Respiratory Substrates of Campylobacter Jejuni

NON-TECHNICAL SUMMARY: Campylobacter jejuni is a food-borne pathogen that causes millions of cases of diarrheal illness in the US every year. Our lab is investigating factors that allow for C. jejuni live in poultry (chickens and turkeys), which are the major mode of transmission. <P>APPROACH: We will be using a combination of classical microbiological methods, biochemistry, genetics and molecular techniques to achieve our goals. Key genes and metabolic pathways will be targeted for mutagenesis using molecular and genetic techniques. The resulting strains will then be characterized using standard microbiological and biochemical techniques to determine the relevant phenotypes. Finally, we will be using chicken colonization assays to determine the importance of the targeted metabolic pathways in host persistence.

<P>PROGRESS: 2006/10 TO 2007/09 <BR>
The role of 8 respiratory enzymes, a metal transporter and a regulatory protein have been assigned to the general physiology of Campylobacter jejuni. This has been accomplished through the mutation of 23 different genes in the C. jejuni genome and biochemical characterization of the resulting strains. 11 of these strains have been tested for host colonization (the chicken), and 8 show decreased ability to colonize the host.
<BR><BR>
IMPACT: 2006/10 TO 2007/09<BR>
The identification of 8 genes that decrease the ability of Campylobacter jejuni to colonize chickens helps to direct potential mitigation strategies in decreasing Campylobacter contamination of the food supply.
<BR><BR>

PROGRESS: 2005/10/01 TO 2006/09/30<BR>
All three of the respiratory donors in Campylobacter jejuni have been characterized, mutated and the role of each in host colonization has been tested. Hydrogenase and formate dehydrogenase oxidize the respiratory donors hydrogen and formate, respectively. Addition of either substrate increases the growth rate of C. jejuni in vitro and deletion of either modestly affects the ability of the bacterium to colonize the host (chickens). Deletion of both enzymes has a much more dramatic effect on host colonization. The third respiratory donor enzyme complex characterized is a modified complex I. C. jejuni encodes 12 of the 14 subunits that make up the respiratory enzyme NADH ubiquinone oxidoreductase (also called complex I). The two nuo genes not present in C. jejuni encode the NADH dehydrogenase, and in their place in the operon are the novel genes designated as Cj1575c and Cj1574c. A series of mutants was generated in which each of the12 nuo genes (homologues to known complex I subunits) were disrupted or deleted. Each of the nuo mutants will not grow in amino acid-based media unless supplemented with an alternative respiratory substrate such as formate. Unlike the nuo genes, Cj1574c is an essential gene and could not be disrupted unless an intact copy of the gene was provided at an unrelated site on the chromosome. A nuo deletion mutant can efficiently respire formate but is deficient in á-ketoglutarate respiratory activity when compared to WT. In C. jejuni, á-ketoglutarate respiration is mediated by the enzyme 2-oxoglutarate:acceptor oxidoreductase (OOR), mutagenesis of this enzyme abolishes á-ketoglutarate-dependent O2 uptake and fails to reduce the electron transport chain. The electron acceptor for OOR was determined to be flavodoxin, which was also determined to be an essential protein in C. jejuni. A model is presented in which CJ1574 mediates electron flow into the respiratory transport chain from reduced flavodoxin and through complex I. The importance of complex I is further illuminated by host colonization assay, in which three nuo mutants are significantly deficient.
<BR><BR>
IMPACT: 2005/10/01 TO 2006/09/30<BR>
1. Issue: Campylobacter jejuni has emerged as the most prevalent bacterial food-borne pathogen in the world. There are an estimated 2.5 million cases of campylobacteriosis in the U.S. annually, with a cost of infection between 1.2 and 1.4 billion dollars. 2. What Has Been Done: We have mutated many of the genes for enzymes of the Campylobacter jejuni respiratory chain. We have shown that the lack of many of these enzymes significantly impairs the ability of C. jejuni to grow in the conditions that exist in the chicken intestinal tract. 3. Impact: Reduction in the level of C. jejuni contamination of food supply is a priority in the fight to reduce the personal and economic burden of campylobacteriosis. Enzymes of central metabolism are a logical choice for rational drug design, as they are required for an organism to remain viable. Characterization of the energy metabolism pathways and enzymes of C. jejuni is an important first step in developing specific inhibitors of this food-borne pathogen. Specific inhibitors of C. jejuni could safely be used in food animals, unlike medically important antibiotics, the use of which is currently being severely curtailed in non-human animals.
