cells to its own toxin. We speculate that the ability of EHEC to dampen epithelial cell inflammatory responses to Stx and cytokines facilitates intestinal colonization. Copyright © 2009 the American Physiological Society.
• Roxas, J. L., Viswanathan, V. K., Viswanathan, V. K., Turner, J. R., Royan, S. V., Roxas, J. L., Neish, A. S., Koutsouris, A., Jones, R. M., Hecht, G. A., & Bellmeyer, A. (2009). 118 Enteropathogenic E. coli Non-Lee Encoded Effectors NleH1 and NleH2 Inhibit NF-κB Activation. Gastroenterology, 136(5), A-21. doi:10.1016/s0016-5085(09)60101-3
• Viswanathan, V. K., Hodges, K., & Hecht, G. (2009). Enteric infection meets intestinal function: How bacterial pathogens cause diarrhoea. Nature Reviews Microbiology, 7(2), 110-119.
  More info

  PMID: 19116615;PMCID: PMC3326399;Abstract: Infectious diarrhoea is a significant contributor to morbidity and mortality worldwide. In bacterium-induced diarrhoea, rapid loss of fluids and electrolytes results from inhibition of the normal absorptive function of the intestine as well as the activation of secretory processes. Advances in the past 10 years in the fields of gastrointestinal physiology, innate immunity and enteric bacterial virulence mechanisms highlight the multifactorial nature of infectious diarrhoea. This Review explores the various mechanisms that contribute to loss of fluids and electrolytes following bacterial infections, and attempts to link these events to specific virulence factors and toxins.
• Viswanathan, V. K., Weflen, A., Koutsouris, A., Roxas, J. L., & Hecht, G. (2008). Enteropathogenic E. coli-induced barrier function alteration is not a consequence of host cell apoptosis. American Journal of Physiology - Gastrointestinal and Liver Physiology, 294(5), G1165-G1170.
  More info

  PMID: 18356531;PMCID: PMC3327053;Abstract: Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death. Copyright © 2008 the American Physiological Society.
• Roxas, J. L., Koutsouris, A., & Viswanathan, V. K. (2007). Enteropathogenic Escherichia coli-induced epidermal growth factor receptor activation contributes to physiological alterations in intestinal epithelial cells.. Infection and immunity, 75(5), 2316-2324.
  More info

  PMID: 17339360;PMCID: PMC1865758;Abstract: The diarrheagenic pathogen enteropathogenic Escherichia coli (EPEC) is responsible for significant infant mortality and morbidity, particularly in developing countries. EPEC pathogenesis relies on a type III secretion system-mediated transfer of virulence effectors into host cells. EPEC modulates host cell survival and inflammation, although the proximal signaling pathways have not been well defined. We therefore examined the effect of EPEC on the epidermal growth factor receptor (EGFR), a known upstream activator of both the prosurvival phosphoinositide 3-kinase/Akt and proinflammatory mitogen-activated protein (MAP) kinase pathways. EPEC induced the autophosphorylation of EGFR in intestinal epithelial cells within 15 min postinfection, with maximal phosphorylation being observed at 4 h. Filter-sterilized supernatants of EPEC cultures also stimulated EGFR phosphorylation, suggesting that a secreted component(s) contributes to this activity. EPEC-induced EGFR phosphorylation was blocked by the pharmacological inhibitor tyrphostin AG1478, as well as by EGFR-neutralizing antibodies. Inhibition of EGFR phosphorylation by AG1478 had no effect on bacterial adherence, actin recruitment to sites of attachment, or EPEC-induced epithelial barrier function alteration. EPEC-mediated Akt phosphorylation, however, was inhibited by both AG1478 and EGFR-neutralizing antibodies. Correspondingly, inhibition of EGFR activation increased the apoptosis/necrosis of infected epithelial cells. Inhibition of EGFR phosphorylation also curtailed EPEC-induced ERK1/2 (MAP kinase) phosphorylation and, correspondingly, the production of the proinflammatory cytokine interleukin-8 by infected epithelial cells. Our studies suggest that EGFR is a key proximal signaling molecule during EPEC pathogenesis.
• Hodges, K., Viswanathan, V. K., & Hecht, G. (2006). Physiology of Host{dot plus}Pathogen Interactions. Physiology of the Gastrointestinal Tract, 2, 1163-1187.
• Sharma, R., Tesfay, S., Tomson, F. L., Kanteti, R. P., Viswanathan, V. K., & Hecht, G. (2006). Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenic Escherichia coli on intestinal epithelial cells. American Journal of Physiology - Gastrointestinal and Liver Physiology, 290(4), G685-G694.
  More info

