Source: NORTH CAROLINA STATE UNIV submitted to
RESPIRATORY SUBSTRATES OF CAMPYLOBACTER JEJUNI
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0193537
Grant No.
(N/A)
Project No.
NC06679
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2002
Project End Date
Jun 30, 2007
Grant Year
(N/A)
Project Director
Olson, J.
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Microbiology
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
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71232601100100%
Goals / Objectives
Campylobacter jejuni is a food-born pathogen responsible for over 2 million cases of gastroenteritis per year. C. jejuni typically colonizes an anaerobic region of the intestine, where it grows using a branched respiratory chain to fulfill all of its energy requirements. Four of C. jejuni's respiratory enzymes (sulfite oxidase, formate dehydrogenase, nitrate reductase and DMSO reductase) are molybdenum-containing enzymes. These enzymes have been implicated in the ability of C. jejuni to grow anaerobically, and mutants in formate dehydrogenase, nitrate reductase, and DMSO reductase are all impacted in the ability to colonize chickens. Molybdenum acquisition and processing proteins therefore provide a tempting target for antimicrobials against C. jejuni in the avian host. As well as with the Mo-containing enzymes, the genome also contains the genes encoding a high affinity molybdenum transport system (modABC), 10 genes predicted to synthesize the pterin found in all four of the Mo-enzymes, and a gene (Cj1507c) encodes a protein that shares homology with the Escherichia coli Mo-responsive regulatory protein ModE. Preliminary data of a CJ1507c mutant shows this protein acts as both a repressor and activator of mo-related genes in C. jejuni. We propose to study the role of the Mo-related genes through mutagenesis, in vitro characterization of gene products, and complementation of E. coli deficient mutants. Finally, the importance of these genes and proteins in host colonization will be determined in chickens.
Project Methods
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.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: The work done in the lab has been disseminated via two invited seminars, one at the Triangle Microbial Interactions Meeting, Cary, NC on April 28, 2010, and the second at the University of Georgia, Department of Microbiology, February 10, 2010. In addition, the lab has produced 4 posters presented at local meetings. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: People who come in contact with poultry, swine, or bovine. This includes poultry, swine and beef consumers. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We have made several finding that we hope will be used in the detection and eradication of Campylobacter jejuni and Campylobacter coli, two import human pathogens. First, we have created a strain of C. jejuni that encodes a marker on its chromosome that will allow us to further investigate its transmission. We have also identified a gene that allows Campylobacter coli to overcome oxidative stress Lastly, we have sequenced a strain of C. jejuni that was isolated from a patient with Guillon Barre (an autoimmune disease brought on by preceding infection), and are analyzing this strain to identify the genetic basis for pathogenesis.

Publications

  • No publications reported this period


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The goal of this research is to identify the relative importance of various respiratory donor and acceptor compounds on the physiology of the important human pathogen Campylobacter jejuni. In this period we characterized the role of four donors (hydrogen, formate, succinate and 2-oxoglutarate) and one acceptor (fumarate). The results of these experimtents were published in widely available microbiology journals. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Microbilogists Food Scientists Food Safety experts PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The physiology of Campylobacter jejuni is quite unique, and could be used as a target to reduce or eliminate this important human pathogen from the food supply. For instance, in order to use formate as a respiratory donor, the bacterium relies on the enzyme formate dehyrogenase. Neither humans nor the birds (the normal host of C. jejuni) use metabolize formate nor express a fomrate dehydrogenase, so an inhibitor would not prove toxic to the host. Our studies have confirmed that colonization is reduced when the enzyme is absent.

Publications

  • Weingarten, Rebecca A., Taveirne, Michael E., and Olson, Jonathan W. 2009. The dual-functioning fumarate reductase is the sole succinate:quinone reductase in Campylobacter jejuni and is required for full host colonization. J. bacteriol. 191:5293-5300.
  • Weerakoon, Dilan R., Borden, Nanthen J. , Goodson, Carrie M., Grimes, Jesse, and Olson, Jonathan W. 2009. The Role of Respiratory Donor Enzymes in Campylobacter jejuni Host Colonization and Physiology. Microbial Pathogenesis. 47:8-15


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Our laboratory has demonstrated that, unlike all other organisms studied to date, C. jejuni does not use NADH as a respiratory substrate despite encoding 12 of the 14 genes of the NADH dehydrogenase enzyme typically referred to as complex I. We have also shown that the donor to this complex is a reduce flavodoxin, and at least one substrate used to reduce flavodoxin is 2-oxoglutarate, a Kreb's cycle intermediate. PARTICIPANTS: Jesse Grimes, collaborator, NCSU Poultry Science Rebecca Weingarten, graduate student, NCSU Microbiology Dilan Weerakoon, graduate student, NCSU Microbiology TARGET AUDIENCES: Microbiologists, food scientists, poultry scientists PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Campylobacter jejuni is a significant human pathogen, causes over 2 million cases of gastro-enteritis per year. A better understanding of C. jejuni's metabolic capability could be used to keep the food supply clear of this typically food-borne bacterium.

