Source: UNIV OF MINNESOTA submitted to NRP
WHOLE-GEONOME SEQUENCING AND ANALYSIS OF LAWSONIA INTRACELLULARIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0186399
Grant No.
00-52100-9687
Cumulative Award Amt.
(N/A)
Proposal No.
2000-04534
Multistate No.
(N/A)
Project Start Date
Sep 15, 2000
Project End Date
Sep 30, 2004
Grant Year
2000
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
VETERINARY PATHOBIOLOGY
Non Technical Summary
Lawsonia intracellularis causes proliferative enteritis in swine and is also known to infect other animal species. This organism has a major economic and animal health impact in the United States and other parts of the world. Despite the tremendous loss associated with L. intracelluaris infections, we lack effective diagnostic tests and vaccines for preventing the disease. This is in large part due to a lack of basic understanding of the bacterium, it's requirements for growth in the laboratory, and the molecular basis for its ability to infect animals and cause disease. We have proposed a collaborative project to determine the complete nucleotide sequence of this bacterium. To accomplish this objective, we will utilize a random shotgun sequencing strategy that has been successfully applied in the past for whole genome sequence analysis of other microbial organisms. We will next initiate studies to identify genes that encode potential diagnostic antigens, and apply microarray technology to identify major L. intracellularis genes whose expression is altered during the infection process. The results of our investigations will help in the identification genes responsible for microbial replication, virulence, species-specificity, and ability to evade the immune system. We believe that this comprehensive knowledge of the pathogen's genome will provide all the necessary information required for cost-effective and targeted research on the development of superior diagnostic tests and vaccines against this organism.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113510110070%
3113910110030%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3910 - Cross-commodity research--multiple animal species; 3510 - Swine, live animal;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
We propose to sequence and annotate the complete genome of Lawsonia intracellularis, the causative agent of proliferative enteropathy in swine. Next, we will use this information to develop whole genome microarrays for this organisms in order to provide a rational basis for the development of new and effective diagnostic reagents, vaccines and drugs, but also facilitate future research on important questions relating to virulence and pathogenicity of L. intracellularis.
Project Methods
Small and large-insert library construction will take place as per established procedures in pUC18 and lambda vectors. High-throughput DNA sequencing will be utilized to determined the nucleotide with eight-fold redundancy from the small-insert library. The sequencing of the ends of two large-insert libraries constructed in bacteriophage lambda will provide a scaffolding structure to enable gap filling by constructing new primers and sequencing of the ends of the lambda clones wherever there are sequence gaps. All sequence data management, editing, similarity searching and annotation will be performed with the public-domain computer programs, phredPhrap/Consed, ARTEMIS, and WIT2 that have been written expressly for these purposes and are in routine use in our laboratory. The aligned nucleotide sequences will be visually inspected and all sequence ambiguities will be edited and resolved. The genome will then be annotated based on database similarity searches and the genes assigned with known or putative functional roles. The annotated sequences developed from this study will be deposited in public databases (eg. GenBank, TIGR database) as well as uploaded daily onto our web-site with a built-in search engine. Using this strategy, we will be able to rapidly and completely annotate and analyze the genome of L. intracellularis, as well as enable us and other researchers to utilize the sequence database for facilitating their hypothesis-based studies. Next, we will use this information to develop whole genome microarrays as well as identify specific genes for the development of superior serological diagnostic tests.