<BR><BR>
PROGRESS: 2004/10/01 TO 2005/09/30<BR>
Both formate and hydrogen provide an energetic boost to C. jejuni when growing aerobically, and one (or the other) compound is absolutely required for growth anaerobically when using nitrate as the terminal electron acceptor, which is the first report of anaerobic growth of this organism.. A double mutant has been created in which both the hydrogenase and formate dehydrogenase genes are inactivated, this mutant is unable to use either substrate. All three strains (the hydrogenase mutant, the formate dehydrogenase mutant, and the double mutant) have a diminished ability to colonize chickens, however only formate dehydrogenase and double mutant are significantly different than wild type. There is a synergistic effect between the two mutations, the double mutant colonized at significantly lower levels than either single mutant. The genome sequence of C. jejuni predicts a respiratory chain that begins with the fourteen-subunit proton-pumping enzyme referred to as Complex I. Our first attempt to characterize the C. jejuni nuo operon has been via mutagenesis of the nuo genes. We believe that two of the nuo subunits (designated cj1574c and cj1575c), are essential, as numerous attempts to disrupt or delete the genes for these subunits have been unsuccessful. 10 of the remaining 12 nuo genes (nuoC, muoD, nuoG, nuoI, nuoJ, nuoK, nuoL, nuoM, and nuoN), have been mutated via either insertion or deletion gene-directed mutagenesis. None of these mutants cannot grow on amino acid based media (such as Mueller-Hinton agar), but can grow on these media when supplemented with the respiratory donors hydrogen or formate. Our hypothesis is that the hydrogen and formate pathways (see aim 1) can bypass complex I. The physiological donor to complex I and the role of Cj1574c and Cj1575c are the focus of ongoing research
<BR><BR>
IMPACT: 2004/10/01 TO 2005/09/30<BR>
We have identified 3 respiratory donors and associated dehydrogenases that influence the fitness of Campylobacter jejuni to colonize chickens. Future efforts to eradicate C. jejuni from the food supply may eventually depend on targeting these proteins.
<BR><BR>
PROGRESS: 2003/10/01 TO 2004/09/30<BR>
Significant progress has been made in characterizing the Campylobacter jejuni respiratory chain. The mutant strains reported from the last period have been extensively characterized, three more mutants have been isolated, and the in vitro characterization of complex I is progressing. The hydrogenase mutant (Hyd:cm) grows as well as the parent strain in non-hydrogen containing atmospheres, but has a significantly longer generation time (1.4 hrs) than the parent when grown in H2 -containing atmospheres. Chickens were colonized by both the mutant and the wt C. jejuni strains. The formate dehydrogenase mutant grows much slower than the parent under all conditions. Formate addition to the growth media significantly stimulated the growth of the parent, but not the mutant strain. The mutant was also significantly impaired in its ability to colonize chickens. Thee new respiratory chain mutants were obtained, all three in electron acceptors. These mutants include the bd-type cytochrome c oxidase (CydA:cm), the nitrate reductase (NapA:cm) and the Nitrate reductase (NrfA:cm). Characterization of these mutants is ongoing. The two novel components of Complex I have been purified and we have raised antisera to these proteins. These antisera are currently being used to probe the localization of these two proteins.
<BR><BR>
IMPACT: 2003/10/01 TO 2004/09/30<BR>
We have identified several genes which we believe are are required for the viability and growth of Campylobacter jejuni, which makes them attractive targets for rational dreug design. Other enzymes which are being studied, the presence of which significantly increases C. jejuni growth under the conditions we believe exist in the animal resovoir of this pathogen.
<BR><BR>
PROGRESS: 2003/10/01 TO 2004/09/30<BR>
Significant progress has been made in characterizing the Campylobacter jejuni respiratory chain. The mutant strains reported from the last period have been extensively characterized, three more mutants have been isolated, and the in vitro characterization of complex I is progressing. The hydrogenase mutant (Hyd:cm) grows as well as the parent strain in non-hydrogen containing atmospheres, but has a significantly longer generation time (1.4 hrs) than the parent when grown in H2 -containing atmospheres. Chickens were colonized by both the mutant and the wt C. jejuni strains. The formate dehydrogenase mutant grows much slower than the parent under all conditions. Formate addition to the growth media significantly stimulated the growth of the parent, but not the mutant strain. The mutant was also significantly impaired in its ability to colonize chickens. Thee new respiratory chain mutants were obtained, all three in electron acceptors. These mutants include the bd-type cytochrome c oxidase (CydA:cm), the nitrate reductase (NapA:cm) and the Nitrate reductase (NrfA:cm). Characterization of these mutants is ongoing. The two novel components of Complex I have been purified and we have raised antisera to these proteins. These antisera are currently being used to probe the localization of these two proteins.
