John S Wilbur

Interests

Teaching

Active learning, microbiology, immunology, pathogenic organisms, laboratory techniques, introductory science

Research

Host/pathogen interactions, bacterial genetics

Courses

Scholarly Contributions

Journals/Publications

• Viswanathan, V., Vedantam, G., Wilbur, J. S., Harishankar, A., Cocchi, K., Ramamurthy, S., Roxas, J. L., Rutins, I., Agellon, A., & Sylejmani, G. (2022). Enteropathogenic Escherichia coli regulates host-cell mitochondrial morphology. Gut Microbes, 14(1). doi:10.1080/19490976.2022.2143224
• Viswanathan, V. K., Vedantam, G., Riggs, M. W., Ramamurthy, S., Wilbur, J. S., Byrd, W., Ledvina, H. E., Khirfan, K., & Boedeker, E. C. (2015). The Secreted Effector Protein EspZ Is Essential for Virulence of Rabbit Enteropathogenic Escherichia coli. Infection and Immunity, 83(3), 1139-1149. doi:10.1128/iai.02876-14
• 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
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  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.
• Nikolakakis, K., Amber, S., Wilbur, J. S., Diner, E. J., Aoki, S. K., Poole, S. J., Tuanyok, A., Keim, P. S., Peacock, S., Hayes, C. S., & Low, D. A. (2012). The toxin/immunity network of Burkholderia pseudomallei contact-dependent growth inhibition (CDI) systems. Molecular microbiology, 84(3), 516-29.
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  Burkholderia pseudomallei is a category B pathogen and the causative agent of melioidosis--a serious infectious disease that is typically acquired directly from environmental reservoirs. Nearly all B. pseudomallei strains sequenced to date (> 85 isolates) contain gene clusters that are related to the contact-dependent growth inhibition (CDI) systems of γ-proteobacteria. CDI systems from Escherichia coli and Dickeya dadantii play significant roles in bacterial competition, suggesting these systems may also contribute to the competitive fitness of B. pseudomallei. Here, we identify 10 distinct CDI systems in B. pseudomallei based on polymorphisms within the cdiA-CT/cdiI coding regions, which are predicted to encode CdiA-CT/CdiI toxin/immunity protein pairs. Biochemical analysis of three B. pseudomallei CdiA-CTs revealed that each protein possesses a distinct tRNase activity capable of inhibiting cell growth. These toxin activities are blocked by cognate CdiI immunity proteins, which specifically bind the CdiA-CT and protect cells from growth inhibition. Using Burkholderia thailandensis E264 as a model, we show that a CDI system from B. pseudomallei 1026b mediates CDI and is capable of delivering CdiA-CT toxins derived from other B. pseudomallei strains. These results demonstrate that Burkholderia species contain functional CDI systems, which may confer a competitive advantage to these bacteria.
• Roxas, J. L., Wilbur, J. S., Zhang, X., Martinez, G., Vedantam, G., & Viswanathan, V. K. (2012). The enteropathogenic Escherichia coli-secreted protein EspZ inhibits host cell apoptosis. Infection and immunity, 80(11), 3850-7.
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  The diarrheagenic pathogen enteropathogenic Escherichia coli (EPEC) limits the death of infected enterocytes early in infection. A number of bacterial molecules and host signaling pathways contribute to the enhanced survival of EPEC-infected host cells. EspZ, a type III secreted effector protein that is unique to EPEC and related "attaching and effacing" (A/E) pathogens, plays a role in limiting host cell death, but the precise host signaling pathways responsible for this phenotype are not known. We hypothesized that EspZ contributes to the survival of infected intestinal epithelial cells by interfering with apoptosis. Consistent with previous studies, scanning electron microscopy analysis of intestinal epithelial cells infected with an EPEC espZ mutant (ΔespZ) showed increased levels of apoptotic and necrotic cells compared to cells infected with the isogenic parent strain. Correspondingly, higher levels of cytosolic cytochrome c and increased activation of caspases 9, 7, and 3 were observed for ΔespZ strain-infected cells compared to wild-type (WT) EPEC-infected cells. Finally, espZ-transfected epithelial cells were significantly protected from staurosporine-induced, but not tumor necrosis factor alpha (TNF-α)/cycloheximide-induced, apoptosis. Thus, EspZ contributes to epithelial cell survival by mechanisms that include the inhibition of the intrinsic apoptotic pathway. The enhanced survival of infected enterocytes by molecules such as EspZ likely plays a key role in optimal colonization by A/E pathogens.
