Performing Department
Veterinary Pathobiology
Non Technical Summary
Campylobacter species, especially C. jejuni, are leading causes of foodborne diarrheal illness in the United States. Active surveillance data provided by the Foodborne Diseases Active Surveillance Network known as FoodNet suggests that about 14 cases of Campylobacter induced gastroenteritis are diagnosed each year per 100,000 people in the U.S. population. Since many more cases go undiagnosed or unreported, campylobacteriosis is estimated to affect over 1.3 million people every year. The most common sources of Campylobacter in the food supply are chicken and chicken products followed by unpasteurized cow's milk. Based on estimates of the infection of chicken flocks with C. jejuni, up to 90% of chicken flocks in the US are colonized . Because of the high degree of prevalence in chicken flocks, methods aimed at reducing the frequency and/or duration of colonization by C. jejuni are desirable. We recently completed transcriptional profiling studies aimed at assessing genes differentially expressed in the human host versus the chicken host, and we observed that genes designated as Cj0371 and Cj0372 from the first sequenced strain of were upregulated at 42 °C, the body temperature of chickens . In other studies by collaborators (Dr. Alain Stintzi, Ottawa Institute of Systems Biology), these genes were also upregulated in a rabbit illeal loop model (39°C body temp) suggesting that they might be important for survival/colonization in non-human hosts . To test the hypothesis that one or more of the genes upregulated at 42°C might be important for survival/persistence in the chicken host, we created a null mutant for the Cj0372 gene (no functional Cj0372 protein produced) in strain NCTC 11168 and found that the mutant strain is non-motile and invasion defective. Motility and the ability to invade eukaryotic cells are important virulence determinants for C. jejuni. Additionally, the strain lacking Cj0372 produces a large amount of an unidentified secreted substance and so is prone to autoagglutination especially at 42C, because the substance is either produced earlier in the growth cycle or is secreted to higher levels at that temperature. Autoagglutination results in precipitation in vitro and thus is viewed as negative for optimal growth. Mutants lacking Cj0372 are also 30% smaller on average than wild-type C. jejuni . Because these mutants are non-motile we have been unable to look at the in vivo effects of the mutation (successful animal colonization requires motility, but based on the in vitro results, this gene is a potential target to attack with non-antibiotic treatments to reduce and possibly eliminate C. jejuni from infected chickens and livestock species. Although Cj0371 and Cj0372 were originally annotated as conserved hypothetical genes, we have made substantial progress in our understanding of the function of Cj0372 and now believe it is a glutathionylspermidine synthetase (GspS). GspS's have been identified in other bacterial species previously, in particular in E. coli. Until recently, little was known of the importance of these enzymes to bacteria, although a similar enzyme from trypanosome species (trypanothione synthetase-amidase) is being investigated by several groups as a drug target against these organisms. Since the null mutant strain lacking Cj0372 has severe phenotypes, we decided to investigate the effect of a null mutation in the same gene on a more clinically relevant strain and thus chose strain 81-176, a human vaccine candidate strain that causes severe and often bloody diarrhea. In this new project, we want to extend and expand our studies of both of these mutated strains in order to determine how the mutation of a single gene like Cj0372 can impact very different pathways. A more complete understanding of how the Cj0372 gene functions will allow us to focus future studies on potentially new and more effective ways to control and/or eliminate C. jejuni from the chicken host and/or from poultry derived food products.
Animal Health Component
0%
Research Effort Categories
Basic
85%
Applied
10%
Developmental
5%
Goals / Objectives
In previous studies we created and characterized a mutant C. jejuni strain deficient in an unknown hypothetical protein that appeared to be important for survival and/or maintenance in non-human hosts including the chicken. We will complete the examination of the Cj0372 mutant strains and determine mechanism(s) to explain the observed phenotypes. Aim 1, Assay the recombinant Cj0372 protein to determine if it is a GspS using assays developed for E. coli GspS. Verify that the C. jejuni protein has GspS function in order to better understand why loss of this protein results in the observed phenotypes. In Aim 2, we will sequence the genomes of both the wild-type parental strains and the mutant strains to verify that the only mutations impacting the observed phenotypes are those resulting from the creation of the null mutations and that there are no other mutations that could be responsible. In Aim 3, we will assess the transcriptional changes of the Cj0372 mutant strains and their respective parental strains using microarrays to determine what effect this mutation has on the expression of other genes in the mutants. This will allow us to investigate possible mechanisms to explain why the loss of the GspS enzyme results in a loss of motility and the other observed phenotypes. In Aim 4 we will quantify the severity of the phenotypes observed in 81-176 with those in 11168. With specific phenotypes quantified more precisely, we will have a more informed approach to the analysis of the microarray data as well as the sequencing data. In Aim 5, we will determine the identity of the secreted substance that accumulates in Cj0372 mutant strains. In particular, does the identity of this substance help explain the observed phenotypes and/or mechanism underlying the observed phenotypes? In Aim 6, we will use the antibodies we have for Cj0372 to determine the location of this protein in C. jejuni, i.e., does its location suggest a mechanism of action?