Progress 09/15/00 to 09/30/04

Outputs
The goal of this project was to determine the complete DNA sequence of the chromosome of Lawsonia intracellularis, the causative agent of proliferative enteropathy in swine and other domestic animals. Whole genome sequence of the strain PHE/MN1-00 was successfully determined using random shot gun sequencing approach with 8x genome coverage. Sequence fragments were assembled into one circular chromosome of 1,457,619 base pairs and three plasmids of 27,048, 39,794 and 194,553 base pairs, respectively. The chromosome and plasmids together constitute a total of 1,719,014 base pairs with an average G+C content of 33.08%. The genome carries 1,345 potential open reading frames (ORFs) with an average size of 1,063 base pairs accounting for 83% of the entire genome. The remaining 17% of the sequence is comprised of 2 complete rRNA operons (16S-23S-5S), 52 tRNA genes representing all 20 amino acids, and a relatively small number of non-coding elements. The contribution of a significant portion of the genome in the biochemistry and cell biology of this organism remains to be determined as almost half of the potential ORFs translate into hypothetical proteins with no known functions. Among the important findings of genome annotation and analyses is the identification of L. intracellularis orthologs for the genes coding for and those involved in the assembly of flagellar apparatus proteins including flagellum filament, hook, and a basal body which provides a facile means of developing specific reagents to delineate their role in virulence and infectivity. This finding is noteworthy because flagellar structures are often highly immunogenic and antibodies against these surface structures may lead to bacterial opsonization and killing in the infected host. Therefore the flagellar genes identified in L. intracellularis genome may be of great value in applications to develop diagnostic and immunoprophylactic reagents. L. intracellularis genome possesses orthologs of almost all genes required for a highly complex mechanism of protein translocation characterized as type III secretion system. In other gram negative bacteria, genes of type III secretion system are typically associated with the pathogenicity islands. L. intracellularis type III secretion system genes were found in two clusters on the chromosome. The two clusters include yscV, yscS, yscT, yscU, sctR, sctN and yscC, yscJ, yscL, respectively. The genome also contains a few orthologs of type I secretion system including genes for secA, secY, secG, secF, secD, yajC, yidC along with TatC and TatD genes. L. intracellularis has several virulence related candidate genes including hemolysin, catalase, alpha-amylase, metaloproteases, and nucleases. The genome also carries ortholog for Cu, Zn-cofactored superoxide dismutase which has been implicated in bacterial virulence and protection against exogenous oxidative damage.

Impacts
The knowledge of complete genome sequence and the overall coding potential of L. intracellularis has greatly enhanced our understanding of previously unknown biochemical pathways, structural components and potential virulence mediators of this bacterium that have significant implications in future development of improved diagnostic and immunogenic reagents to facilitate better control of L. intracellularis infection in pigs and other species.

Publications

  • Beckler, D.C., V. Kapur, and C.J. Gebhart. 2004. Molecular epidemiology of Lawsonia intracellularis. Leman Swine Conference, St. Paul, MN.
  • Beckler, D., A. Amonsin, V. Kapur and C. Gebhart. 2004. Multiple-locus variable number tandem repeat analysis for the differentiation of L. intracellularis isolates. Proc. Am. Assoc. Swine Vet., Ames, IA.
  • Nuntaprasert, A, K. Kaur, C.J. Gebhart and V. Kapur. 2004. Expression and purification of a flagellar protein of Lawsonia intracellularis. Proc. 85th Conf. Res. Workers An. Dis., Chicago, IL, November 14-16.
  • Kapur et al., 2005. Genome Sequence of Lawsonia intracellularis, the causative agent of proliferative enteropathy, identifies numerous genes of relevance to diagnosis, virulence and immunoprophylaxis. Manuscript in preparation.


Progress 01/01/01 to 12/31/01

Outputs
We are now making rapid progress towards the completion of project goals. The major breakthrough in the project achieved during this period has been the preparation of Lawsonia intracellularis genomic DNA with very low levels of contaminating eukaryotic DNA that results from growth within an intracellular environment. Random shotgun libraries have been prepared and the shotgun sequencing phase is well underway. The project has already identified numerous interesting leads from a diagnostic test and vaccine development standpoint.

Impacts
1. The project is expected to identify all of the genes in the genome of Lawsonia intracellularis. 2. The project is expected to identify potential diagnostic antigens and vaccine targets in the genomic sequence of this bacterium. 3. The project is anticipated result in the development of Lawsonia intracellularis microarrays which will serve as a community resource.

Publications

  • No publications reported this period