Progress 08/01/06 to 07/31/10
Outputs
OUTPUTS: The goals of the proposal were to characterize three sets of factors of Campylobacter jejuni involved in different biological processes required for commensal colonization of the gastrointestinal tracts of 1-day old chicks. These three classes of colonization factors include: DocA and Cj0358, two putative cytochrome c peroxidases (CCPs); the Liv amino acid transport system; and FlgP, FlgQ, and FlhG, three proteins required for flagellar motility. In analysis of the DocA and Cj0358 proteins, a docA mutant demonstrated a severe colonization defect at all inocula tested. However, a cj0358 mutant displayed only a signficant colonization defect when a low inoculum was used for infections. Further analysis revealed that both proteins are located in the periplasm and bind heme, which are characteristics of CCPs. Both DocA and Cj0358 have peroxidase activity, but are not required for survival to hydrogen peroxide. Based on a homologous operon in E. coli, the liv operon of C. jejuni is hypothesized to encode six proteins that form a transport system for leucine, isoleucine, and valine. The LIv system in E. coli is composed of LivJ and LivK (two periplasmic binding proteins), LivM and LivH (two inner mebrane permeases) , and LivF and LivG (two cytoplasmic ATPases). C. jejuni mutants were generated that lacked a single liv gene or mutant lacking both genes encoding proteins with paired functions (e.g,. a livJ and livK double mutant). Mutants lacking LivJ and LivK demonstrated defects in efficient colonization of the chick gastrointestinal tract. However, mutation of the other liv genes did not appear to be defective for colonization. To understand the function of the Liv system, amino acid transport assays were performed. Unlike the E. coli Liv system, the C. jejuni Liv system was found to be required for transport of leucine, but not isoleucine or valine. Preliminary analysis indicated that FlgP and FlgQ are likely involved in flagellar rotation as C. jejuni lacking each protein express wild-type levels of flagellar genes and produce normal levels of flagella organelles, but are non-motile. By immunoblot analyses, FlgP was found to localize to the outer membrane and this localization was dependent on FlgQ. Wild-type C. jejuni normally produces a single flagellum at one or both poles of the bacterium. A C. jejuni flhG mutant was found to produce multiple flagella at a pole, which causes a decrease in motility. Furthermore, FlhG was found to perform a role in controlling proper septation as a flhG mutant produced minicells, indicating that bacterial cell division was occurring at polar regions in the mutant rather than at the midpoint of the cell. An flhG mutant with a point mutation in an ATPase domain resulted a severe inhibition of septation and elongation of the bacteria to lengths 10-times longer than wild-type bacteria. These outputs were disseminated by 7 publications, participation in multiple meetings with seminar and poster presentations, and invited seminars at multiple universities. PARTICIPANTS: David R. Hendrixson, Ph.D. (PI): Dr. Hendrixson designed all experiments related to this project and supervised the progress of the project. Dr. Hendrixson also trained two graduate students who worked on aspects of this project. Deborah A. Ribardo, Ph.D.: Dr. Ribardo is a research scientist and lab manager who participated in most of the experiments related to analysis of the Liv system. Dr. Ribardo also assisted in the training of two graduate students who worked on this project. Lacey K Bingham-Ramos, Ph.D.: Dr. Bingham-Ramos was a graduate research assistant who worked on experiments related to understanding the role of DocA and Cj0358 in promoting growth of C. jejuni in the intestinal tract. By accomplishing these and other studies, Dr. Bingham-Ramos completed her Ph.D. requirements. Murat Balaban: Mr. Balaban is a graduate research assistant who participated in the analysis of FlhG. These and other studies are part of his Ph.D. thesis which will be completed within the next few months. Shawn Sommerlad: Mr. Sommerlad was a research assistant who participated on the projects related to the Liv system, FlgP, and FlgQ. TARGET AUDIENCES: The target audience for this research project includes the biomedical science community with an emphasis on those individuals interested in the biology of food-borne pathogens and Campylobacter jejuni. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have provided a better understanding of the colonization factors of Campylobacter jejuni required for efficient colonization of the chick gastrointestinal tract. This information will be valuable for considering potential targets for vaccination or therapeutics to reduce the ability of C. jejuni to colonize the natural poultry host in agricultural settings. DocA and Cj0358 were found to both have characteristics of CCPs, including being localized to the periplasm, existing in a heme-bound state, and possessing peroxidase activity. However, these proteins do not appear to be required for survival to exogenous hydrogen peroxide. Furthermore, since a catalase mutant, which is severely sensitive to hydrogen peroxidase, was not as defective as a docA mutant for colonization, DocA likely provides a biological function that is not related to protection to hydrogen peroxide during chick colonization. The requirements of the Liv amino acid transport system of C. jejuni for wild-type levels of colonization of the chick intestinal tract was localized to two of the six proteins of this system, LivJ and LivK. All proteins of the Liv system were found to be required for leucine transport into C. jejuni. Because only LivJ and LivK are required for colonization, LivJ and LivK likely bind to and assist in the transport of some other essential factor into C. jejuni to promote optimal growth in the chick intestinal tract. This study suggest that analogs of leucine or other factors may possibly be used to develop an antimicrobial that could be transported in vivo in a LivJ- or LivK-dependent manner to inhibit growth of C. jejuni in the chick intestinal tract. FlgP and FlgQ are proteins required for motility in C. jejuni. However, these proteins are not required for flagellar gene expression or biosynthesis. Because these proteins are found only in a subset of motile bacteria, our findings suggest that flagellar motor functions in C. jejuni may be more complex than in other bacteria. Finding that FlhG limits C. jejuni in producing only one flagellum per a pole supports similar findings in other polarly-flagellated bacteria for the role of FlhG in imparting numerical controls of flagellar biosynthesis. However, finding that FlhG also controls the location of septation is a new function for any FlhG protein. These results may have large impacts on the role of FlhG in other bacteria in possibly participating in other activities other than controlling flagellar biosynthesis.
Publications
- Joslin, S. N. and Hendrixson, D. R. (2008) Analysis of the FlgR response regulatory suggest integration of diverse mechanisms to activate an NtrC-like protein. J Bacteriol 190:2422-2433.
- Hendrixson, D. R. (2008) Restoration of flagellar biosynthesis by varied mutational events in Campylobacter jejuni. Mol Microbiol 70:519-536.
- Joslin, S. N. and Hendrixson, D. R. (2009) Activation of the FlgSR two-component system of Campylobacter jejuni by the flagellar export apparatus. J. Bacteriol 191:2656-2667.
- Balaban, M., Joslin, S. N., and Hendrixson, D. R. (2009) FlhF and its GTPase activity are required for distinct processes in flagellar gene regulation and biosynthesis in Campylobacter jejuni. J. Bacteriol 191:6602-6611.
- Sommerlad, S. M. and Hendrixson, D. R. (2007) Analysis of the roles of FlgP and FlgQ in flagellar motility of Campylobacter jejuni. J. Bacteriol 189:179-186.
- Bingham-Ramos, L. K. and Hendrixson, D. R. (2008) Characterization of two cytochrome c peroxidases of Campylobacter jejuni involved in promoting commensal colonization of poultry. Infect Immun 76:1105-1114.
- Ribardo, D.A., Bingham-Ramos, L. K., and Hendrixson, D. R. (2010) Functional analysis of the RdxA and RdxB nitroreductases of Campylobacter jejuni reveals importance of RdxA in metronidazole susceptibility. J. Bacteriol 192:1890-1901.
