Browsing by Subject "Campylobacter jejuni"
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Item Analysis of Bacterial-Host Interactions Between Campylobacter jejuni and the Avian Host During Commensalism(2009-06-15) Bingham-Ramos, Lacey Kathleen; Hendrixson, David R.Campylobacter jejuni is a leading cause of bacterial enteritis in humans throughout the world. In contrast to the disease seen in humans upon infection, C. jejuni promotes an asymptomatic, intestinal colonization of many animals, especially avian species, to result in commensalism. The primary route of transmission to humans is through the consumption or handling of undercooked poultry meats, making C. jejuni of particular importance to the agricultural industry. The direct interplay between C. jejuni and the natural avian host was examined to better understand the interactions that contribute to commensalism. We analyzed the colonization dynamics of C. jejuni over 28 days and identified a previously uncharacterized prolonged, robust colonization of the bursa of Fabricius, a major lymphoid organ. C. jejuni localized to the mucus layer lining the epithelium of the bursal lumen, with no invasion of or damage to host tissue apparent. However, C. jejuni was detected invading the cecal epithelium of chicks but only at day 1 post-infection, which may contribute to the observed transient, infection of the spleen and liver. Additionally, certain colonization factors of C. jejuni were shown to promote persistence in specific organs. Mutants lacking catalase and the cytolethal distending toxin demonstrated a reduction in levels in the bursa but not the ceca during prolonged colonization, whereas an unencapsulated mutant showed a global colonization defect of all organs. These findings suggest that persistent colonization of the bursa and the ceca, and the ability of the avian host to largely confine C. jejuni to mucosal surfaces may be specific for the development of commensalism. Separate analyses of additional colonization factors of C. jejuni revealed the importance of two putative cytochrome c peroxidases (CCP), DocA and Cjj0382, in promoting efficient cecal colonization. Further analysis of DocA and Cjj0382 revealed that both proteins have typical characteristics of CCPs, as they are periplasmic proteins with heme-dependent peroxidase activity. Our data suggest that although DocA and Cjj0382 have characteristics of CCPs, they likely perform different physiological functions for the bacterium during colonization. Overall, this study enhances our understanding of the interactions between C. jejuni and a natural host that contribute to the development of commensalism.Item Butyrate Sensing by Campylobacter jejuni Impacts Bacterial-Host Interactions(2020-08-01T05:00:00.000Z) Goodman, Kyle Nicholas; Winter, Sebastian E.; Hendrixson, David R.; Sperandio, Vanessa; Pfeiffer, Julie K.The intestinal microbial ecosystem aids the host in digestion and nutrition by breaking down goods and providing beneficial vitamins and metabolites, including short-chain fatty acids (SCFAs) and lactate. Due to the abundance and intestinal distribution of these metabolites, bacterial pathogens can use them as biogeographical cues to discriminate among different regions of the host intestines. Indeed, Campylobacter jejuni, a commensal bacterium of the lower intestinal tract of avian species and a leading cause of bacterial diarrheal disease in humans, recognizes intestinal niches that support growth by sensing molecular cues produced by the microbiota. How C. jejuni senses and responds to microbiota-generated SCFAs and organic acids is not understood. Herein, I identified and characterized the C. jejuni BumSR two-component signal transduction system (TCS) that specifically directs a response to butyrate. Deletion of either C. jejuni bumS or bumR abolishes butyrate-modulated transcriptional changes in gene expression. Analysis of ΔbumS and ΔbumR mutants in a chick model of commensalism indicated that bumR is important for early colonization. This contrasts with a human volunteer infection study that demonstrated bumR is essential for infection of humans. Mutational analyses of genes within the BumSR regulon in the natural avian host revealed additional colonization factors including peb3, a putative glycoprotein adhesin/substrate binding protein, and Cjj0580, a putative d- and tri-carboxylate transporter. Through multiple biochemical assays, I discovered that BumS lacks kinase activity in vitro but possesses specific phosphatase activity towards BumR. These activities are not directly influenced by butyrate, suggesting that other metabolites, perhaps resulting from butyrate catabolism, are the direct cues sensed by BumS to modulate butyrate-dependent responses. By site-directed mutagenesis, I identified residues in the