monash_86733.pdf (13.57 MB)
Proteomics analysis of outer membrane proteins of leptospira interrogans
thesisposted on 2017-02-17, 02:29 authored by Zhang, Kunkun
Leptospirosis is a zoonotic disease caused by spirochetes of the genus Leptospira. The symptoms of leptospirosis in humans range from an infection with mild, influenza-like symptoms to a severe syndrome with multi-organ failure and high mortality. Outer membrane proteins are believed to be the first point of interaction between the bacteria and the host. One of the main aspects of leptospiral virulence is the ability of the bacteria to survive and multiply in the host. Therefore, we set out to investigate the interactions in vitro between leptospiral outer membrane proteins and the environment in the host, by using proteomic approaches. In the first results chapter, a simple and reproducible total membrane preparation method of leptospiral cells was evaluated. Following this method, the total membrane proteome changes of Leptospira interrogans serovar Manilae were analyzed using 2-D gel electrophoresis, upon interaction with extracellular matrix (ECM) proteins or in response to up-shifted osmolarity. However, no proteins were detected significantly up- or down-regulated in either condition. The total membrane proteome of a mutant in lipL32, which encodes the most abundant lipoprotein on the outer surface of virulent leptpospiral strains, was also analyzed using 2-D gel electrophoresis. No proteins were detected compensating for the lack of LipL32. Furthermore, LipL32 and other membrane proteins were confirmed as binding laminin, one of the major ECM component proteins, in ligand blot. The second results chapter presents the analysis of the total membrane proteome of L. interrogans serovar Copenhageni incubated with guinea pig serum. This approach detected the binding of the serum protein Apolipoprotein A-I (ApoA-I) to leptospiral cells. Leptospiral binding to ApoA-I was confirmed in multiple strains using human serum as a source of ApoA-I. A search for leptospiral ApoA-I interacting proteins identified several candidate proteins, including the outer membrane lipoprotein LipL71. However, a severely truncated LipL71 mutant of L. interrogans bound ApoA-I 2.5-fold more than wild type and the other less truncated LipL71 mutant. Furthermore, the severely truncated mutant was attenuated in a hamster model of leptospirosis, while the less truncated mutant retained virulence. Whole genome sequencing detected no other mutations in the attenuated strain. These results indicate that the interaction between Leptospira and ApoA-I is affected by LipL71 and that this interaction may play a prominent role in leptospiral infection. A model was also suggested by this study as to how LipL71 might participate in pathogenesis of leptospirosis by decreasing ApoA-I binding and consequently escape from the host innate immune system. In the final results chapter, a comparison was made of the antibody response induced in hamsters by an attenuated LPS mutant, M1352, with that induced by killed bacteria. This approach highlighted four outer membrane proteins, LigB, Loa22, OmpL36 and LA1939, which might be responsible for cross-serovar immunoprotection. Recombinant proteins of Loa22, OmpL36 and LA1939 were cloned and purified from E. coli. All the recombinant proteins showed immunoreactivity in the immunoblot assays probing with the sera of hamsters and also of humans diagnosed with leptospirosis. However, neither individual recombinant protein nor a combination of all three proteins conferred protection in a hamster model of infection.