Browsing by Subject "Flagella"
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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.Item Multi-Step Trafficking Pathway for Regulation of the Protein Composition of the Chlamydomonas Flagellar Membrane during Cilium-Generated Signaling(2013-01-16) Lara, Carmen Isabel Hernandez; Snell, William J., Ph.D.The primary cilium is a signaling organelle found on most cell types in vertebrates. It is a distinct compartment for membrane and cytoplasmic proteins that mediate multiple sensory processes. Little is known about the cellular and molecular mechanisms that determine regulated ciliary localization of membrane or soluble proteins. Our laboratory uses the biflagellated green alga Chlamydomonas as a model system to study flagellar adhesion-induced signaling. During fertilization, Chlamydomonas mating type plus and mating type minus gametes undergo flagellar adhesion through the interaction of flagellar adhesion receptor activities (flagellar agglutinins) encoded by the SAG1 (plus gametes and SAD1 (minus gametes) genes, activating a cAMP-dependent signaling pathway that ultimately leads to cell fusion and zygote formation. Our lab recently developed a Chlamydomonas strain expressing a SAG1-HA transgene, making it possible to study directly the properties of the SAG1 gene product. We found that SAG1 is cleaved soon after its synthesis to generate a C-terminal 65kDa fragment (SAG1-HA-C65), which we showed is an integral membrane protein. In my studies here, I describe new findings on the properties of SAG1-HA-C65 and its trafficking into and from the flagellar membrane. I found that in resting gametes SAG1-HA-C65 was distributed over the entire surface of the cell body plasma membrane, with little in the flagellar membrane indicating that SAG1-HA-C65 was excluded from the flagellar membrane. After flagellar adhesion-induced gamete activation or after activation of gametes with di-butyryl cAMP, SAG1-HA-C65 rapidly became enriched in the flagellar membrane concomitant with becoming concentrated around the bases of the flagella at the apical end of the cell. Moreover, cytoplasmic microtubules were required for SAG1-HA-C65 apical accumulation and for its significant delivery to the flagella during signaling. Apical concentration and flagellar enrichment of SAG1-HA-C65 occurred in cells conditionally depleted of the anterograde intraflagellar transport motor protein, kinesin-2. Furthermore, studies on the fate of SAG1-HA-C65 showed that it was shed into the medium in a detergent soluble membrane fraction during flagellar adhesion. This work provides new insights into membrane protein trafficking that regulates the protein composition of the ciliary/flagellar membrane.Item The Salmonella flagellar antigen g ... as a potential carrier of "foreign epitopes"(Texas Tech University, 1993-12) Masten, Barbara JeanSalmonella flagella filaments are polymers of a protein termed, 'flagellin'. We are interested in expressing medically important epitopes at the site(s) of naturally occurring B-cell epitopes expressed by flagellin. To determine the molecular basis for expression of the epitopes by which the Salmonella phase-1 g... series flagellin antigens are distinguished, 17 members (S. adelaide [fliC9^, S. berta [fliC9% S. budapest [fliC9^, S. califomia [fUCd^rJt]^ S. chaco [fliCdrrt]^ S. danysz [fliCd"^, S. derby [fliC9f\, S. dublin [fliC9P], S. enteritidis [fliCQrrf], S. essen [fliCd'T^, S. jena [fiiCdn^, S. monschaui [fliC^^, S. montevideo [fliCd'T^^, S. moscow [fliC9Q], S. oranienberg [ftiC^, S. mstock [fliC9P% and S. senftenberg [fliC9S^) of this series were selected and their fliC (the stmctural gene for phase-1 flagellin) genes sequenced. Comparison of the flagellin amino acid sequences showed complete homology in the N-terminal (region I, II and III) and C-terminal (region VIII) segments of the proteins. Differences in amino acids were found throughout the central portion (regions IV, V, VI and VII) of the flagellins. No localized area substituted to specify subfactor epitopes could be identified, suggesting the subfactors of the g... series are conformational at the molecular level. The amino acids comprising each of the subfactor epitopes were not definable by sequence analysis. Results of epitope mapping, by two approaches, support the view that the g... series phase-1 flagellin B-ceil epitopes are conformational. Based upon amino acid comparison, the fliC gene of S. califomia, S. monschaui and S. oranienberg may be the gene of choice for substitution(s)since it may tolerate amino acid interchanges in region V better than the fliC gene of other members of the g... series. Hydrophobicity and surface exposure plots suggest three potential sites, two within region IV and one within region VI, for epitope substitution within the flagellin of any g... series member Phylogenetic analysis and comparative analysis shows S. california, S. monschaui and S. oranienberg to be the most diverse g... series serovars. A combination of spontaneous mutation, insertion and deletion most likely generated the antigenic polymorphism seen with salmonellar g... series flagellins.