Coxiella burnetii infects domestic and wild animals and is distributed worldwide. Human disease, referred to as Q-fever, occurs by contact with infected animals or animal products. The pathogen is an obligately parasitic bacterium that causes both acute and chronic disease. Knowledge of the mechanisms by which C. burnetii causes disease is limited. A primary reason for the lack of information is the inability to do studies that identify, compare and characterize genetic differences between virulent (phase I) and attenuated (phase II) strains. A spontaneous chromosomal deletion characterizes the Nine Mile phase II strain. It is hypothesized that this deleted DNA accounts for the loss of virulence. To test this, genetic recombination must be developed for Coxiella.
The aims are 1) develop transformation in Coxiella, 2) optimize the system by use of a reporter plasmid, and 3) reconstruct partial or full virulence in phase II organisms.
In aim 1, organisms will be transformed to ampicillin resistance using a mixture of plasmids en masse via electroporation. These plasmids carry the C. burnetii autonomous replication sequence (ars) and a ColE1 origin. Electroporated C. burnetii will then be used to infect fibroblasts. After a long period of selection in ampicillin, cultures will be expanded, DNA extracted, and the presence of the amp-r gene confirmed by Southern hybridizations. After cloning by limiting dilutions technique, clones will be confirmed for the presence and expression of amp-r genes. The ampicillin and host cell systems used will thus select, from mixtures, the most stable plasmid construct and recombinant. The gene's location, chromosomal or plasmid, will also be determined. Finding a successful vector will then justify its further use for development as a transformation reporter plasmid. This will be done by inserting a C. burnetii heat shock promoter with a reporter gene (luciferase) into that vector (aim 2). Use of the latter will enable baseline studies of transformation to be completed in a much shorter time. Studies will then proceed to genetic reconstruction of virulence in antigenic phase II (avirulent) recipients by homologous recombination of phase I DNA into the bacterial chromosome (aim 3). Transformants possessing full or intermediate virulence will be selected by passage through A/J mice. Survivors recovered from mouse spleens and identified by recognition of specific DNA sequences will be cloned and characterized. Individual genes then suspected to be important for virulence will be studied by knockout mutations. The proposed experiments are the first to examine C. burnetii by genetic transformation.
| Miller, J D; Curns, A T; Thompson, H A (2004) A growth study of Coxiella burnetii Nine Mile Phase I and Phase II in fibroblasts. FEMS Immunol Med Microbiol 42:291-7 |
| Miller, Jeffrey D; Thompson, Herbert A (2002) Permeability of Coxiella burnetii to ribonucleosides. Microbiology 148:2393-403 |
| Hoover, T A; Culp, D W; Vodkin, M H et al. (2002) Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii nine mile strain. Infect Immun 70:6726-33 |
| Suhan, M L; Thompson, H A (2000) Expression of beta-lactamase in Coxiella burnetii transformants. FEMS Microbiol Lett 184:303-6 |