  PMID: 16322091;Abstract: Enteropathogenic Escherichia coli (EPEC) virulence requires a type III secretion system (TTSS) to deliver effector molecules in host cells. Although the TTSS is crucial to EPEC pathogenesis, its function in EPEC-induced inflammation is not known. The aim of this study was to investigate the role of the TTSS in EPEC-induced inflammation. HT-29 intestinal epithelial cells were infected with wild-type (WT) EPEC or select mutant strains or exposed to corresponding filter-sterilized supernatants (SN), and interleukin-8 (IL-8) secretion was determined by ELISA. EPEC SN stimulated significantly greater IL-8 production than EPEC organisms. Flagellin, as well as a TTSS-independent >50-kDa nonflagellin protein, was found to significantly contribute to this response. Dose-response studies showed that increasing concentrations of WT SN proportionally increased IL-8, whereas increasing multiplicity of infection of EPEC inversely correlated with IL-8 secretion, suggesting that EPEC dampens this host response. Infection with ΔescN (nonfunctional TTSS) markedly increased IL-8 compared with WT, indicating that a functional TTSS is required for this anti-inflammatory property; complementation of escN restored the attenuated response. Mutation of espB also enhanced the IL-8 response, and complementation returned IL-8 to near WT levels, suggesting involvement of this effector. The anti-inflammatory effect extends to both bacterial and host-derived proinflammatory stimuli, since prior infection with EPEC suppressed the IL-8 response to tumor necrosis factor-α, IL-1β, and enterohemorrhagic E. coli flagellin. These findings indicate that EPEC-induced inflammation is a balance between pro- and anti-inflammatory proteins; extracellular factors, including flagellin and an unidentified TTSS-independent, >50-kDa protein, trigger inflammation while intracellular TTSS-dependent factors, including EspB, attenuate this response. Copyright © 2006 the American Physiological Society.
• Tomson, F. L., Viswanathan, V. K., Kanack, K. J., Kanteti, R. P., Straub, K. V., Menet, M., Kaper, J. B., & Hecht, G. (2005). Enteropathogenic Escherichia coli EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of the tight junction barrier. Molecular Microbiology, 56(2), 447-464.
  More info

  PMID: 15813736;Abstract: EspG, a secreted effector of enteropathogenic Escherichia coli (EPEC), as well as its homologue Orf3, has been shown to disrupt microtubules (MTs) in fibroblasts and non-polarized epithelial cells. The roles of MTs and the effects of MT disruption in these cell types differ significantly. The aim of this study was to investigate the effects of EspG on polarized, host target intestinal epithelial cells. Immunofluorescent labelling of tubulin showed that EPEC caused progressive fragmentation and loss of the MT network in cells harbouring attached organisms. Immunoblots of proteins extracted from EPEC-infected cells showed a corresponding loss of α-tubulin. Type III secretion system (TTSS)-deficient strains had no effect on MT suggesting TTSS dependence. Mutation of espG, but not espF or map, ablated EPEC's effects on MTs for up to 6 h. Ectopic expression of EspG in HeLa cells caused MT disruption. While deletion of espG alone had no effect on the EPEC-induced decrease in transepithelial electrical resistance (TER), mutation of both espG and orf3 significantly delayed the kinetics of this response. Complementation of the double mutant with espG alone restored the kinetics of TER drop to that of wild type. Herein, we describe a previously unrecognized phenotype for the EPEC effectors EspG and Orf3. © 2005 Blackwell Publishing Ltd.
• Tomson, F. L., Koutsouris, A., Viswanathan, V. K., Turner, J. R., Savkovic, S. D., & Hecht, G. (2004). Differing roles of protein kinase C-ζ in disruption of tight junction barrier by enteropathogenic and enterohemorrhagic Escherichia coli. Gastroenterology, 127(3), 859-869.
  More info