Project Methods
For Specific Aim 1, we have already cloned Cj0372 from strain 11168 and expressed the protein for antibody production. Although we did not need active enzyme to create antibodies, we need to purify protein with optimal enzymatic function in order to fully assay for enzyme activity. Methods describing the assay for the E. coli enzyme have only recently been published, and we plan to follow this new assay to measure function of the C. jejuni enzyme. The manuscript describing the function of the E. coli enzyme demonstrated that two other putative E. coli gsp genes encoded phosphatases but not glutathionylspermidine synthetases. In Aim 2, we will sequence the genomes of both the wild-type parental strains and the mutant strains using standard sequencing methods (454) to verify that the only mutations impacting the observed phenotypes are those resulting from the creation of the null mutations and that there are no other mutations that could be responsible for the observed phenotypes. This is necessary since we have not been able to successfully complement the Cj0372 mutation. While we believe that this is because the mutation impacts the uptake of DNA and/or impacts the homologous recombination process we cannot rule out the possibility of a secondary mutation without sequencing the genome. Complementation is the gold standard for assigning phenotypes to a single gene mutation. For Aim 3, we will assess the transcriptional changes of the Cj0372 mutant strains and their respective parental strains using microarrays. This will allow us to investigate possible mechanisms to explain why the loss of the GspS enzyme results in a loss of motility and other observed phenotypes. There is no demonstrated effect on motility in E. coli mutants lacking GspS (KN Delaney and DS Threadgill, unpublished) and thus there is no a priori way to know whether this is an outcome expected for loss of this enzyme in C. jejuni. We previously identified Cj0372 as a gene upregulated at 42 °C using microarrays, and many or all of the genes impacting motility in C. jejuni have been identified so it should be straightforward to determine whether the loss of Cj0372 directly impacts transcription of motility-related genes or whether the effect is indirect. Microarray analysis will also allow us to determine if other genes/pathways may be impacted in the mutant strains thus helping explain the mechanism(s) of action. In Aim 4 we will quantify the severity of the phenotypes observed in 81-176 with those in 11168. With specific phenotypes quantified more precisely, we will have a more informed approach to the analysis of the microarray data as well as the sequencing data. To approach the question of the importance of additional glutathione-related genes to the observed phenotypes, we will generate a targeted mutation in those genes in 81-176. If the presence of these glutathione-related genes is responsible for the worsened outcomes in the 81-176 mutant strain, we anticipate removal of these genes would ameliorate the defects back to what is seen with the 11168 mutant strain. If however the glutathione-related genes do not play a major role in the observed worsened outcomes, we expect no or minimal amelioration in the double mutant. For Aim 5, the secreted substance that accumulates in Cj0372 mutant strain will be purified and identified through biochemical methods. We will first determine whether the substance is sensitive to digestion by proteases, nucleases and polysaccharide lyases. This will help to suggest the nature of the substance and what type of assistance to seek for identification. For example core facilities that specialize in proteomics are not always adept at polysaccharide analysis, so having a better idea about the biochemical nature of the unknown substance will guide us to the appropriate expertise. For Aim 6, we will use the antibodies we have for Cj0372 with both electron microscopy and fractionation techniques to establish protein location. The location of this protein in the cell will help to suggest how the loss of this single protein is causing the pleotropic effects we observe. In particular, we will address what other protein or proteins may interact directly with Cj0372 based on its cellular location. This could be enhanced by the microarray data indicating particular pathways that are impacted in the mutant strains.