<BR><BR>
IMPACT: 2003/10/01 TO 2004/09/30<BR>
We have identified several genes which we believe are are required for the viability and growth of Campylobacter jejuni, which makes them attractive targets for rational dreug design. Other enzymes which are being studied, the presence of which significantly increases C. jejuni growth under the conditions we believe exist in the animal resovoir of this pathogen.
<BR><BR>
PROGRESS: 2002/10/01 TO 2003/09/30<BR>
In the period from 10-1-02 to 9-30-02, significant progress has been made in all three of the objectives from the project Respiratory Substrates of Campylobacter jejuni. Objective I: Characterization of the phenotypes of respiratory chain components: Two of the respiratory chain components have been successfully mutated, the hydrogenase and formate dehydrogenase. The mutant strains (termed hyd:cm and fdh:cm) have been partially characterized, each is unable to oxidize its substrate (hydrogen and formate, respectively). Furthermore, the each mutant has been extensively assayed for growth (see objective 3). Other respiratory chain components have been cloned and interrupted with the antibiotic resistance cassette, but we have been unable to recover mutants. These components include the pyruvate:ferredoxin oxidoreductase and the uncharacterized genes cj1574c and cj1575c. We hypothesis that these genes/proteins are required for Campylobacter jejuni viability, which would explain why our mutagenesis procedures have not been fruitful. Objective II: Purify and characterize cj1574c and cj1575c. Both genes have been cloned separately and together, and expressed in a E. coli expression strain. The recombinant proteins are expressed at very high levels, however solubility has been a problem. We have been able to purify both components to near homogeneity from the E. coli extracts, and these proteins are currently being used as antigens to raise antibodies in rabbits. Unfortunately, we feel that the recombinant proteins are not suitable for further biochemical characterization, as they have been re-solubilized, so have most likely lost any associated co-factors (or were never fully assembled in the heterologous host). UV/visible spectra of the purified recombinant protein do not reveal any features characteristic of an associated co-factor. Objective III. We have determined what we feel are minimal requirements to a media that will support C. jejuni growth. We have also determined that the respiratory substrates we are studying, hydrogen, formate, and pyruvate, significantly improve the C. jejuni growth when supplied in this media. Both hydrogen and formate do no stimulate the growth in the hyd:cm and fdh:cm mutants. We have also been able to show that the addition of hydrogen to the growth environment will significantly alter the range of Oxygen that C. jejuni will tolerate. Normally, C. jejuni is considered a microaerophile, with a growth range in oxygen from 2-15%. Addition of hydrogen extends the oxygen tolerance up to 25%, relieving the microaerophillic requirement.
<BR><BR>
IMPACT: 2002/10/01 TO 2003/09/30<BR>
We have identified two genes which we believe are are required for the viability and growth of Campylobacter jejuni, which makes them attractive targets for rational dreug design. Two other enzymes which are being studied, the presence of which significantly increases C. jejuni growth under the conditions we believe exist in the animal resovoir of this pathogen.
<BR><BR>

PROGRESS: 2002/07/01 TO 2002/09/30<BR>
Though the time period covered in this progress report (07/01/2002 - 09/30/2002) is quite short, significant progress has been made in setting up a functioning laboratory suitable for the investigation of Campylobacter jejuni physiology. The lab is now fully equipped to perform all of the experiments required to meet the objectives stated in the project outline. For example, we are now routinely growing C. jejuni cultures, and have optimized the energy-limited defined media to the point that we believe we have identified the minimal requirements for C. jejuni growth. Experiments to determine the range of potential growth substrates are ongoing. The lab is fully functional with regard to DNA cloning and manipulation, and we have successfully cloned four of the C. jejuni genes targeted in the project outline: hydA, fdhA, cj1574c, and cj1575c. Two of these genes (hydA and fdhA) are being modified (interrupted with drug resistance cassettes) for the purpose of mutagenizing these genes in C. jejuni. The genes cj1574c and cj1575c have been cloned into E. coli expression vectors, and the corresponding proteins have been partially purified, also an objective of this project.
<BR><BR>
IMPACT: 2002/07/01 TO 2002/09/30<BR>
The expected impact of the project has not changed.