Progress 01/15/09 to 01/14/13
Outputs
OUTPUTS: Aim 1: The capsule biosynthesis locus is a large locus that consists of over 25 genes in C. jejuni 81-176. To better understand transcriptional organization of the locus, we performed reverse transcriptase-PCR using primer sets to detect cotranscription of adjacent genes. By using this approach, we provided evidence for co-transcription of 18 consecutive genes ranging from kpsE at the 5 end to cjj1420 at the 3 end. We also uncovered another genetic mechanism that may influence expression of the capsule locus and biosynthesis of the capsule. In the original bocR mutant, we discovered that the capsule locus from genes cjj1435 to hddC had been lost from the chromosome. Excision of the capsule locus appeared to occur at a direct repeat located at the ends of this excised region. Aim 2: We discovered a link between capsule biosynthesis production and the production of phosphodonors for the FlgR response regulator to activate expression of flagellar genes. Mutations in the capsule biosynthesis locus not only result in reductions in capsular polysaccharide production but also reduce activation of FlgR proteins and flagellar gene expression. We used a screening procedure to identify mutants with reduced FlgR activation and then examined mutants for reductions in capsular polysaccharide production. By this method, we uncovered four previously unidentified genes that are required for wild-type levels of capsular polysaccharide production. Aim 3: To begin to understand factors of C. jejuni required for production of the capsule, we developed three potential screening procedures to identify these genes involved in producing the capsular polysaccharide or transporting the polysaccharide to the bacterial surface. One approach is based on a difference in appearance of C. jejuni after growth on media. Mutants defective in capsule production demonstrate a different growth morphology than wild-type strains. Second, we uncovered mutants with transposon insertions in some capsular biosynthesis genes that are partially reduced for expression of flagellar genes. By using a reporter strain of C. jejuni that has an altered FlgR protein that appears to be responsive to non-cognate phosphodonors, we identified genes within the capsule locus that had transposon insertions that cause reductions in flagellar gene expression. Finally, we began developing a screening procedure using serotype-specific bacteriophages that recognize capsular polysaccharide as a target for adsorption and eventual lysis of host bacteria. Use of these bacteriophages will help in identifying C. jejuni mutants that are resistant to phage infection through reductions in capsular polysaccharide production. PARTICIPANTS: David R. Hendrixson is the principal investigator. Dr. Hendrixson supervised all work in the project. Deborah A. Ribardo is a research scientist. Dr. Ribardo performed studies characterizing the transcriptional organization of the capsule locus and developed one of the assays to potentially identify mutants with alterations in capsule production. Dr. Ribardo performed studies to isolate capsular polysaccharide from potential mutants and began work on phage infections. Joseph Boll was a reserach assistant and is currently a graduate student who made many of the capsule mutants and identified a second assay to potentially identify genes involved in capsule production. Joseph Boll also found that capsular polysaccharide mutants show reduced activation of the FlgR response regulator, likely due to decreased phosphoramidate pools in these mutants. Dr. William Cody is a post-doctoral research fellow. Dr. Cody performed initial studies to develop procedures for performing bacteriophage infections with C. jejuni. 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. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We provided a better understanding of the transcriptional organization of the capsule locus. This information will be valuable for future experiments in examining potential promoters that are involved in transcription of genes required for production of the capsular polysaccharide. These promoters will likely be the direct targets of potential transcriptional regulators that influence expression of the capsule in C. jejuni. The finding that the capsule locus in a mutant of C. jejuni had excised by a recombination event between two direct repeats suggest potential interesting recombinogenic mechanisms may occur in the capsule locus within a strain of C. jejuni to cause loss or rearrangement of capsule genes to alter the genetic makeup of the capsule locus. We also discovered a potentially important link between the capsular biosynthesis pathway and signal transduction pathways in C. jejuni. This finding may suggest that the process of capsular biosynthesis may have global effects on multiple aspects of the biology of the bacterium. Thus, by better understanding capsular biosynthesis and development of inhibitors to block this pathway, potential therapeutics to eliminate capsular polysaccharide biosynthesis may also target synthesis of other important factors of C. jejuni. We anticipate by using a combination of a transposon mutant library of C. jejuni and serotype-specific bacteriophages that promote infection of strains in a capsule-dependent manner, we will be able to identify mutants lacking genes essential for capsule production. We believe that we will not only identify genes within the capsular polysaccharide biosynthesis locus, but possibly genes outside this locus that were not previously known to impact capsular polysaccharide production.
