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 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(2011-08-18T18:30:56Z) Balaban, Murat; Hendrikson, 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 Membrane remodeling in epsilon proteobacteria and its impact on pathogenesis(2012-05) Cullen, Thomas Wilson; Trent, Michael Stephen; Whiteley, Marvin; Harshey, Rasika M.; Stevens, Scott W.; O'Halloran, Terry J.Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines.