Coxiella burnetii is a gram-negative g-proteobacterial species that causes Q fever, an acute debilitating flu-like illness. People contract Q fever from contaminated material from farm animals or from material in slaughterhouses and meat packing plants. It is estimated that as many as 1010 organisms can be shed at parturition. Although Q fever is self-limiting in most cases, a chronic, difficult to treat endocarditis - associated form f the disease can develop in a small percentage of recovered individuals. A large outbreak of Q fever continues in a small heavily agricultural region in the Netherlands, where more than 4,000 individuals have contracted the disease since 2007. C. burnetii is extremely stable in the environment as it is extremely resistant to desiccation. In addition, when C. burnetii infects host cells, it resides in an intracellular vacuole that has many properties of phagolysosomes. Indeed, the organism is only metabolically active at the intravacuolar pH of 4.5 and is resistant to the antimicrobial defenses usually found in mature phagolysosomes. The molecular mechanisms of resistance to environmental stresses are poorly understood. Recent advances in axenic culture of C. burnetii and emerging genetic techniques make it now possible to pursue experimental approaches that were not possible up until now. The work described in this proposal is aimed at finding out the molecular basis of the extreme desiccation resistance of C. burnetii. In order to do this three specific aims are proposed. The first specific aim is to identify small molecular weight osmo-protectant compatible solutes that should be produced or accumulated upon osmotic stress. Upon desiccation, the bacteria must first adapt to increased osmolarity usually by synthesis or accumulation of solutes such as trehalose, glycine betaine or proline. This phenomenon has not been explored for C. burnetii. We will expose C. burnetii to hyperosmotic conditions, isolate low molecular weight solutes and examine the contents of the extracts by 13C-NMR. The second specific aim is to identify C. burnetii genes that are regulated by hyperosmotic conditions, oxidative damage, and by desiccation. Usually even if bacteria are tolerant of hyperosmotic conditions, they remain sensitive to desiccation. Desiccation sensitivity is thought to be due to oxidative damage to proteins and DNA from high intracellular concentrations of Fe. Transcriptome analysis by RNAseq will be used to identify C. burnetii genes that are upregulated by exposure of C. burnetii to all three stresses. Finally the third aim is to use genetic approaches to identify genes required for desiccation resistance in C. burnetii. This will be accomplished by screening for transposon-induced mutants that have lost the ability to tolerate desiccation. The proposed work will provide the first set of information about how C. burnetii resists osmotic stress and desiccation.
The proposed work is aimed at understanding how Q fever, a common world-wide flu-like disease, caused by Coxiella burnetii, is transmitted from farm animals to people. These bacteria are extremely resistant to drying in the environment and survival under dry conditions is thought to be important for their ability to be transmitted to people. The proposed work is aimed at elucidating the mechanism of desiccation resistance.