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Progress 08/01/08 to 07/31/09
Outputs
OUTPUTS: Aim 1: We have completed all work described in the proposal and in previous Annual Accomplishments Reports regarding the characterization of two putative cytochrome c peroxidases (CCPs), DocA and Cj0358, of C. jejuni required for efficient colonization of the gastrointestinal tracts of poultry. We have collaborated with Dr. David Kelly of the Department of Molecular Biology and Biotechnology at the University of Sheffield to understand if these proteins are required for aspects of C. jejuni metabolism. Through this collaboration, we have found that the cj0358 mutant, but not the docA mutant, displayed a slight growth defect with fumarate as the terminal electron acceptor in microaerobic and oxygen-limited conditions. Aim 2: We are continuing to characterize the role of the liv operon of C. jejuni in promoting colonization of the gastrointestinal tract of chicks. This operon encodes six proteins that presumably form a system for branched-chain amino acid transport into C. jejuni required for wild-type levels of colonization of poultry. Each component of the system is encoded by two genes of the operon, thus creating possible functional redundancy. As described previously, we found that deletion of both genes encoding the periplasmic amino acid binding proteins, livJ and livK, results in a significant defect in colonization of 1-day old chicks at an inoculum of 10000 cfu and deletion of each single gene results in a significant defect in colonization at an inoculum of 100 cfu. To determine if branched-amino acid biosynthesis was also important for colonization of poultry, we deleted ilvE from C. jejuni. This mutant only displays a colonization defect when administered at 100 cfu. We have begun to perform in vitro transport amino acid pathways with isoleucine, leucine, and valine. Our preliminary analysis indicates that the Liv system of C. jejuni transports isoleucine and leucine, but not valine. We are currently exploring if transport by the Liv system of C. jejuni is able to transport other amino acids. Aim 3: We have completed the studies analyzing the role of flgP and flgQ in motility as previously described in the Annual Report of 2007. We have extended our studies by identifying flagellar proteins that may interact with FlgP or FlgQ. We have analyzed various flagellar mutants and found less outer membrane-association of FlgP in a flgH mutant which lacks the outer membrane flagellar pore. We are currently conducting studies to understand how this localization contributes to flagellar rotation and motility. We are continuing to analyze the role of flhG in flagellar motility. We have remade a flhG mutant of C. jejuni to ensure expression of downstream genes and this mutant is less motile than the wild-type strain and expresses all flagellar genes at wild-type levels. Examination of wild-type and flhG mutant strains by electron microscopy revealed that lack of flhG results in bacteria producing an increased number of flagella at the bacterial pole. Whereas the wild-type strain normally produced a single flagellum at one or both poles, the flhG mutant commonly produced up to four flagella per pole. PARTICIPANTS: David R. Hendrixson is the prinicipal investigator. Dr. Hendrixson supervised all work in the project and performed experiments characterizing the FlgP and FlgQ proteins. Deborah A. Ribardo is a Research Scientist. Dr. Ribardo performed all experiments analyzing the liv operon. Lacey Bingham-Ramos is a Graduate Research Assistant. Ms. Bingham-Ramos performed all research activities regarding Cj0358 and DocA. Murat Balaban is a Graduate Research Assistant. Mr. Balaban performed all research activies regarding FlhG. Both Ms. Bingham-Ramos and Mr. Balaban are trainees at the University of Texas Southwestern Medical Center in the laboratory of Dr. Hendrixson who were completing work for their PhD thesis in 2008-2009. Ms. Bingham-Ramos successfully completed and defended her PhD thesis in April 2009. Dr. David Kelly is a principal investigator at the University of Sheffield in the United Kingdom who performed growth assays of wild-type and mutant strains lacking Cj0358 and DocA. TARGET AUDIENCES: Target audiences for this work are basic science researchers interested in understanding the molecular biology of Campylobacter jejuni. In addition, individuals interested in understanding the control and transmission of food-borne pathogens in agriculture are target audiences. The research program outlined in this proposal was central to the reserarch projects of two graduate research assistants who have completed or are completing their PhD thesis. