Coxiella burnetii is the causative agent of both acute Q fever and chronic endocarditis in humans. Both plasmid and chromosomal differences correlate with the type of disease (acute or chronic) caused by an isolate suggesting that the different isolates have different virulence determinants. We have designed experiments to extend our knowledge of C. burnetii genomic and plasmid DNA and their relationship to disease. Unique plasmid regions will be analyzed by cloning, restriction enzyme, Southern hybridization and sequencing analyses. We will also identify the plasmid origin of replication for studies on transformation and mutagenesis of C. burnetii. Plasmid encoded surface proteins (possible virulence factors) will be identified by looking for protease-sensitive surface proteins produced by maxicells containing pUC/C. burnetii plasmid constructs and also by TnphoA mutagenesis. We will identify chromosomal encoded surface antigens employing both cosmid and 1 ZapII gene libraries in E. coli, by screening for colonies/plaques reacting with C. burnetii-specific antisera and monoclonal antibodies to isolates with differing disease potential. The distribution of antigens in C. burnetii isolates will be determined using surface iodination, PAGE, and western analyses. To understand the role of gene in virulence and intracellular growth of C. burnetii, we have designed experiments to evaluate gene function directly within the organism. We will use previously cloned C. burnetii chromosomal and plasmid genes as specific probes to assay their transcriptional activation at different times and under different conditions, in vitro and in vivo. Concurrently, we will examine qualitative differences in C. burnetii RNA production. Genes specifying unique or differentially expressed transcripts produced at different time points in vitro or post-infection will be cloned and sequenced. Proteins being synthesized under these conditions will also be evaluated by 1 and 2 -D PAGE. In this fashion we will develop an understanding of gene expression in C. burnetii during the host-parasite interaction. We also will examine plasmids and the chromosome for alternative virulence functions; factors that enhance intracellular survival and growth and thus pathogenesis. We will clone genes encoding key metabolic functions as well as those shown to be important for intracellular survival of other organisms. These genes will be cloned from C. burnetii employing the methods of PCR, mutant complementation and using homologous genes as probes. And because the development of genetic exchange systems is vital to an understanding of rickettsial genetic mechanisms, we will continue to develop transformation and mutagenesis systems for C. burnetii using electroporation and antibiotic selection in cell cultures.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020190-10
Application #
3129693
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1983-07-01
Project End
1996-05-31
Budget Start
1992-06-01
Budget End
1993-05-31
Support Year
10
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Washington State University
Department
Type
Schools of Arts and Sciences
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Rivera-Amill, V; Kim, B J; Seshu, J et al. (2001) Secretion of the virulence-associated Campylobacter invasion antigens from Campylobacter jejuni requires a stimulatory signal. J Infect Dis 183:1607-16
Howe, D; Mallavia, L P (2000) Coxiella burnetii exhibits morphological change and delays phagolysosomal fusion after internalization by J774A.1 cells. Infect Immun 68:3815-21
Lin, Z; Mallavia, L P (1999) Functional analysis of the active partition region of the Coxiella burnetii plasmid QpH1. J Bacteriol 181:1947-52
Howe, D; Mallavia, L P (1999) Coxiella burnetii infection increases transferrin receptors on J774A. 1 cells. Infect Immun 67:3236-41
Lin, Z; Mallavia, L P (1998) Membrane association of active plasmid partitioning protein A in Escherichia coli. J Biol Chem 273:11302-12
Mo, Y Y; Seshu, J; Wang, D et al. (1998) Synthesis in Escherichia coli of two smaller enzymically active analogues of Coxiella burnetii macrophage infectivity potentiator (CbMip) protein utilizing a single open reading frame from the cbmip gene. Biochem J 335 ( Pt 1):67-77
Lin, Z; Yang, S; Mallavia, L P (1997) Codon usage and nucleotide composition in Coxiella burnetii. Gene 198:171-80
Afseth, G; Mallavia, L P (1997) Copy number of the 16S rRNA gene in Coxiella burnetii. Eur J Epidemiol 13:729-31
Seshu, J; McIvor, K L; Mallavia, L P (1997) Antibodies are generated during infection to Coxiella burnetii macrophage infectivity potentiator protein (Cb-Mip). Microbiol Immunol 41:371-6
Heinzen, R A; Howe, D; Mallavia, L P et al. (1996) Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii. Mol Microbiol 22:9-19

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