  PMID: 15362041;Abstract: Background & Aims: Enteropathogenic Escherichia coli and enterohemorrhagic E. coli harbor highly homologous pathogenicity islands yet show key differences in their mechanisms of action. Both disrupt host intestinal epithelial tight junctions, but the effects of enteropathogenic E. coli are more profound than those of enterohemorrhagic E. coli. The basis for this is not understood. The atypical protein kinase C isoform, protein kinase C-ζ, associates with and regulates the tight junction complex. The aim of this study was to compare the role of protein kinase C-ζ in the disruption of tight junctions after infection with enteropathogenic E. coli and enterohemorrhagic E. coli. Methods: Model intestinal epithelial monolayers infected by enteropathogenic E. coli or enterohemorrhagic E. coli were used for these studies. Results: Neither bisindolylmaleimide nor Gö6976, which block several protein kinase C isoforms but not protein kinase C-ζ, protected against the decrease in transepithelial electrical resistance after enteropathogenic E. coli infection. Rottlerin at concentrations that block novel and atypical isoforms, including protein kinase C-ζ, significantly attenuated the decrease in transepithelial electrical resistance. The specific inhibitory peptide, myristoylated protein kinase C-ζ pseudosubstrate, also significantly decreased the enteropathogenic E. coli-associated decrease in transepithelial electrical resistance and redistribution of tight junction proteins. In contrast to enteropathogenic E. coli, the level of protein kinase C-ζ enzyme activity stimulated by enterohemorrhagic E. coli was transient and minor, and protein kinase C-ζ inhibition had no effect on the decrease in transepithelial electrical resistance or the redistribution of occludin. Conclusions: The differential regulation of protein kinase C-ζ by enteropathogenic E. coli and enterohemorrhagic E. coli may in part explain the less profound effect of the latter on the barrier function of tight junctions.
• Viswanathan, V. K., Koutsouris, A., Lukic, S., Pilkinton, M., Simonovic, I., Simonovic, M., & Hecht, G. (2004). Comparative analysis of EspF from enteropathogenic and enterohemorrhagic Escherichia coli in alteration of epithelial barrier function. Infection and Immunity, 72(6), 3218-3227.
  More info

  PMID: 15155623;PMCID: PMC415647;Abstract: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related intestinal pathogens that harbor highly similar pathogenicity islands known as the locus of enterocyte effacement (LEE). Despite their genetic similarity, these two pathogens disrupt epithelial tight junction barrier function with distinct kinetics. EHEC-induced reduction in transepithelial electrical resistance (TER), a measure of barrier function disruption, is significantly slower and more modest in comparison to that induced by EPEC. The variation in bacterial adherence only partially accounted for these differences. The LEE-encoded effector protein EspF has been shown to be critical for EPEC-induced alterations in TER. EspF from both EPEC and EHEC is expressed and secreted upon growth in tissue culture medium. The mutation of EHEC cesF suggested that the optimal expression and secretion of EHEC EspF required its chaperone CesF, as has been shown for EPEC. In contrast to EPEC espF and cesF, mutation of the corresponding EHEC homologs did not dramatically alter the decrease in TER. These differences could possibly be explained by the presence of additional espF-like sequences (designated U- and M-espF, where the letter designations refer to the specific cryptic prophage sequences on the EHEC chromosome closest to the respective genes) in EHEC. Reverse transcription-PCR analyses revealed coordinate regulation of EHEC U-espF and the LEE-encoded espF, with enhanced expression in bacteria grown in Dulbecco-Vogt modified Eagle's medium compared to bacteria grown in Luria broth. Both EHEC espf and U-espF complemented an EPEC espF deletion strain for barrier function alteration. The overexpression of U-espF, but not espF, in wild-type EHEC potentiated the TER response. These studies reveal further similarities and differences in the pathogenesis of EPEC and EHEC.
• Viswanathan, V. K., Lukic, S., Koutsouris, A., Miao, R., Muza, M. M., & Hecht, G. (2004). Cytokeratin 18 interacts with the enteropathogenic Escherichia coli secreted protein F (EspF) and is redistributed after infection. Cellular Microbiology, 6(10), 987-997.
  More info

