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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2021)
Campylobacter jejuni is the leading bacterial cause of severe infectious diarrhea worldwide, and prior infection with the pathogen is highly correlated with the acute neuromuscular paralysis of Guillain-Barré Syndrome. As a zoonotic bacterium, C. jejuni successfully colonizes multiple host animal species harmlessly, survives transmission in the natural environment, and as a widespread food-borne pathogen, causes gastroenteritis in humans. Stress survival mechanisms are important to understand because they enable this success, and may lead to new food safety strategies and interventions to eliminate the harm caused by C. jejuni. Here, the global response of C. jejuni to hyperosmotic stress—which is encountered in hosts, the environment, and food processing—was characterized by whole-genome transcriptional profiling. This identified important physiological responses to hyperosmotic stress, and in particular, identified the importance of cross-induction of heat shock and oxidative stress response proteins. Single-cell and single-colony comparisons also identified prevalent stress phenotypic heterogeneity within the population. Contributing to the phenotypic variation, high frequency multifarious mutations in two purine biosynthesis genes were identified via whole-genome sequencing of colonial isolates. These mutations differentially affected tolerance to a variety of stresses, and different mutations were important for enhanced intracellular survival in epithelial cells, which is correlated with virulence in humans. This genetic variation in the population also contributed to enhanced biofilm formation, and conferred differential niche-preference behavior to individual mutation-bearing bacteria. Together, these high frequency mutations contributed to novel adaptive properties of C. jejuni, and thus a mechanism of success was identified. A hyperosmotic stress-upregulated gene, cj1533c, was also recognized as a novel determinant of hyperosmotic and oxidative stress resistance. Preliminary characterizations of cj1533c via mutational and proteomic analyses suggested a critical role for Cj1533c, a putative ATPase, in the coordination of multiple stress-related cellular pathways. Lastly, new genetics tools, fluorescent probes, and proteomics techniques were adapted for used in future C. jejuni research. Collectively, a number of unique C. jejuni success mechanisms were identified via their importance for hyperosmotic stress tolerance.
Campylobacter jejuni is a leading cause of foodborne bacterial gastroenteritis in both developed and developing nations. Although C. jejuni is a common environmentally acquired pathogen, it is quite fastidious, rapidly losing viability in aerobic conditions. Genome sequence analyses have failed to identify classical virulence factors, making the pathogenic success of C. jejuni a mystery. Mutational analysis described herein identified novel metabolic factors that are important for infection of human epithelial cells as well as generation of oxidative stress in C. jejuni during aerobic incubation. Investigation of a novel operon, fdhTU, induced during C. jejuni epithelial infection, showed that FdhTU positively regulates formate dehydrogenase. Subsequent analyses found that fdhTU and formate dehydrogenase are important for recovery of C. jejuni from epithelial cells. Further work showed that intracellular C. jejuni are undergoing oxidative stress, and that neutralization of oxidative stress with sulfite or catalase could significantly enhance recovery of C. jejuni following epithelial cell infection. Analyses of other respiratory dehydrogenases failed to identify other systems important for recovery of C. jejuni from epithelial cells, but did identify a role for gluconate dehydrogenase in reducing necrosis in T84 epithelial cells in a reactive oxygen species- and calpain-dependent manner. In addition to the importance for epithelial cell infection, metabolic features were also found to be involved in causing oxidative stress in C. jejuni under aerobic conditions. C. jejuni was found to produce H₂O₂ when incubated in aerobic but not microaerobic conditions at 37ºC but not 4ºC, with formate dehydrogenase and sulfite oxidoreductase dependent respiration important for H₂O₂ production. Sulfite and cysteine could reduce C. jejuni loss of viability in aerobic conditions in a manner dependent on the sulfur assimilation pathway protein Atps. Atps was identified as important for aerobic survival, H₂O₂ resistance, and in reducing H₂O₂ produced by formate dehydrogenase dependent respiration. Characterization of the role of multiple respiratory systems in C. jejuni, a bacterial model that shares little with other common pathogenic bacteria, has identified a central role of respiration in epithelial cell infection and environmental survival.
Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis in the developed world. Althoughillness is usually self-limiting, immunocompromised individuals are at risk for infections recalcitrant toantibiotic treatment. Prior infection with C. jejuni also correlates with serious sequelae such as Guillain-Barrésyndrome. The success of C. jejuni as a zoonotic pathogen indicates it can adapt to varied conditionsencountered during pathogenesis, despite apparent fastidiousness in the lab. Understanding how C. jejunisurvives in common reservoirs may allow development of strategies to limit survival in infection reservoirs orduring pathogenesis, and greatly reduce the impact of C. jejuni-mediated disease. A two-component regulatorysystem, (CprRS; Campylobacter planktonic growth regulation) was previously identified in a screen for genesthat may be required for adaptation to the host. Subsequent characterization of CprRS has contributed tounderstanding of two themes related to C. jejuni survival: environmental gene regulation and biofilmformation. The CprR response regulator was essential for viability, and while the CprS sensor kinase wasdispensable, a ΔcprS mutant showed significant phenotypic differences from WT. Initial characterization ofΔcprS using phenotypic and proteomic means provided evidence that CprRS affects phenomena related tobiofilm formation. Further characterization of CprRS was undertaken through transcriptomics of ΔcprS,molecular analysis of CprR, and promoter analysis. The CprRS regulon suggests that the system may controlaspects of the cell envelope, including expression of the HtrA periplasmic protease. Finally, subsequentanalysis of the biofilm-enhanced ΔcprS mutant, together with epistatic analyses and analysis of WT C. jejuniunder stress conditions, has provided insight into C. jejuni biofilm initiation, maturation, and physiology. Aspecific role for flagella in biofilm initiation was demonstrated, and lysis and extracellular DNA release duringbiofilm maturation was also observed. Furthermore, evidence that the C. jejuni biofilm lifestyle confers stresstolerance that is not present in planktonic counterparts was obtained. Characterization of CprRS has thuscontributed to knowledge of both physiological and regulatory themes that provide C. jejuni, a pathogenwhich diverges from paradigms set out in model bacteria, with its surprising resilience during zoonosis, andhas also identified novel targets for infection control.
Bacteria are found in almost all conceivable environments, and some species can survive many different conditions. The ability to detect environmental conditions and respond with appropriate changes to gene expression is essential to survival. Bacteria sometimes express genes involved in horizontal gene transfer when encountering a stressful environment. Horizontal gene transfer has an important role in the evolution of prokaryotic genomes. Rhodobacter capsulatus produces a mediator of horizontal gene transfer called the gene transfer agent (GTA). The R. capsulatus GTA is a bacteriophage-like particle that transfers ~4 kb of double stranded genomic DNA using a transduction-like mechanism. Previously, two proteins encoded outside the GTA gene cluster, GtaI and CtrA, were found to regulate GTA expression. GtaI and GtaR are LuxI-type and LuxR-type quorum sensing proteins, respectively. CtrA and CckA are homologues of the response regulator and sensor kinase, respectively, of the Caulobacter crescentus CtrA/CckA signal transduction system. In this thesis, I studied the interactions between these regulatory proteins, environmental conditions and GTA in R. capsulatus. I found that growth conditions had opposite effects on GTA and ctrA expression, but no effect on gtaR expression, and phosphate limitation decreased expression of ctrA. Knockout experiments revealed that GtaI and GtaR affect ctrA, gtaR and GTA expression. Results from GtaR-DNA binding experiments were consistent with a model in which GtaR directly regulates its own expression but indirectly regulates ctrA and GTA expression. These studies also identified GtaR binding sequences. I found that in R. capsulatus CtrA did not regulate its own transcription, contrary to what occurs in C. crescentus. My research also showed that GTA expression was affected by at least one other unidentified system. Promoter deletion studies of ctrA, gtaR and GTA genes identified sequences that may be involved in GtaI-, GtaR-, CtrA-, and/or growth condition-based regulation. Overall, these studies contribute to the understanding of how bacteria detect multiple environmental signals and respond with changes to gene expression.
Master's Student Supervision (2010 - 2020)
Campylobacter jejuni (C. jejuni), a zoonotic commensal that is pathogenic in humans, is one of the most common bacterial causes of food borne illness worldwide. To assess how C. jejuni responds to the metabolome of a commensal host (chickens) versus a disease susceptible host (humans), differences in gene expression was evaluated after C. jejuni exposure to cell-free extracts prepared from chicken cecal and human fecal matter. RNA sequencing identified 12 genes with >2 fold difference in expression when C. jejuni was exposed to human fecal extracts in comparison to chicken cecal extract. 10 of these genes appear to be involved in iron uptake, of which 7 (CJJ81176_1649 to 1655) were part of one iron uptake system. This system likely acquires chelated iron not recognized by other iron uptake systems since measurement of total iron content showed that human fecal extracts contained ~4.5X more iron than chicken cecal extract. Homologs of the CJJ81176_1649 to 1655 proteins were identified in alpha, epsilon and gamma proteobacteria, and mapping of the homologous proteins to representative bacterial genomes showed that gene order and operon structure were well preserved for homologs of the entire CJJ81176_1649 to 1655 gene cluster. The widespread prevalence of the entire gene cluster putatively suggests that the proteins encoded by CJJ81176_1649 to 1655 represent a complete iron uptake system. The CJJ81176_1649 iron transporter and the p19 (CJJ81176_1650) periplasmic iron binding proteins have been previously characterized, but the downstream genes have not been directly studied and functions are predicted by homology. Deletion of CJJ81176_1651 to 1655 and the overlapping CJJ81176_1656 gene in this study rendered C. jejuni more sensitive to iron depletion than wild type, comparable to that of the p19 mutant. Furthermore, this iron uptake system appears to be involved in adaptation to low pH, but at the cost of increased sensitivity to hydrogen peroxide stress. This work demonstrates that the heretofore understudied, but widely conserved, CJJ81176_1649 to 1656 iron uptake system may be involved in host colonization by uptake of chelated iron more abundantly present in the human intestinal environment than that of the chicken cecum.