Source: JAMES MADISON UNIVERSITY submitted to
BORDETALLA AVIUM: GENOME SEQUENCE AND EDUCATIONAL TOOL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0203362
Grant No.
2002-35600-15687
Project No.
VAR-2005-03751
Proposal No.
2005-03751
Multistate No.
(N/A)
Program Code
23.2
Project Start Date
Dec 1, 2004
Project End Date
Dec 31, 2006
Grant Year
2005
Project Director
Temple, L.
Recipient Organization
JAMES MADISON UNIVERSITY
(N/A)
HARRISONBURG,VA 22807
Performing Department
(N/A)
Non Technical Summary
Genomics offers a unique opportunity for making exponential progress in our understanding of the biology, host specificity, and pathogenicity determinants of Bordetella avium. Members of the Bordetella avium species are important pathogens in the poultry industry world-wide. Understanding of the molecular biology of this bacterium has lagged significantly behind that of the subspecies, B. pertussis, B. bronchiseptica, and B. parapertussis, due mainly to three reasons. Firstly, strains now grouped into this species were speciated and identified as the causative agents of bordetellosis in turkeys only within the last 25 years. Secondly, until very recently it was thought that B. avium lacked many of the important virulence factors of the subspecies members, thus indicating that virulence mechanisms found in B. avium were likely to differ substantially from the others. More recent sequence data indicate that B. avium has many of the same virulence factors, although these are not typically detected by DNA hybridization or antigenic screening. These recent observations validate the study of B. avium pathogenesis in its natural host, results of which may be useful to understanding mechanisms of disease causation and host specificity of all Bordetellae. Finally, B. avium lacks an easily visible phenotype to detect and characterize the virulent vs. non-virulent phases. Sequencing the genome of the avian Bordetella and identifying unique genes would allow us to make quantum leaps in understanding the biology and pathogenic mechanisms of this organism.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31132301100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3230 - Turkey, live animal;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Long term goals are 1. to characterise the basis for pathogenicity and host specificity in Bordetella avium. Our understanding of the mechanisms of pathogenicity in B. avium has been hampered by the lack of a phenotypic marker for virulence. Genomics technologies offer alternative strategies for elucidating factors in disease causation and thus rational design of vaccines and treatments. 2. to enhance science education for undergraduate students at Drew University by facilitating original research projects, classes, and collaborations that incorporate disciplinary and interdisciplinary exercises in genomics, examination of evolutionary relatedness of genes within and beyond the genus, and searches for genomic differences within the genus. Specific Objectives are 1. To sequence, analyse and annotate the genome of one of the major U.S. poultry pathogens, B. avium and to expand existing microarrays of Bordetellae to facilitate inter-genic comparisons. (b) providing data for original research projects by individuals and groups, (c) placing students in internships at labs in research institutions with scientists working with Bordetellae, and (d) hosting an annual seminar on genomics at James Madison University.
Project Methods
The chromosomal sequence of B. avium was obtained with funding already spent from this grant. This work was accomplished by the pathogens sequencing unit, Wellcome Trust Sanger Centre, UK. The remaining work to be funded by the grant includes microarray analysis, performed by collaborators at Stanford University, as described in the original documents. In brief, we will grow B. avium under differential modulating conditoins and test a subset of genes derived from analysis of the sequence, for expression under these different conditions. We will also do whole genome comparison, using a pan-Bordetella array already constructed. The remainder of the work is both research and educationally motivated. Students at James Madison University and in the labs of collaborators have employed a variety of experimental tools to research questions about B. avium and a family of bacteriophages of B. avium, including DNA cloning, sequencing, and restriction analysis, PCR for identification of sequences or for cloning, Southern blotting, lambda library construction and screening, protein purification, gene expression studies using reporter gene activity and RT-PCR, chemical and biological mutagenesis, transduction, transformation, complementation studies, and other standard biochemical and microbiological techniques. Having the entire chromosomal sequence of B . avium will greatly enlarge the kinds of questions that can be asked by these students. For example, whereas in the past we have been limited in screening for mutants to two phenotypes, motility and hemagglutination, now we will be able to pick likely target genes from the DNA sequence and proceed with constructing mutants in those genes and in constructing expression clones for study of the proteins. We will also learn much about the number and variety of prophage present in the sequenced strain and can apply that information to studying bacteriophage biology in a wide variety of other B. avium strains. Such information will help us to know whether these bacteriophage are related to disease causation, are important determinants of host specificity, or have been instrumental in horizontal gene transfer. Finally, the genomic sequence will provide information for countless data-analysis research projects, focusing, for examples, on molecular evolution of bacterial genes. Two projects are already underway as a result of the genomic analysis. Both involve use of standard microbiological and molecular biology techniques to prepare deletions in genes discovered by searching for potential virulence factors in the genomic sequence.