Publications
- Gilbreath, J. J., Cody, W. L., Merrell, D. S., and Hendrixson, D. R. (2011) Change is good: variations in common biological mechanisms in the epsilonproteobacterial genera Campylobacter and Helicobacter. Microbiol. Mol. Bio. Rev. 75:84-132.
- Balaban, M. and Hendrixson, D. R. (2011) Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni. PLoS Pathog. 7:e1002420.
- Boll, J. M. and Hendrixson, D. R. (2011) A specificity determinant for phosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphate. Proc Natl Acad Sci USA. 108:20160-20165.
- Lertsethtakarn, P., Ottemann, K. M, and Hendrixson, D. R. (2011) Motility and chemotaxis in Campylobacter and Helicobacter. Annu. Rev. Microbiol. 65:389-410.
- Ribardo, D. A. and Hendrixson, D. R. (2011) Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport. J. Bacteriol. 193:6233-6243.
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Progress 01/15/11 to 01/14/12
Outputs
OUTPUTS: Aim 1: As described in previous reports, we have completed our analysis of transcription of the capsular polysaccharide locus of C. jejuni 81-176. We found that 18 genes are co-transcribed in this locus. Aim 2: We found that inactivation of certain capsular polysaccharide genes likely decrease the phosphoramidate pool of C. jejuni. This finding is significant as we discovered that the putative decrease in phosphoramidate likely decreases crosstalk to and phosphorylation of some response regulators, such as FlgR. Decreased activation of FlgR leads to decreased expression of the sigma54-dependent flagellar genes and reduced motility, which is essential for infection of poultry to promote commensal colonization and infection of humans to promote disease. Aim 3: As described in previous reports, we have developed a screening procedure to identify transposon mutants lacking capsular polysaccharide production by identifying those that survive bacteriophage infection. In C. jejuni, the capsular polysaccharide produced by certain serotypes functions as a receptor for lytic bacteriophages. We are proceeding with this screening procedure to identify those transposon mutants defective in capsule biosynthesis. We will then create mutants lacking these genes and then test the mutants for capsular polysaccharide production, colonization of the chick gastrointestinal tract, and interactions with human intestinal epithelial cells. PARTICIPANTS: David R. Hendrixson is the principal investigator. Dr. Hendrixson supervised all work in the project. Deborah A. Ribardo is a research scientist. Dr. Ribardo performed studies to isolate capsular polysaccharide from potential mutants and began work on phage infections. Joseph M. Boll is a graduate research assistant who found that capsular polysaccharide mutants show reduced activation of the FlgR response regulator, likely due to decreased phosphoramidate pools in these mutants. 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. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have discovered that capsular polysaccharide biosynthesis produces phosphoramidate as an intermediate, which can influence signal transduction systems in C. jejuni. We found that phosphorylation and activation of the FlgR response regulator, which is essential for motility and infection of hosts, was reduced in certain capsule mutants. This finding suggests that capsular biosynthesis can affect signaling pathways in C. jejuni by providing a phosphodonor that is available to activate some response regulators. Thus, the development of potential therapeutics to eliminate capsular polysaccharide biosynthesis may also target signaling pathways that reduce or abolish synthesis of important virulence or colonization factors of C. jejuni. We anticipate by using a combination of a transposon mutant library of C. jejuni and serotype-specific bacteriophages that promote infection of strains in a capsule-dependent manner, we will be able to identify mutants lacking genes essential for capsule production. We believe that we will not only identify genes within the capsular polysaccharide biosynthesis locus, but possibly genes outside this locus that were not previously known to impact capsular polysaccharide production.
Publications
- Balaban, M. and Hendrixson, D. R. (2011) Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni. PLoS Pathog. 7:e1002420.
