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The characterisation of putative virulence determinants of Leptospira interrogans.
thesisposted on 15.02.2017, 04:40 by King, Amy McKarral
Leptospirosis is a zoonotic disease found worldwide. It is a systemic infection that affects a large variety of hosts including humans, cattle, dogs and rodents. The disease is acute and can be mild, but severe leptospirosis can occur, resulting in multi-organ failure and death. The transmission and maintenance of pathogenic Leptospira in the environment is facilitated by animal hosts; leptospires colonise the proximal renal tubules of animal hosts and are excreted in the urine. Infection occurs through contact with infected animals or contaminated water and soil. Knowledge of virulence-associated factors, in acute leptospirosis and renal colonisation, is imperative for the development of vaccines to reduce transmission. The aim of the work presented in this thesis was to characterise putative virulence-associated factors of L. interrogans. The leptospiral outer membrane is an important interface between host and pathogen, and surface exposed proteins are of interest as they may be important for virulence though adhesion, dissemination and/or colonisation. The first results chapter characterises outer membrane lipoprotein LipL41 and its role in acute leptospirosis. The virulence of a lipL41 mutant in L. interrogans serovar Pomona was assessed in the hamster model of acute leptospirosis and the mutant was found to retain virulence. It was found that lipL41 is transcribed with a small gene lep, found 28 bp downstream. A lep mutant in L. interrogans serovar Manilae had only 10% expression of LipL41 compared to wild type. The transcription of lipL41 in the lep mutant was not affected, and it was hypothesised that Lep is a LipL41 chaperone stabilising LipL41 expression, and in the absence of Lep, LipL41 is degraded. The second results chapter investigated the protein-protein interaction between LipL41 and Lep. The proteins were shown to interact via co-purification experiments with recombinant proteins, and protein cross-linking of leptospiral cells analysed by immunoblotting with specific antisera. The function of LipL41 was also investigated. The ability of LipL41 to bind to haemin was assessed, but evidence suggested that it did not bind to haemin. LipL41 was also assessed for its ability to bind to extracellular matrix components. The final results chapter describes an htpG mutant in L. interrogans serovar Manilae. HtpG (high temperature protein G) is a molecular chaperone homologous to Hsp90 and is widely conserved in bacteria. The htpG mutant was highly attenuated in the hamster model of acute leptospirosis. Complementation of this mutant completely restored virulence. Phenotypic assays were performed to determine the basis for attenuation in the htpG mutant, including growth under heat, oxidative and osmotic stress, analysis of protein profiles, and sensitivity to serum complement. Although the mechanism of attenuation of this mutant could not be established, HtpG is one of the few known essential factors for acute leptospirosis. Further characterisation of HtpG will provide insight into leptospiral pathogenesis and potentially identify novel virulence mechanisms.