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Campylobacter jejuni is a leading cause of food-borne bacterial gastroenteritis resulting in a mild to severe, bloody diarrheal disease in humans. However, C. jejuni promotes a natural, harmless commensal colonization of the gastrointestinal tracts of poultry and other animals. This ability of C. jejuni to colonize agriculturally important animals creates a major opportunity for the bacterium to contaminate the human food supply during the slaughtering and processing of livestock. Identifying and understanding factors of C. jejuni that are involved in colonization of poultry may reveal new targets for antimicrobial agents that may be able to be used to eliminate C. jejuni infections in poultry, thereby creating safer meat products for human consumption and reducing C. jejuni disease in humans. We have performed a genetic screen and identified over twenty proteins of C. jejuni involved in commensal colonization of the chick gastrointestinal tract in a 1-day old chick model of infection. C. jejuni mutants lacking any of these factors are defective for colonization of the chick gastrointestinal tract. Through a series of biochemical and genetic experiments, we propose to functionally characterize three sets of colonization factors we have identified to determine how these systems contribute to the ability of C. jejuni to colonize chicks. These colonization factors we will analyze include: 1) the putative DocA and Cj0358 cytochrome c peroxidases; 2) the putative Liv amino acid transport system; and 3) three proteins, FlgP, FlgQ, and Cj0063c that are involved in flagellar motility. The main objectives of the proposed research is to better understand how these proteins aid the bacterium in promoting a commensal relationship with poultry. In addition, we will explore the possibility that these proteins could be new targets for the development of antimicrobial agents.
NON-TECHNICAL SUMMARY: Diarrheal disease in humans due to the bacterium Campylobacter jejuni most often is related to consuming or handling contaminated meat products. Understanding how this bacterium is able to grow in agriculturally important animals may lead to new methods for eliminating the bacterium from animals to make safer meat products for human consumption. The purpose of this study is to further characterize factors of Campylobacter jejuni that we have identified to be important for the bacterium to promote growth in animals. By analyzing these factors, we aim to better understand how these proteins function in the bacterium which may lead to the development of new approaches for eliminating the bacterium from animals to make safer meat products
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APPROACH: By using a series of biochemical approaches including bacterial fractionation and immunoblotting, we will be able to determine the location in the bacterium where many of these proteins reside. To determine the function of the DocA and Cj0358 cytochrome c peroxidases, we will employ a heme-staining assay and a hydrogen peroxide resistance assay. Each component of the Liv transport system will be examined in transport assays with radioactively-labelled amino acids to determine more specifically which amino acids are transported to the cytoplasm to aid in growth. We will use transcriptional reporter assays, electron microscopy, and motility assays to determine the roles of FlgP, FlgQ, and Cj0063c in flagellar motility.
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PROGRESS: 2007/08 TO 2008/07<BR>
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. <BR>
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. <BR>
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. <BR>
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IMPACT: 2007/08 TO 2008/07<BR>
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.
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PROGRESS: 2006/08/01 TO 2007/07/31<BR>
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.ƒnƒnOur 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.
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IMPACT: 2006/08/01 TO 2007/07/31<BR>
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.