Surface?proteomics?of?Leptospira?interrogans?serovar?Pomona

Abstract

Leptospirosis is a re-emerging zoonosis of global distribution including Thailand. It caused by pathogenic Leptospira. The pathogenesis is not clear. Surface-exposed proteins (PSEs) are first part for interactions with host cells, immune system and should be potential targets for vaccine development. To date, many researches study only individual PSEs but the studies of surface proteomic is less. The study of surface proteomic will be important information for pathogenesis studies and search for vaccine candidate. This study aimed to identify surface proteomics of pathogenic Leptospira by surface biotinylation and surface shaving using proteinase K that has been used to identify surface proteins in several bacteria combined with Liquid chromatography tandem-mass spectrometry (LC-MS/MS). Intact leptospiral proteins were labeled with biotin (Sulfo-NHS-SS-Biotin). Biotinylated proteins were purified though avidin column before further identification by LC-MS/MS. For surface shaving, Intact leptospiral proteins were treated with proteinase K (ProK) at optimal concentration. The supernatant (shaved protein) were identified by LC-MS/MS. The membrane integrity of leptospires were investigated during experiment in both methods by fluorescence and western blot of periplasmic protein FlaA1 and outer membrane protein OmpL1 or surface-exposed protein OmpL47 in eluted protein sample from biotinylation and in pellet cells of shaving. Proteins were predicted localization by subcellular localization tools including PSORTb, CELLO and SOSUI-GramN. These tools predicted as total 510 outer membrane proteins (OMPs) including 214 OMPs from both of surface biotinylation and shaving method, 66 of which were identified as hypothetical proteins, 222 OMPs from biotinylation only and 74 OMPs from surface shaving method. 8 OMPs. All replicate experiments of both methods identified 8 proteins in common including LipL71 (LIC11003), DUF3383 domain-containing protein/Phage-related protein (LIC12615), LipL45 (LIC11643), LolA outer membrane lipoprotein carrier protein (LIC12545), LipL41 (LIC12966), cheA1 chemotaxis protein histidine kinase-like kinase (LIC13522), FlaA-1 flagellar filament sheath protein (LIC10788), and conserved hypothetical protein (LIC10175). In addition, known PSEs including OmpL1, OmpL47, OmpL37, OmpL41, LipL71 and LigA were found in this study. Therefore, OMPs were found by surface biotinylation and surface shaving. Therefore, this study obtained surface-OMPs information of Leptospira interrogans serovar Pomona. There were at least 66 hypothetical proteins identified as putative surface-exposed OMPs. These proteins are interesting targets to be confirmed as PSEs and further study on their roles in pathogenic leptospires.