conserved H box that are required for BumS phosphatase activity. Consistent with previous work, phospho-BumR exhibits enhanced binding of target promoters in electrophoretic mobility shift assays, indicating that phosphorylated BumR likely has higher affinity to bind DNA at target promoters in vivo to either enhance or repress gene expression. Overall, this highlights BumSR as a non-canonical and first-identified TCS that directs a response to butyrate to modulate colonization gene expression through a phosphatase-dependent mechanism.Item Characterization of the Activation of the FlgSR Two-Component System in Campylobacter Jejuni(2009-06-17) Joslin, Stephanie Nicole; Hendrixson, David R.Epidemiological studies indicate that Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide. This organism has the ability to live as a commensal or a pathogen, depending on the host with which it is associated. While colonization of the gastrointestinal tract of many avian and mammalian species results in a harmless commensal relationship, human infection can cause diarrheal disease. In both scenarios flagellar motility is crucial for promoting optimal host interactions, as non-motile C. jejuni colonize the gastrointestinal tracts of commensal hosts at levels significantly lower than motile isolates and are incapable of causing disease in humans. The means by which C. jejuni regulates flagellar gene transcription and assembly differ from the well-studied pathways in species of Salmonella, E. coli, and Vibrio. Previous studies found that C. jejuni requires the flagellar export apparatus, sigma54, and a two-component regulatory system comprised of the FlgS sensor kinase and the FlgR response regulator to activate transcription of the middle and late sigma54-dependent flagellar genes. The FlgR response regulator is an NtrC-like protein that can be divided into three domains: an N-terminal domain that is phosphorylated by FlgS, a central sigma54 interaction domain, and a C-terminal domain of unknown function. Characterization of FlgR was accomplished by generating constructs that lack the N- or C-terminal domains of the protein and the site of phosphorylation. Through genetic and biochemical analyses, we found that both the N- and C-terminal domains have suppressive functions that prevent FlgR activation of sigma54-dependent flagellar gene transcription in the absence of FlgS. Our data also indicate that unlike other NtrC-family proteins, the C-terminus of FlgR does not bind DNA and is dispensable for FlgR activity. The FlgS sensor kinase activates FlgR through phosphorylation, but little was known about its activation prior to these studies. We have identified the site of FlgS autophosphorylation and demonstrated that formation of the flagellar export apparatus and the presence of at least one other flagellum-associated protein is required for autoactivation of this protein. This study provides insight into the unusual regulation of the FlgSR two-component system and its role in activating sigma54-dependent flagellar gene transcription.Item Determinants Influencing Polar Flagellar Biosynthesis and Cell Division in Campylobacter jejuni(2011-08-18) Balaban, Murat; Hendrixson, David R.Campylobacter jejuni is a worldwide leading cause of bacterial gastrointestinal disease. The natural habitat of this organism is the gastrointestinal tracts of warm-blooded animals, especially poultry, where the bacterium promotoes a harmless commensal colonization. The abundance of C. jejuni in poultry creates a risk for food-borne infections to human populations. Flagellar motility by C. jejuni is required to colonize both human and animal hosts. For motility, C. jejuni produces amphitrichous flagella, resulting in the formation of a single flagellum at both poles. This work explored factors that regulate numerical and spatial parameters for amphitrichious flagellation. Two factors that have been identified to control flagellar placement and numbers in polarly-flagellated bacteria are the FlhF GTPase and the FlhG ATPase. FlhF has been shown to be required for regulation of flagellar gene expression and flagellar placement in some Pseudomonas and Vibrio species. Characterization of FlhF in C. jejuni was accomplished by creating point mutants in C-terminal GTPase domain of FlhF to decrease its GTPase activity. GTPase mutants, unlike mutants that lack FlhF, did not have a significant reduction in sigma54-dependent flagellar gene expression. Instead, a significant proportion of the population produced flagella at lateral sites or produced multiple flagella at a pole, whereas wild-type bacteria produced single polar flagella. Further experiments suggested that FlhF functions downstream of the FlgSR-flagellar export apparatus (FEA) pathway to activate sigma54-dependent flagellar gene expression. Thus, our