Progress 12/01/04 to 12/31/06

Outputs
Bordetella avium, a pathogen of poultry, is a distinct species in the Bordetella genus, compared with those bordetellae that infect mammals, B. bronchiseptica, B. pertussis, and B. parapertussis. In order to better understand the evolutionary relatedness of Bordetella species and further the understanding of disease causation, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3.7 megabasepairs (Mbp) of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence from B. avium of virulence factors previously reported were confirmed, and the genetic bases for growth characteristics were elucidated. Over 1100 genes present in B. avium but not B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type II secretion system adjacent to two very large genes for secreted proteins, unique genes for LPS biosynthesis, and a unique fimbrial biogenesis gene cluster. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that has been previously shown to be involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation w as confirmed experimentally for five of these. We compared 20 strains of B. avium using comparative genomic hybridization. These strains were isolated from both sick poultry and apparently well wild birds. Results from these studies showed that the strains differ in the presence or absence of certain prophage sequences, a toxin gene, and a gene encoding fimbrae, which are often implicated in virulence. A major portion of the funding went to educational activities. We were able to send 11 undergraduate interns to work for a summer in the labs of 9 different investigators, world-wide. While there were many good learning experiences had by the interns, one experience stood out from the others in production of data. A Drew University student worked at University of British Columbia and found, through bioinformatics, the gene for a potential autotransporter in B. avium; this is a class of proteins frequently involved in disease causation. She cloned and expressed the gene, and showed that the gene was active under laboratory conditions in B. avium. We have constructed a mutant of the gene and are currently assessing what phenotypic effect the loss of this gene has on the bacterium. The funds were also used to support an annual "Genomics" seminar. Dr. Duncan Maskell, Cambridge University, presented the first lecture at Drew Universtiy. Dr. Claire Fraser, president of TIGR, presented a seminar at JMU, and Dr. Nicholas Thompson, Wellcome Trust Sanger Centre, presented the third in the series.

Impacts
The genome sequence is serving to support work in several laboratories In addition to the undergraduate work of Temple, investigators at University of Buffalo, North Carolina State University, Stanford University, University of Guelph, and the USDA installation in Ames, Iowa, are actively involved in studies made possible by the genomic information. We believe that the understanding of virulence factors in this organism will lead to vaccine development, much needed according to official of the turkey industry. Outcomes for undergraduate students are not always easy to measure, but it is notable that of the 11 interns supported by this funding, all are in graduate or medical schools, or they are working in the biomedical industry. At least 25 other undergraduates have worked or are currently working on independent research projects based on the genomic information. This is an excellent model to illustrate the use of bioinformatics to develop and test hypotheses, and we have used it in classroom settings, as well. Finally, the results will support funding efforts in the future with more direct applications to the health and well-being of commercially grown turkeys as we get closer to developing an effective vaccine for this disease.

Publications

  • . Sebaihia, M., A. Preston, D. J. Maskell, H. Kuzmiak, T. D. Connell, N. D. King, P. E. Orndorff, D. M. Miyamoto, N. R. Thomson, D. Harris, A. Goble, A. Lord, L. Murphy, M. A. Quail, S. Rutter, R. Squares, S. Squares, J. Woodward, J. Parkhill, and L. M. Temple. 2006. Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis and B. parapertussis reveals extensive diversity in surface structures associated with host interaction. J. Bacteriol.188:6002-15