  PMID: 15339273;Abstract: Enteropathogenic Escherichia coli (EPEC) pathogenesis requires the delivery of effector proteins into host cytosol by a type III secretion system. The effector protein EspF, while critical for disruption of epithelial barrier function through alteration of tight junctions, is not required for bacterial viability or attachment. Yeast two-hybrid analyses revealed host intermediate filament (IF) protein cytokeratin 18 (CK18) as an interacting partner of EspF. This was confirmed by co-immunoprecipitation of extracts from EPEC-infected epithelial cells. EPEC infection increased detergent-soluble CK18 amounts without significantly altering CK18 expression. The adaptor protein 14-3-3 binds to CK18 and modulates its solubility. EPEC infection promoted CK18/14-3-3 interactions, corresponding to the increase of CK18 in the soluble fractions. 14-3-3 also co-immunoprecipitated with EspF, suggesting that CK18, 14-3-3 and EspF may form a complex in infected cells. The 14-3-3ζ isoform was co-immunoprecipitated with CK18, suggesting the involvement of specific signalling pathways. Immunofluorescence studies revealed a dramatic alteration in the architecture of the IF network in EPEC-infected epithelial cells. IF fragmentation, evident at 2 h post infection, progressed to a collapse of this network at later time points. The secretion mutant (ΔescN) failed to alter CK18 solubility and IF morphology, while deletion of espF partially impaired the ability of EPEC to induce CK18 modifications. These results suggest that modifications in 14-3-3 interactions and IF network, modulated by type III secreted proteins, may be an important step in EPEC pathogenesis. © 2004 Blackwell Publishing Ltd.
• Viswanathan, V. K., Sharma, R., & Hecht, G. (2004). Microbes and their products - Physiological effects upon mammalian mucosa. Advanced Drug Delivery Reviews, 56(6), 727-762.
  More info

  PMID: 15063587;Abstract: A dynamic array of interactions occurs between pathogens and host mucosal surfaces. The signature molecules unique to microbial pathogens allow the mammalian immune system to recognize them as non-self. This recognition, mediated by the toll-like receptor proteins, results in innate immune responses targeted against the invading organism. Pathogens also elaborate a variety of proteins that actively engage host signaling pathways and subvert them to facilitate their growth and dispersal. These interactions, developed over a long evolutionary period, have been specialized to exquisite detail. These proteins and toxins are either secreted into the medium or directly delivered into host cells by specialized secretion systems. An array of host function alterations is mediated by microbial pathogens including inflammatory responses, secretory responses, alteration of host cytoskeleton, disruption of epithelial tight junctions and apoptosis. The signaling axes involved in these interactions are potential targets for therapeutic strategies against infectious microbes. © 2004 Elsevier B.V. All rights reserved.
• Berkes, J., Viswanathan, V. K., Savkovic, S. D., & Hecht, G. (2003). Intestinal epithelial responses to enteric pathogens: Effects on the tight junction barrier, ion transport, and inflammation. Gut, 52(3), 439-451.
  More info

  PMID: 12584232;PMCID: PMC1773546;Abstract: The effects of pathogenic organisms on host intestinal epithelial cells are vast. Innumerable signalling pathways are triggered leading ultimately to drastic changes in physiological functions. Here, the ways in which enteric bacterial pathogens utilise and impact on the three major physiological functions of the intestinal epithelium are discussed: alterations in the structure and function of the tight junction barrier, induction of fluid and electrolyte secretion, and activation of the inflammatory cascade. This field of investigation, which was virtually non-existent a decade ago, has now exploded, thus rapidly expanding our understanding of bacterial pathogenesis. Through increased delineation of the ways in which microbes alter host physiology, we simultaneous gain insight into the normal regulatory mechanisms of the intestinal epithelium.
• Viswanathan, V. K., & Hecht, G. (2003). Enteropathogenic Escherichia coli induced alterations in epithelial barrier function. Archives of Hellenic Medicine, 20(SUPPL. A), 26-29.
  More info

  Abstract: Enteropathogenic Escherichia coli (EPEC), a causative agent of infantile diarrhea, attaches to intestinal epithelial cells, subverts their function, and produces the characteristic "attaching and effacing (A/E) lesion". One mechanism by which EPEC causes diarrhea is by disrupting epithelial barrier function and elevating transcellular permeability. These result from an altered distribution of tight junction proteins such as occludin and claudin, as well as by the contraction of the peri-junctional actomyosin ring. While the signal transduction cascades leading to tight junction protein re-distribution remain to be elucidated, we have demonstrated that the contraction of the actomysin ring results from a calcium-dependent activation of myosin light chain kinase. EPEC pathogenesis requires a type III secretion system that directly injects effector proteins into host cells. One of the effector proteins, EspF, is critically required to cause barrier function alterations in a dose-dependent fashion. EspF has three proline-rich repeat sequences, and these have been predicted to interact with host proteins. Curiously, a clone deleted for these repeat sequences continued to complement an EspF deletion strain, suggesting that the proline-rich sequences are not critical for EspF-dependent barrier function alteration. The precise mechanism by which EspF induces barrier function alterations and the requirement of other secreted proteins for mediating this effect remain to be determined.
• Viswanathan, V. K., & Hecht, G. (2000). Innate immunity and the gut. Current Opinion in Gastroenterology, 16(6), 546-551.
  More info