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have published our findings regarding the requirements of DocA and Cj0358 for promoting colonization of the gastrointestinal tracts of poultry by C. jejuni. Through our collaboration with Dr. Kelly, we have acquired additional information regarding a potential role of Cj0358 in metabolism of C. jejuni in vitro. These results suggest that Cj0358 may influence the ability to grow on certain substrates such as fumarate and confirm that DocA and Cj0358 are likely involved in different processes in C. jejuni. We can now use this information as a basis to explore other potential metabolic pathways that may use Cj0358 for optimal growth to potentially explain the colonization defect seen in 1-day old chicks upon infection with the cj0358 mutant. In continuing to understand important issues regarding C. jejuni metabolism, we have analyzed the liv operon, which encodes genes presumably required for the transport of branched-chain amino acids into C. jejuni. Since the liv operon is required by C. jejuni for colonization of the gastrointestinal tracts of chicks, we hypothesize that the bacterium needs to acquire branched-chain amino acids such as leucine, isoleucine, or valine for optimal in vivo growth. We have found that this system appears to transport leucine and isoleucine, but not valine. These results are different from the analogous system in E. coli and suggest that the systems may have different specificities among bacteria. Because an ilvE mutant which is presumably required for biosynthesis of branched-chain amino acids has a colonization defect when administered at low inoculum to 1-day old chicks, we speculate that the transport of certain branched chain amino acids in vivo is not enough to satisfy the requirements for these amino acids and that C. jejuni must be able to synthesize these amino acids as well. We have preliminary findings suggesting that the FlgP protein which is required for motility is dependent on FlgH for optimal outer membrane-localization. We conclude that FlgP may form an important novel structure with FlgH or at the site nearby FlgH in the outer membrane to allow for rotation of flagella. Analysis of flhG revealed that the encoded protein controls flagellar biosynthesis to ensure that only one flagellum is formed at each pole. Since flhG is found in many other bacteria that produce polar flagella, we can use C. jejuni as a model system to understand how FlhG controls flagellar number in a diverse group of bacteria. Through continued analysis of FlgP, FlgQ, and FlhG, we are providing insights into flagellar biosynthesis in multiple polarly-flagellated bacteria.
Publications
- No publications reported this period
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Progress 08/01/07 to 07/31/08
Outputs
OUTPUTS: Specific Aim 1: As described in the proposal, we have characterized the requirement of two putative cytochrome c peroxidases (CCPs), DocA and Cj0358, of C. jejuni for efficient colonization of the gastrointestinal tracts of poultry. We have completed work described in the original proposal and in the annual report for 2007 for this aim. Our findings have been published in Infection and Immunity in March 2008. One hypothesis for the requirement of these proteins for the growth of C. jejuni in the chick intestinal tract is that the CCPs may aid in metabolism of certain substrates. We are collaborating with Dr. David Kelly at the Unversity of Sheffield in the United Kingdom who is an expert regarding metabolism of C. jejuni. His laboratory is assisting us in determining if a C. jejuni mutant lacking docA or cj0358 is defective for growth under specific conditions. Specific Aim 2: We are characterizing the role of the liv operon of C. jejuni in promoting colonization of the gastrointestinal tract of chickens by the bacterium. This operon is hypothesized to encode six genes that may function together to bind and transport branched-chain amino acids into the bacterium. Redundancy is likely in this system as two genes encode the amino-acid binding proteins, two genes encode the inner membrane permeases, and two genes encode the ATPase proteins. As mentioned in the proposal, mutants that contain a single deletion or a deletion of two similar genes may have to be generated to determine the function of components of this operon in amino-acid transport. We have generated a collection of mutants that contain all the appropriate deletions and have performed colonization experiments in 1-day old chicks to determine which genes of this operon are specifically required for colonization. In addition, we have generated a mutant lacking ilvE which encodes a protein in the biosynthesis pathway for making branched-chain amino acids. By using this mutant along with the mutants defective in transport of branched-chain amino acids, we can determine if C. jejuni requires biosynthesis of these amino acids, transport of these amino acids, or both for growth in the intestinal tract of