Publications

  • Weingarten, Rebecca A., Grimes, Jesse L, and Jonathan W. Olson. 2008 The Role of Campylobacter jejuni Respiratory Oxidases and Reductases in Host Colonization Appl. Environ. Microbiol. 74:1367-1375.
  • Weerakoon, Dilan R. and Olson, Jonathan W. 2008 The Campylobacter jejuni NADH:ubiquinone oxidoreductase (complex I) utilizes flavodoxin rather than NADH. J. Bacteriol. 190:915-925.


Progress 10/01/06 to 09/30/07

Outputs
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.

Impacts
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.

Publications

  • Weingarten, Rebecca A., Goodson, Carrie M., Borden, Nathan J., Grimes, Jesse and Olson, Jonathan W. The Role of C. jejuni alternative respiratory components on host colonization. (Abstract). Program and Abstract Book 107th General meeting of the American Society for Microbiology. May 23, 2007 Toronto, Canada.
  • Weerakoon, Dilan R. and Olson, Jonathan W. Complex I of Campylobacter jejuni. (Abstract) Program and Abstract Book, 107th General meeting of the American Society for Microbiology. May 23, 2007 Toronto, Canada.
  • Taveirne, Michael and Olson, Jonathan W. Molybdenum Transport in Campylobacter jejuni. (Abstract). Program and Abstract Book, 107th General meeting of the American Society for Microbiology. May 22, 2007 Toronto, Canada.


Progress 07/01/02 to 06/30/07

Outputs
Target Audience: People who come in contact with poultry, swine, orbovine. This includes poultry, swine and beef consumers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Michael Taveirne was supported by NIFA and recieved his PhD as a result of this progect. 5 NC State undergraduate students have also done research projects in the laboratory during this period. How have the results been disseminated to communities of interest? Yes, through the journal articles described. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The Mo-related genes have been characterize and the result have been published.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Issmat I Kassem, Mahesh Khatri, Malak A Esseili, Yasser M Sanad, Yehia M Saif, Jonathan W Olson and Gireesh Rajashekara. 2012. Respiratory proteins contribute differentially to Campylobacter jejuni's survival and in vitro interaction with hosts' intestinal cells. BMC Microbiology. 12:258
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Crespo MD, Olson JW, Altermann E, Siletzky RM, Kathariou S. 2012. Chromosomal tet(O) harboring-regions in Campylobacter coli from turkeys and swine. Applied and Environmental Microbiology 78:8488-8491.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Snipen, L., Wassenaar, T., Alterman, E., Olson, J. Katharious, S., Lagesen, K., Knochel, S., Ussery, D. and Meinersmann, R.J. 2012. Analysis of co-evolving genes in Campylobacter jejuni and Campylobacter coli. Microbial Informatics and Experimentation 2(8) 1-11.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Gardner, Susan P. and Olson, Jonathan W. 2012. Barriers to horizontal gene transmission in Campylobacter jejuni. Advances in Applied Microbiology, 79:19-42.


Progress 10/01/05 to 09/30/06

Outputs
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 α-ketoglutarate respiratory activity when compared to WT. In C. jejuni, α-ketoglutarate respiration is mediated by the enzyme 2-oxoglutarate:acceptor oxidoreductase (OOR), mutagenesis of this enzyme abolishes α-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.

Impacts
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.

Publications

  • No publications reported this period


Progress 10/01/04 to 09/30/05

Outputs
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

Impacts
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.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
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.

Impacts
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.

Publications

  • No publications reported this period


Progress 10/01/02 to 09/30/03

Outputs
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.

Impacts
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.

Publications

  • Olczak, A.A., Seyler, R.W. Olson, J.W., and Maier, R.J. 2003. Association of Helicobacter pylori antioxidant activities with host colonization efficiency. Infection and Immunity 71:580-583.
  • Mehta, N., Olson, J.W., and Maier, R.J. 2003. Characterization of Helicobacter pylori nickel metabolism accessory proteins needed for the maturation of both urease and hydrogenase. J. Bacteriol. 185:726-734.
  • Maier, R.J. Olson, J.W. and Olczak, A. 2003. Hydrogen Oxidizing Capabilities of Helicobacter hepaticus and in vivo Availability of the Substrate. J. Bacteriol. 185: 2680-2682.


Progress 07/01/02 to 09/30/02

Outputs
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.

Impacts
The expected impact of the project has not changed.

Publications

  • Olson, J.W., and Maier R.J. 2002. Molecular Hydrogen as an Energy source for Helicobacter pylori. In press, Science