  PMID: 17031136;Abstract: The intestinal epithelium encounters a unique environment consisting of microbes, both commensals and pathogens, as well as dietary nutrients and antigens. This complex composition necessitates the presence of a dynamic system of defense to contain both pathogenic and commensal bacteria within the lumen yet allow for nutrient absorption. Tight junctions provide protection of the intercellular spaces while other surface molecules, such as intestinal trefoil factor, help to maintain the structural integrity of the epithelium. Other more active processes, including upregulated expression and activation of antimicrobial peptides and enhanced fluid secretion, provide a second level of innate defense. Despite providing the interface between an exuberant immune system and a highly antigenic lumenal environment, the intestinal epithelium must remain quiescent. As such, several novel antiinflammatory mechanisms were recently identified. Studies that elaborate the various aspects of these pathways are discussed in this review. © 2000 Lippincott Williams & Wilkins, Inc.
• Viswanathan, V. K., Green, J. M., & Nichols, B. P. (1995). Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli. Journal of Bacteriology, 177(20), 5918-5923.
  More info

  PMID: 7592344;PMCID: PMC177419;Abstract: The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB undergoes inactivation upon incubation at 37°C, which is prevented by the presence of chorismate or PabA; glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the K(m) values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.

Proceedings Publications

• Viswanathan, V. (2016, Spring). Session Chair. In Digestive Diseases Week.
  More info

  Session Type: Research ForumSession Title: Gut Infections and Microbial PathogenesisSession Date & Time: May 24, 2016 from 10:00 AM to 11:30 AM

Presentations

• Halpern, M. D., Viswanathan, V., Ridgeway, K., Anett, G., Phanindra, B., Jagadish, N., Ramamurthy, S., Carothers, K., & Calton, C. M. (2022, May). Secondary Bile Acid Producing Bacteria Enhance Necrotizing Enterocolitis in a Neonatal Rat Model. . Digestive Disease Week. San Diego, CA: American Gastroenterological Association.
• Viswanathan, V. K. (2020, Summer). Pathogenic Escherichia coli manipulate intestinal epithelial cell structure and function. Gut Group, University of Arizona.
• Viswanathan, V. K. (2021, March). Pathogenic Escherichia coli manipulate mitochondrial structure & function. University of Illinois Gastroenterology Seminar. Online seminar: University of Illinois.
• Viswanathan, V. K. (2014, Fall). Enteropathogenic Escherichia coli targets the mitochondria. MicroLunch. University of Arizona.
• Viswanathan, V. K., & Vedantam, G. (2018, May). Rho Gtpase Inhibition Contributes to Enteropathogenic Escherichia Coli Esph-Induced Desmosomal Perturbations. Digestive Diseases Week. Washington DC: American Gastroenterological Association.
• Viswanathan, V. K., Viswanathan, V. K., Campos, S. K., Campos, S. K., Vedantam, G., Vedantam, G., Riggs, M. W., & Riggs, M. W. (2018, May). Dissection of the Contributions of Enteropathogenic Escherichia Coli Secreted Effector Protein ESPZ to Virulence. Digestive Diseases Week. Washington DC: American Gastroenterological Association.
  More info

  Invited oral presentation for DDW conference
• Viswanathan, V. K. (2017, March 7th). Microbial Puppetry: Enteropathogenic Escherichia coli manipulates intestinal epithelial cell function. UA Infectious Diseases Grand Rounds. Banner University of Arizona Health Sciences 6th Floor Conference Room: Division of Infectious Diseases.
• Viswanathan, V. K. (2017, March). Microbial Puppetry: Enteropathogenic Escherichia coli manipulates intestinal epithelial cell function. University of Nevada Life Sciences Seminar. University of Nevada, Las Vegas: University of Nevada, Las Vegas.
• Viswanathan, V. (2016, April). Dynamic regulation of host cell survival by enteropathogenic Escherichia coli. Invited Speaker. Chicago: University of Illinois, Chicago.
• Viswanathan, V. (2014, Fall). Environmental Adaptation of Enterohemorrhagic E. coli. MicroLunch.
• Viswanathan, V. K. (2013, April). "E. coli pathogenesis and innate immunity against enteric pathogens ". ISU-Health Research Initiative Infectious Diseases Symposium 2013. Ames, Iowa: Iowa State University.
  More info