• Mattison, K., Wilbur, J. S., So, M., & Brennan, R. G. (2006). Structure of FitAB from Neisseria gonorrhoeae bound to DNA reveals a tetramer of toxin-antitoxin heterodimers containing pin domains and ribbon-helix-helix motifs. The Journal of biological chemistry, 281(49), 37942-51.
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  Neisseria gonorrhoeae is a sexually transmitted pathogen that initiates infections in humans by adhering to the mucosal epithelium of the urogenital tract. The bacterium then enters the apical region of the cell and traffics across the cell to exit into the subepithelial matrix. Mutations in the fast intracellular trafficking (fitAB) locus cause the bacteria to transit a polarized epithelial monolayer more quickly than the wild-type parent and to replicate within cells at an accelerated rate. Here, we describe the crystal structure of the toxin-antitoxin heterodimer, FitAB, bound to a high affinity 36-bp DNA fragment from the fitAB promoter. FitA, the antitoxin, binds DNA through its ribbon-helix-helix motif and is tethered to FitB, the toxin, to form a heterodimer by the insertion of a four turn alpha-helix into an extensive FitB hydrophobic pocket. FitB is composed of a PIN (PilT N terminus) domain, with a central, twisted, 5-stranded parallel beta-sheet that is open on one side and flanked by five alpha-helices. FitB in the context of the FitAB complex does not display nuclease activity against tested PIN substrates. The FitAB complex points to the mechanism by which antitoxins with RHH motifs can block the activity of toxins with PIN domains. Interactions between two FitB molecules result in the formation of a tetramer of FitAB heterodimers, which binds to the 36-bp DNA fragment and provides an explanation for how FitB enhances the DNA binding affinity of FitA.
• Ayala, P., Wilbur, J. S., Wetzler, L. M., Tainer, J. A., Snyder, A., & So, M. (2005). The pilus and porin of Neisseria gonorrhoeae cooperatively induce Ca(2+) transients in infected epithelial cells. Cellular microbiology, 7(12), 1736-48.
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  Purified pili and porin from Neisseria quickly mobilize calcium (Ca(2+)) stores in monocytes and epithelial cells, ultimately influencing host cell viability as well as bacterial intracellular survival. Here, we examined the Ca(2+) transients induced in human epithelial cells during infection by live, piliated N. gonorrhoeae. Porin induced an influx of Ca(2+) from the extracellular medium less than 60 s post infection. The porin-induced transient is followed by a pilus-induced release of Ca(2+) from intracellular stores. The timing of these events is similar to that observed using purified proteins. Interestingly, the porin-induced Ca(2+) flux is required for the pilus-induced transient, indicating that the pilus-induced Ca(2+) release is, itself, Ca(2+) dependent. Several lines of evidence indicate that porin is present on pili. Moreover, pilus retraction strongly influences the porin- and pilus-induced Ca(2+) fluxes. These and other results strongly suggest that the pilus and porin cooperate to modulate calcium signalling in epithelial cells, and propose a model to explain how N. gonorrhoeae triggers Ca(2+) transients in the initial stages of pilus-mediated attachment.
• Wilbur, J. S., Chivers, P. T., Mattison, K., Potter, L., Brennan, R. G., & So, M. (2005). Neisseria gonorrhoeae FitA interacts with FitB to bind DNA through its ribbon-helix-helix motif. Biochemistry, 44(37), 12515-24.
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  The fit locus, encoding two proteins, FitA and FitB, was identified in a genetic screen for Neisseria gonorrhoeae determinants that affect trafficking across polarized epithelial cells. To better understand how the locus may control these activities, we have undertaken a biochemical analysis of FitA and FitB. FitA is a DNA-binding protein with a putative ribbon-helix-helix (RHH) motif. Purified FitA forms a homodimer that binds a 150 bp fit promoter sequence containing the translational start site. A putative beta strand mutant of FitA, FitA(R7A), is unable to bind this DNA, supporting further that FitA is a RHH protein. FitB interacts with FitA to form a 98 kDa complex. FitA/B binds DNA with a 38-fold higher affinity than the FitA homodimer. In DNase I footprint assays, FitA/B protects a 62-bp region within the fit promoter containing the predicted -10 sequence and an 8-bp inverted repeat, TGCTATCA-N(12)-TGATAGCA. FitA/B(His) is able to bind to either half-site alone with high affinity.