- Boll, J. M. and Hendrixson, D. R. (2011) A specificity determinant for phosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphate. Proc Natl Acad Sci USA. 108:20160-20165.
- Gilbreath, J. J., Cody, W. L., Merrell, D. S., and Hendrixson, D. R. (2011) Change is good: variations in common biological mechanisms in the epsilonproteobacterial genera Campylobacter and Helicobacter. Microbiol. Mol. Bio. Rev. 75:84-132.
- Lertsethtakarn, P., Ottemann, K. M, and Hendrixson, D. R. (2011) Motility and chemotaxis in Campylobacter and Helicobacter. Annu. Rev. Microbiol. 65:389-410.
- Ribardo, D. A. and Hendrixson, D. R. (2011) Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport. J. Bacteriol. 193:6233-6243.
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Progress 01/15/10 to 01/14/11
Outputs
OUTPUTS: Aim 1: The capsule biosynthesis locus is a large locus that consists of over 25 genes in C. jejuni 81-176. We have completed our analysis of the transcriptional organization of the capsule biosynthesis locus as described in previous reports. We found evidence of co-transcription of at least 18 consecutive genes of this locus Aim 2: We discovered a link between capsule biosynthesis production and the production of phosphodonors for the FlgR response regulator to activate expression of flagellar genes. Mutations in the capsule biosynthesis locus not only result in reductions in capsular polysaccharide production but also reduce activation of FlgR proteins and flagellar gene expression. We used a screening procedure to identify mutants with reduced FlgR activation and then examined mutants for reductions in capsular polysaccharide production. By this method, we uncovered four previously unidentified genes that are required for wild-type levels of capsular polysaccharide production. Aim 3: We are in the process of developing a screening procedure using serotype-specific bacteriophages that recognize capsular polysaccharide as a target for adsorption and eventual lysis of host bacteria. Use of these bacteriophages will help in identifying C. jejuni mutants that are resistant to phage infection through reductions in capsular polysaccharide production. Mutants discovered by these approaches will be thoroughly analyzed for capsule biosynthesis, colonization of the chick gastrointestinal tract, and interactions with human intestinal epithelial cells. These results have been disseminated at scientific research meetings. PARTICIPANTS: David R. Hendrixson is the principal investigator. Dr. Hendrixson supervised all work in the project. Deborah A. Ribardo is a research scientist. Dr. Ribardo performed studies to isolate capsular polysaccharide from potential mutants. Dr. William Cody is a post-doctoral research fellow. Dr. Cody performed initial studies to develop procedures for performing bacteriophage infections with C. jejuni. 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. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have discovered a potentially important link between the capsular biosynthesis pathway and signal transduction pathways in C. jejuni. This finding may suggest that the process of capsular biosynthesis may have global effects on multiple aspects of the biology of the bacterium. Thus, by better understanding capsular biosynthesis and development of inhibitors to block this pathway, potential therapeutics to eliminate capsular polysaccharide biosynthesis may also target synthesis of other important factors of C. jejuni. The use of serotype-specific bacteriophages that require C. jejuni capsular polysaccharides as receptors for phage absorption and subsequent infection and lysis of the bacterial cell will facilitate a screening procedure to identify genes of C. jejuni required for capsule production. By using a transposon mutant library of C. jejuni, we can identify mutants lacking capsule production by exposing the library to the bacteriophage. Mutants that are resistant to phage infection due to loss of capsule production should not be infected by the phage and survive. Genes interrupted in the unencapsulated mutants can then be identified. By using this approach, we may be able to identify genes both within and outside the capsule biosynthesis locus that influence capsular polysaccharide production. Therefore, we will be able to identify a broad set of genes, some of which may not be initially obvious, that are necessary for capsule biosynthesis. A large advantage of this screening procedure is that by using bacteriophages of different serotype specificities, we will be able to conduct screens on many different serotypes to identify mutants defective for capsular biosynthesis across species with diverse capsular biosynthesis loci.