  Symposium Event on April 22-23rd, 2013 featuring experts and collaborators from the three investigative areas of Vaccines/Diagnostics/Therapeutics (VDT), Pathogenesis and Host Responses (PHR), and Transmission/Epidemiology/Diagnostics (TED).
• Viswanathan, V. K. (2013, March). Role of EspZ in Enteropathogenic E. coli virulene. Frontiers in Immunobiology & Immunopathogenesis Symposium. Tucson, Arizona: Department of Immunobiology.
• Viswanathan, V. -. (2011). Host-pathogen relationships: It s complicated. University of Arizona, Department of Plant Sciences Seminar. Tucson, AZ.
• Viswanathan, V. -. (2011). Invited speaker, Department of Immunobiology, University of Arizona. May 2011. Seminar - Immunobiology Department. Tucson, AZ.
• Viswanathan, V. -. (2011). The Enteropathogenic Escherichia coli Type III-Secreted Protein EspZ Inhibits Host Cell ApoptosisDigestive Diseases WeekGI INFECTIONS: MOLECULAR MECHANISMS. American Gastroenterological Association Research Forum 1. Chicago.

Poster Presentations

• Vedantam, G., & Viswanathan, V. (2016, July). Clostridium difficile Infection Surveillance in a Tertiary Medical Center Reveals Significant Strain Variations. 13th Biennial Conference of the Anaerobe Society of the Americas. Nashville, TN: Anaerobe Society of the Americas.
• Vedantam, G., & Viswanathan, V. (2016, March). Multiple Student Poster Presentations (6 total). Immunobiology Annual Conference, Bio5, University of Arizona. University of Arizona: Dept. of Immunobiology, University of Arizona.
• Vedantam, G., & Viswanathan, V. (2016, May). Flagellar Glycosylation in CLostridium difficile. American Gastroenterological Association - Digestive Diseases Week. San Diego, CA: American Gastroenterological Association.
• Viswanathan, V. (2014, Jan-Dec). E. coli Pathogenesis. VARIOUS.
  More info

  Over 10 posters were presented by graduate and undergraduate students in my laboratory in various venues including (1) UBRP Annual Symposium; (2) Immunobiology Symposium, (3) ACBS Research Symposium, and (4) ASM Regional Conference.
• Vedantam, G. -., Anwar, F., Viswanathan, V. -., Mallozzi, M., Wolk, D., Ampel, N., & Noon, A. (2013, April). Characterization of low-toxin producing infections in Southern Arizona hospitals. 53ed Annual Regional Meeting, American Society for Microbiology. Tucson, AZ.
• Vedantam, G. -., Viswanathan, V. -., Chu, M., Mallozzi, M., & Roxas, B. (2013, April). Analysis of surface polysaccharides in epidemic-associated strains of Clostridium difficile. 53rd Annual Regional Meeting of the American Society for Microbiology. Tucson, AZ.
• Viswanathan, V. K., & Vedantam, G. -. (2013, August,). The EPEC effector EspZ promotes colonization of rabbits and suppresses host cell apoptosis in vivo. FASEB Summer Research Conferences, Gastrintestinal Tract XV. Steamboat Springs, Colorado: Federation of American Society for Experimental Biologists.
  More info

  Other authors:JS Wilbur, Wyatt Byrd, Ed Boedeker
• Viswanathan, V. K., Viswanathan, V. K., Vedantam, G. -., & Vedantam, G. -. (2013, August,). Quantitative global phosphoproteomic analysis of human colonic epithelial cells infected with enteropathogenic E. coli. FASEB Summer Research Conferences, Gastrintestinal Tract XV. Steamboat Springs, Colorado: Federation of American Society for Experimental Biologists.
  More info

  Other authors:Jennifer Roxas, Bryan Roxas, Gayatri Vedantam, Katheryn Ryan, Jessica Franco.

Others

• Viswanathan, V. K. (2013). Shigella takes the temperature. Gut microbes.
  More info

  For bacteria, temperature is a fairly reliable cue for entry into a vertebrate host. Many pathogenic bacteria sense elevated temperatures (37°C) and turn on virulence and metabolic genes appropriate for survival and growth within the host. A recent study demonstrated that the expression of a heme uptake protein in Shigella dysenteriae and pathogenic Escherichia coli was dependent on an RNA thermometer.
• Vedantam, G., & Viswanathan, V. (2011, September). Invited Editorial: Unlocking the gates to inflammatory bowel disease.