Delineating host-pathogen interaction of pathogenic Leptospira spp.

Abstract

Delineating host-pathogen interaction of pathogenic Leptospira spp. Abstract Leptospirosis is a highly infectious, global, zoonotic disease affecting the majority of the mammalian species. Leptospirosis is caused by pathogenic bacteria, Leptospira spp., with more than >250 serovars identified. Cattle are one of the most susceptible hosts where the infection is commonly caused by serovar Hardjobovis. Bovine leptospirosis (BL) causes severe reproductive disorders and is a significant public health risk. Commercially available BL vaccines are typically bacterin and considered limited as they are serovar-specific and confer temporary protection. Bacterial outer membrane proteins (OMPs) are extensively studied as potential vaccine candidates for infectious diseases due to their ability to stimulate robust immune responses and induce cross-protective immunity. Here, seven novels OMPs from L. borgpetersenii serovar Hardjobovis L550 were identified using a reverse vaccinology approach. Four OMP genes were successfully cloned, expressed and purified as recombinant proteins. Subsequent functional in vitro binding assays showed that these OMPs could adhere to various host components and two OMPs of 37 and 49 kDa with significant binding results were re-assigned as ‘Leptospiral adhesin’ (Lsa) together with molecular weights as Lsa37 and Lsa49. Immunological evaluation of antibody titres against the OMPs in cattle bulk milk suggests these proteins are expressed by the bacteria and interact with the host immune system with two OMPs, OmpL1 and rLBL0375 exhibiting better discrimination with disease status. Several leptospiral OMPs exhibit molecular diversity through comparative sequence analysis. Here, two groups of OMP variants, OmpL1 and Lsa49 across five pathogenic genomospecies were selected via phylogenetic analysis to evaluate their functional binding diversities towards various host components. OmpL1 exhibited significant binding variation against various host components, compared to Lsa49. The diversity is strongly correlated with variations on predicted OmpL1 surface-exposed loops contributing to functional loss and gain via molecular evolution, which resulted in binding preferential towards specific host molecules. Immunological evaluation of cattle sera showed that these OMPs are expressed and exposed to the host immune system, and had a strong association against one another. This suggests that these OMPs may have similar structural epitopes that allow antibody binding, and indicates conserved immunogenicity across species. In a final study, we investigated whether the ruminant gastrointestinal (GI) tract was a carriage site for Leptospira. Through PCR surveys of ruminant gingival and rectal tissues, the presence of leptospires was identified as extremely low, thus indicating the GI tract does not appear to be an important leptospire carriage site. In conclusion, here we have identified several novel bovine leptospire OMPs, which may be useful vaccine or diagnostic components for bovine leptospirosis in the future. Additionally, the functional diversity between leptospiral OMP variants identifies OMP genetic evolution resulting in addition or loss of binding function, highlighting the complex host-pathogen interaction of leptospirosis. Lastly, this study does not suggest a role for the ruminant GI tract in leptospire carriage, indicating disease transmission through this route is unlikely.