poultry. Specific Aim 3: We have completed the studies analyzing the role of flgP and flgQ in motility as previously described in last year's annual report.We are continuing to determine the role of flhG in flagellar motility of C. jejuni. We have compared a wild-type C. jejuni strain with an flhG mutant for flagellar biosynthesis and expression of flagellar genes. We have made a mutant of C. jejuni that makes a FlhG protein presumably defective for ATP-binding and hydrolysis and have analyzed this mutant for motility, flagellar biosynthesis, and expression of flagellar genes. We have purified the C. jejuni wild-type and mutant FlhG proteins for in vitro biochemical studies and antibody generation. Studies have been performed to localize FlhG to a specific compartment in C. jejuni. Information regarding these studies has been published, presented at annual microbiology meetings, or presented in presentations to scientific colleagues at other research institutes. PARTICIPANTS: David Hendrixson is the Principal Investigator on the project. He supervises all research activities. Lacey Bingham-Ramos is a graduate research assistant who performed all work described in Specific Aim 1. Deborah Ribardo is a research scientist who performed all work described in Specific Aim 2. Murat Balaban is a graduate research assistant who performed all work described in Specific Aim 3. Dr. David Kelly is a collaborator at the University of Sheffield in the United Kingdom who is assisting in determining if specific mutants described in Specific Aim 1 are defective for certain aspects of C. jejuni metabolism. Dr. Kelly is receiving no salary support from this grant. Because Ms. Ramos and Mr. Balaban are graduate research assistants, this grant is providing a training opprotunity to educate graduate students in basic science research. TARGET AUDIENCES: This work has been presented to the Natioan Research Initiative Food Safety Project Director Meeting, the Department of Microbiology at Texas A&M Health Sciences University, and at the 2008 Canadian Campylobacter meeting in Montreal, Canada. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have published our findings regarding the requirements of DocA and Cj0358 for promoting colonization of the gastrointestinal tracts of poultry by C. jejuni. Through our collaboration with Dr. Kelly, we have acquired some additional information regarding a potential role of Cj0358 in C. jejuni. A mutant lacking cj0358 displays a growth defect in microerobic conditions in the presence of formate and oxygen-limited conditions in the presence of fumarate. These results suggest a potentially important role of Cj0358 in the metabolism of C. jejuni in vivo. In continuing to understand important issues regarding C. jejuni metabolism, we have been characterizing the requirement of the liv operon, which encodes genes presumably required for the transport of branched-chain amino acids into C. jejuni, for colonization of the gastrointestinal tracts of chicks by the bacterium. We have found that mutants lacking livJ, livK, or both livJ and livK show defects in colonization of chickens. These genes encode the amino-acid binding components of the system, suggesting that the bacterium may have to acquire amino acids such as leucine, isoleucine, and valine for efficient growth in chickens. However, mutants lacking other components of the operon are not defective for colonization. Furthermore, a mutant defective for biosynthesis of these branched-chain amino acids is defective for colonization when administered at a low inoculum to chickens. These combined studies contribute valuable information that may be able to be used to target important pathways required by the bacterium for growth in the intestinal tracts of poultry. In addition, our findings regarding the role of the FlhG protein in flagellar motility of C. jejuni provides more understanding into the biosynthesis of an important organelle required for efficient colonization of poultry. We have found that FlhG is a cytoplasmic protein that is required for flagellar biosynthesis, but it is not required for expression of flagellar genes. A mutant that abolishes the putative ATPase activity of FlhG appears to be motile and expresses flagellar genes but may be altered in the correct number of flagella produced by the bacterium. These studies will continue to provide unique requirements of C. jejuni for generation of an essential colonization factor.
Publications
- Bingham-Ramos, L. K. and Hendrixson, D. R. (2008) Characterization of two cytochrome c peroxidases of Campylobacter jejuni involved in promoting commensal colonization of poultry. Infect Immun 76:1105-1114.
- Joslin, S. N. and Hendrixson, D. R. (2008) Analysis of the FlgR response regulatory suggest integration of diverse mechanisms to activate an NtrC-like protein. J Bacteriol 190:2422-2433.