• Hopper, S., Vasquez, B., Merz, A., Clary, S., Wilbur, J. S., & So, M. (2000). Effects of the immunoglobulin A1 protease on Neisseria gonorrhoeae trafficking across polarized T84 epithelial monolayers. Infection and immunity, 68(2), 906-11.
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  We previously demonstrated that the Neisseria IgA1 protease cleaves LAMP1 (lysosome-associated membrane protein 1), a major integral membrane glycoprotein of lysosomes, thereby accelerating its degradation rate in infected A431 human epidermoid carcinoma cells and resulting in the alteration of lysosomes in these cells. In this study, we determined whether the IgA1 protease also affects the trafficking of Neisseria gonorrhoeae across polarized T84 epithelial monolayers. We report that N. gonorrhoeae infection of T84 monolayers, grown on a solid substrate or polarized on semiporous membranes, also results in IgA1 protease-mediated reduction of LAMP1. We demonstrate that iga mutants in two genetic backgrounds exited polarized T84 monolayers in fewer numbers than the corresponding wild-type strains. Finally, we present evidence that these mutants have a statistically significant and reproducible defect in their ability to traverse T84 monolayers. These results add to our previous data by showing that the IgA1 protease alters lysosomal content in polarized as well as unpolarized cells and by demonstrating a role for the protease in the traversal of epithelial barriers by N. gonorrhoeae.
• Hopper, S., Wilbur, J. S., Vasquez, B. L., Larson, J., Clary, S., Mehr, I. J., Seifert, H. S., & So, M. (2000). Isolation of Neisseria gonorrhoeae mutants that show enhanced trafficking across polarized T84 epithelial monolayers. Infection and immunity, 68(2), 896-905.
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  Initiation of a gonococcal infection involves attachment of Neisseria gonorrhoeae to the plasma membrane of an epithelial cell in the mucosal epithelium and its internalization, transepithelial trafficking, and exocytosis from the basal membrane. Piliation and expression of certain Opa proteins and the immunoglobulin A1 protease influence the transcytosis process. We are interested in identifying other genetic determinants of N. gonorrhoeae that play a role in transcellular trafficking. Using polarized T84 monolayers as a model epithelial barrier, we have assayed an N. gonorrhoeae FA1090 minitransposon (mTn) mutant bank for isolates that traverse the monolayer more quickly than the isogenic wild-type (WT) strain. From an initial screen, we isolated four mutants, defining three genetic loci, that traverse monolayers significantly more quickly than their WT parent strain. These mutants adhere to and invade cells normally and do not affect the integrity of the monolayer barrier. Backcrosses of the mutations into the WT FA1090 strain yielded mutants with a similar fast-trafficking phenotype. In two mutants, the mTns had inserted 370 bp apart into the same locus, which we have named fit, for fast intracellular trafficker. Backcrosses of one of these mutants into the MS11A genetic background also yielded a fast-trafficking mutant. The fit locus contains two overlapping open reading frames, fitA and fitB, whose deduced amino acid sequences have predicted molecular weights of 8.6 and 15.3, respectively. Neither protein contains a signal sequence. FitA has a potential helix-turn-helix motif, while the deduced sequence of FitB offers no clues to its function. fitA or fitB homologues are present in the genomes of Pseudomonas syringae and Rhizobium meliloti, but not Neisseria meningitidis. Replication of the MS11A fitA mutant in A431 and T84 cells is significantly accelerated compared to that of the isogenic WT strain. In contrast, growth of this mutant in liquid media is normal. Taken together, these results strongly suggest that traversal of N. gonorrhoeae across an epithelial barrier is linked to intracellular bacterial growth.