data suggested that FlhF and its GTPase activity are required for distinct processes in flagellar gene regulation. FlhG has been shown to control flagellar numbers in Pseudomonas and Vibrio species. We examined flhG mutants and confirmed that FlhG regulates flagellar numbers. C. jejuni flhG mutants also demonstrated a minicell phenotype, which is the result of division erroneously occurring at polar regions. Further examination revealed that FlhG and the flagellar base components compose a novel division inhibition system to spatially prevent polar division and encourage septation at the cellular midpoint for symmetrical division. This work greatly extends our understanding of factors that govern spatial and numerical patterns of polar flagellation and has identified an unprecedented system to spatially regulate division in bacteria.Item Identification and Characterization of Flagellar Co-expressed Determinants (Feds) of Campylobacter jejuni(2013-10-23) Barrero-Tobon, Angelica M.; Hansen, Eric J.; Hendrixson, David R.; Alto, Neal; Shiloh, MichaelCampylobacter jejuni is the leading cause of bacterial gastroenteritis in humans throughout the world. In contrast to infection of humans, C. jejuni is a commensal organism of the intestinal tracts of wild and agriculturally-significant animals and avian species. Flagellar motility is the only virulence and colonization factor proven to be required for infection of human volunteers to promote disease and infection of poultry for commensalism. Expression of many flagellar genes is dependent on two alternative sigma factors, σ54 and σ28. Μany rod and hook genes are dependent on σ54 for expression, whereas σ28 is involved in the expression of the major flagellin and other filament genes. We investigated the σ28 regulon and identified five genes that are dependent on σ28 and flagellar components for maximal expression, but are not required for motility. One gene, ciaI, has previously been shown to function in intracellular survival after invasion of human intestinal epithelial cells. The four remaining genes, which we annotated as fedA-fedD (for flagellar co-expressed determinants), encode proteins that have not been characterized. Mutants lacking any one of these feds or ciaI demonstrated a reduced commensal colonization capacity in a natural chick model of colonization. Similar to the σ28-dependent gene product FspA1, a subset of these Feds is secreted by the bacterium in a flagellar-dependent manner. To further investigate the secretion requirements of these σ28-dependent proteins (FedB, CiaI and FspA1), we examined putative flagellar chaperones, flagellar components and other aspects of flagellar biosynthesis such as flagellar protein glycosylation for a role in secretion of Feds. We discovered that, like in other motile organisms, the FliJ chaperone is required for secretion of flagellar components in general, and that FliS is likely the chaperone for the major flagellin, FlaA. However, FliS and other putative flagellar chaperones are not required for secretion of the Feds. We also discovered that secretion of the Feds occurs during or just after hook biosynthesis, suggesting that construction of a hook is required for maximal secretion of these proteins via the flagellum. In addition, in the absence of the flagellar cap or flagellin glycosylation, we observed an increase in secretion of FedB, CiaI and FspA1, suggesting a possible inverse correlation between the amount of Fed proteins secreted via the flagella and length of the flagellar filament. Furthermore, we have identified N- and C-terminal intramolecular determinants within FedB and CiaI that are required for maximal secretion. Based on how other flagellar proteins are secreted, these findings indicate that a flagellar Type III secretion system (T3SS)-specific signal sequence is likely found at the N-terminus, and that an unidentified chaperone may bind to the C-terminus. Both of these factors appear to be required for maximal flagellar-dependent secretion of the Feds. We also examined the importance of secretion of Feds during commensal colonization and invasion of human colonic epithelial cells in vitro. Gentamicin-protection assays revealed that secretion of CiaI is not required for invasion of T84 cells. Furthermore, preliminary studies using a chick model of commensal colonization showed that secretion of FedB is important for colonization of the chick intestinal tract. However, whereas CiaI is required for colonization, secretion of CiaI was not important for colonization of the chick cecum. In summary, our work provides evidence that the flagellar system is a global regulatory system that coordinates production of flagella with colonization and virulence determinants, some of which are secreted in a flagellar-dependent manner, to promote