- Hendrixson, D. R. (2008) Restoration of flagellar biosynthesis by varied mutational events in Campylobacter jejuni. Mol Microbiol 70:519-536.
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Progress 08/01/06 to 07/31/07
Outputs
Specific Aim 1 As described in the original proposal, our goal is to determine the activities of two putative cytochrome c peroxidases (CCPs), DocA and Cj0358, which are required for efficient colonization of the gastrointestinal tracts of poultry. We have extensively compared C. jejuni mutants lacking DocA or Cj0358 in their ability to promote colonization of 1-day old chicks at various inoculating doses. The docA mutant displays a 50- to 20,000-fold reduction in colonization of the chick ceca at day 7 post-infection depending on the inoculum doses. The cj0358 mutant displays a 10-fold colonization defect that is only observed at low inocula; higher inocula diminish this colonization defect. We have performed significant experimentation to characterize DocA and Cj0358 as bacterial CCPs. Through immunoblotting analyses and translational fusion analyses with a phoA reporter gene, both proteins are localized to the periplasm of C. jejuni. In addition, the periplasmic
proteins bind heme and these heme moieties display a peroxidase activity that can be detected by a chemiluminescent assay. These characteristics are common features of typical bacterial CCPs. We then demonstrated that the peroxidase activity of DocA or Cj0358 is not required for survival to exogenous hydrogen peroxide in vitro. We also determined that the cytoplasmic catalase is a major factor in promoting resistance to hydrogen peroxide. However, compared to the docA mutant, the catalase mutant only shows a 50-fold defect in colonization regardless of the inocula dose. These results indicate that resistance to hydrogen peroxide or other oxidative stresses may not be significant for C. jejuni to colonize poultry.Our results suggest that DocA and, to a lesser extent, Cj0358 may have other important physiological roles that are necessary for C. jejuni to promote colonization of chickens. Experiments are ongoing to define these possible alternative roles. A manuscript is
currently in preparation for submission to Infection and Immunity that will detail these findings. Specific Aim 3 We have performed significant experimentation to define the roles of FlgP and FlgQ for flagellar motility in C. jejuni. Mutants lacking flgP or flgQ are not defective for flagellar gene expression or biosynthesis. Instead, these mutants display paralyzed flagellar phenotypes, suggesting that FlgP and FlgQ are required for flagellar rotation. By using immunoblotting analyses, we have determined that FlgP localizes to the outer membrane. Furthermore, the localization or stability of FlgP is dependent on FlgQ. These results indicate that FlgQ may have a chaperone activity for FlgP. Experiments are ongoing to determine if FlgP and FlgQ form a complex and to determine a more precise role of FlgP for flagellar rotation. These findings were published in the Journal of Bacteriology in January 2007.
Impacts
We have provided further insight in the roles of four proteins of C. jejuni that are required by the bacterium to promote optimal levels of colonization of poultry. Understanding the function of these proteins for the bacterium in initiating the colonization process will have the potential to develop therapeutic agents against these factors. Administration of these potential therapeutics to poultry flocks in agriculture could block the ability of C. jejuni to grow in the animals. The levels of C. jejuni may then be reduced in poultry which would have the ultimate result in making safer meat products for human consumption to reduce the amount of C. jejuni dairrheal disease in humans. Secondly, understanding the roles of these factors in C. jejuni may provide insight into the activities of similar proteins in other important bacterial pathogens of humans. For instance, FlgP and FlgQ have been found in other bacteria and appear to be involved in motility. Thus, C. jejuni
could be a model for studying these two proteins with the potential to understand how motility is accomplished in a broad range of bacteria.
Publications
- Sommerlad, S. M. and Hendrixson, D. R. (2007) Analysis of the Roles of FlgP and FlgQ in Flagellar Motility of Campylobacter jejuni. J. Bacteriol. 189:179-186.
- Bingham-Ramos, L. K. and Hendrixson, D. R. (2007) Characterization of two putative cytochrome c peroxidases of Campylobacter jejuni that are involved in commensal colonization. (submitted to Infect. Immun.)
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