Publications
- No publications reported this period
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Progress 01/15/09 to 01/14/10
Outputs
OUTPUTS: Aim 1: The capsule biosynthesis locus is a large locus that consists of over 25 genes in C. jejuni 81-176. To better understand transcriptional organization of the locus, we performed reverse transcriptase-PCR using primer sets to detect cotranscription of adjacent genes. By using this approach, we provided evidence for co-transcription of 18 consecutive genes ranging from kpsE at the 5' end to cjj1420 at the 3' end. Co-transcription of the other genes outside this genetic region remains to be fully characterized. We also uncovered another genetic mechanism that may influence expression of the capsule locus and biosynthesis of the capsule. In the original bocR mutant, we discovered that the capsule locus from genes cjj1435 to hddC had been lost from the chromosome. Excision of the capsule locus appeared to occur at a direct repeat located at the ends of this excised region. Aim 2: In addition to the unencapsulated mutants we previously generated (kpsM, cjj1430, and hddA), we have generated mutants in ten other genes within the capsule locus of 81-176. These mutants will be analyzed for capsule biosynthesis, colonization of the chick gastrointestinal tract, and, adherence to and invasion of human intestinal epithelial cells. Aim 3: For full verification that bocR is involved in expression of genes required for capsular polysaccharide, we generated additional bocR mutants. Analysis of these mutants did not consistently reveal an unencapsulated phenotype associated with bocR mutations. To begin to understand factors of C. jejuni required for production of the capsule, we have potentially developed two screening procedures to identify these genes involved in producing the capsular polysaccharide or transporting the polysaccharide to the bacterial surface. One approach is based on a difference in appearance of C. jejuni after growth on media. Mutants defective in capsule production demonstrate a different growth morphology than wild-type strains. Second, we have uncovered mutants with transposon insertions in some capsular biosynthesis genes that are partially reduced for expression of flagellar genes. By using a reporter strain of C. jejuni that has an altered FlgR protein that appears to be responsive to non-cognate phosphodonors, we identified genes within the capsule locus that had transposon insertions that cause reductions in flagellar gene expression. In addition, we found additional mutants outside the capsule locus that also caused reduced expression of flagellar genes. These latter mutants remain to be examined for defects in capsular polysaccharide biosynthesis. PARTICIPANTS: David R. Hendrixson is the principal investigator. Dr. Hendrixson supervised all work in the project. Deborah A. Ribardo is a research scientist. Dr. Ribardo performed studies characterizing the transcriptional organization of the capsule locus and developed one of the assays to potentially identify mutants with alterations in capsule production. Joseph Boll was a reserach assistant who made many of the capsule mutants and identified a second assay to potentially identify genes involved in capsule production. 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. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have provided a better understanding of the transcriptional organization of the capsule locus. This information will be valuable for future experiments in examining potential promoters that are involved in transcription of genes required for production of the capsular polysaccharide. These promoters will likely be the direct targets of potential transcriptional regulators that influence expression of the capsule in C. jejuni. The finding that the capsule locus in a mutant of C. jejuni had excised by a recombination event between two direct repeats suggest potential interesting recombinogenic mechanisms may occur in the capsule locus within a strain of C. jejuni to cause loss or rearrangement of capsule genes to alter the genetic makeup of the capsule locus. We have begun to systematically mutate all genes within the capsule biosynthesis locus. Analysis of each mutant will provide information if the genes encode proteins involved in production of a core polysaccharide in the capsule, a variable modification of the capsule, or transport of the capsule to the bacterial surface. Considering the inconsistency of bocR mutants to demonstrate lack of capsular polysaccharide production, we began developing methods to use in screening procedures to identify mutants that fail to produce capsule. We have discovered that strains with mutations in genes required for capsule production appear to demonstrate an altered growth morphology when grown on media. In addition, we have discovered that certain mutations in genes required for capsule production influence activation of a form of the FlgR response regulator that is more responsive to non-cognate phosphodonors. These phosphodonors are likely to be certain phosphorylated components of the capsule. These phosphorylated components were likely not produced in mutants lacking specific wild-type genes in these capsule mutants. These mutants remain to be further characterized for their exact roles in producing the capsular polysaccharide. These procedures will facilitate analysis of a large number C. jejuni mutants to identify those genes required for wild-type capsular polysaccharide production.
Publications
- No publications reported this period
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