maximal fitness during colonization and virulence.Item The Regulation of Flagellar Biosynthesis and Cell Division in Campylobacter jejuni(2016-04-05) Gulbronson, Connor James; Hansen, Eric J.; Hendrixson, David R.; Norgard, Michael V.; Alto, NealFlagellar biosynthesis is one of the rare processes known to be spatially and numerically regulated in polarly-flagellated bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. FlhG also influences spatial regulation of division, which is essential for viability and is typically regulated by the Min system in most bacteria. However, C. jejuni lacks the Min system, but appears to utilize FlhG and components of the flagellar MS and C ring to influence spatial regulation of division. We utilized a variety imaging techniques to quantify the in vivo effects of mutations in C. jejuni and used purified proteins to assay the in vitro enzymatic activity of FlhG and FlhF (a GTPase) to determine the influence these factors have on both regulation of flagellar biogenesis and spatial regulation of division. We found that unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni instead, other regions of C. jejuni FlhG were discovered to be involved in numerical regulation of flagellar biogenesis. Mutations in the α6 and α7 helices of FlhG were found to influence aspects of FlhG biology, spatial regulation of division, and numerical regulation of flagellar biogenesis. We also found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. In this work, we propose a model in which FlhF in a GTP-bound ('active') state promotes the formation of the MS and C rings at the aflagellated pole after a division event. We then hypothesize that MS and C ring proteins influence FlhG localization to stimulate FlhF GTPase activity and, by extension, numerical regulation of flagellar biogenesis and spatial regulation of division at poles. Although some aspects of this model have yet to be fully tested, our data could potentially be applied in other polar flagellates to gain a better understanding of numerical regulation of flagellar biogenesis and spatial regulation of division in these organisms.Item Signaling Specificity in a Campylobacter jejuni Two-Component System to Mediate Proper Flagellar Gene Transcription(2013-04-05) Boll, Joseph Michael 1981-; Hansen, Eric J.; Hendrixson, David R.; Norgard, Michael V.; Sperandio, VanessaCampylobacter jejuni is a worldwide leading cause of bacterial gastroenteritis. While infection of humans leads to diarrheal disease, C. jejuni asymptomatically colonizes the intestinal tract of many agriculturally-significant animals, especially poultry. Flagellar motility is essential for Campylobacter jejuni to promote commensal colonization of avian species and for infection of humans to result in disease. Expression of flagellar genes is regulated by alternative σ factors, whose activities are controlled by a regulatory cascade. Previous genetic screens discovered the flagellar type III secretion system (T3SS), the FlgSR two-component regulatory system (TCS), and the FlhF GTPase as requirements to positively regulate expression of σ54-dependent genes that encode flagellar rod and hook proteins. Our laboratory previously proposed that signal transduction through the FlgSR TCS initiates with FlgS detecting formation of the flagellar T3SS and culminates in phosphorylation of the FlgR response regulator and expression of flagellar genes. I investigated this model by determining if any other flagellar components are required for activation of FlgSR and expression of σ54-dependent flagellar genes. I found that mutants lacking the MS ring (FliF), the rotor component of the C ring (FliG), and the rod proteins (FliE, FlgB, FlgC and FlgF) expressed reduced levels of σ54-dependent flagellar genes. These findings suggest a more complex flagellar structure rather than solely the flagellar T3SS is required to activate σ54-dependent gene expression. Due to data generated by additional experimentation, I propose a revised model in which the C. jejuni flagellar T3SS facilitates polymerization of the MS ring and rotor component of the C ring, which together form a cytoplasmic domain that is likely the direct signal sensed by FlgS to activate signal transduction required for flagellar gene expression in C. jejuni. Previous analysis discovered that activation of σ54-dependent flagellar gene expression in C. jejuni is dependent on phosphotransfer through the FlgSR two-component system. Whereas this signaling mechanism results in specific activation of FlgR via its cognate FlgS sensor kinase, I identified a domain of FlgR that possesses an unusual activity in specifically preventing in vivo crosstalk with small phosphodonor metabolites. Through genetic and biochemical analysis, I demonstrated that the metabolite acetyl-phosphate (AcP) serves as an efficient phosphodonor for FlgR lacking its C-terminal domain but not for wild-type FlgR. Additionally, I could reprogram FlgR-dependent flagellar gene expression to respond to the metabolic state of the cell and restore wild-type levels of flagellar gene expression in the absence of FlgS. However, flagellar biosynthesis was not restored to wild-type levels when AcP was the sole phosphodonor for FlgR. This study illustrates how signaling specificity in a TCS ensures a correct output response and highlights the importance of controlling proper signaling between cognate histidine kinase and response regulator pairs in bacterial TCS systems.Item Signals and Sensory Mechanisms that Impact Campylobacter jejuni-Host Interactions(2015-05-21) Luethy, Paul Michael; Sperandio, Vanessa; Hendrixson, David R.; Winter, Sebastian E.; Michael, AnthonyCampylobacter jejuni is a leading cause of bacterial diarrheal disease worldwide and a frequent commensal organism of the intestinal tract of poultry and other agriculturally-important animals. Upon infection of the avian host, C. jejuni likely responds to external stimuli present within the intestinal tract to establish commensalism. The sensing mechanisms and subsequent physiological responses by C. jejuni can be crucial for initial growth and colonization and long-term persistence within the infected host. However, how many of the signals and sensing mechanisms affecting C. jejuni biology are not fully understood. In this work, I explored signal transduction mechanisms and possible in vivo signals that may influence the colonization capacity of C. jejuni. One method C. jejuni employs to monitor environmental stimuli are two-component regulatory systems (TCSs). I analyzed the potential of C. jejuni Cjj81176_1484 (Cjj1484) and Cjj81176_1483 (Cjj1483) to encode a cognate TCS that influences expression of genes possibly important for C. jejuni growth and colonization. Through transcriptome analysis, I discovered that the regulons of the Cjj1484 histidine kinase and the Cjj1483 response regulator contain many common genes, which suggests these proteins likely form a cognate TCS. I found that this TCS generally functions to repress expression of specific proteins with roles in metabolism, iron/heme acquisition, and respiration. Furthermore, the TCS repressed expression of Cjj81176_0438 and Cjj81176_0439, which had previously been found to encode a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract. However, the TCS and other specific genes whose expression is repressed by the TCS were not required for colonization of chicks. I observed that the Cjj1483 response regulator binds target promoters both in unphosphorylated and phosphorylated forms and influences expression of some specific genes independently of the Cjj1484 histidine kinase. I propose that this TCS may sense signals found in the host intestinal tract, wherein repression of genes may be relieved. In addition to characterizing the Cjj1484/Cjj1483 TCS, I explored the role of metabolites that are commonly found in the intestines -- organic acids and short chain fatty acids (SCFAs) -- in C. jejuni commensal colonization. C. jejuni has both acetate and lactate utilization pathways, as well as for acetate production. I observed that acetogenesis mutants incapable of producing acetate were deficient for colonization of the avian intestinal tract early during infection, but not at later points during infection. Furthermore, I found that an acetogenesis mutant was impaired during growth in a defined media containing solely amino acids and organic acids as carbon sources. Transcriptome analysis of the acetogenesis mutant identified the SCFA-induced regulon which contains metabolically important genes, many of which have been implicated in C. jejuni colonization and virulence. In addition, I found that peb1C, which was downregulated in the acetogenesis mutant, was important for colonization of the chick ceca. I further confirmed in vitro that physiological concentrations of the SCFAs acetate and butyrate activated expression of the SCFA-induced regulon whereas the organic acid lactate repressed these genes. I found that in vivo expression of the SCFA-induced regulon was highest in regions of the intestinal tract where SCFAs are present in the greatest concentration. Furthermore, butyrate counteracted the inhibitory effects of lactate when the two compounds were combined in culture in vitro. I propose that C. jejuni senses the concentration of SCFAs and organic acids to discriminate between different regions of the intestinal tract and to coordinate expression of colonization genes in the preferred niche for colonization. In effect, SCFA sensing and signaling allows C. jejuni to home to appropriate sites of the host for colonization and long-term persistence.