Physiological Temperature and Osmotic Changes Drive Dynamic Proteome Alterations in the Leptospiral Outer Membrane and Enhance Protein Export Systems

Leptospirosis, a remerging zoonosis, has no effective
vaccine or
an unambiguous early diagnostic reagent. Proteins differentially expressed
(DE) under pathogenic conditions will be useful candidates for antileptospiral
measures. We employed a multipronged approach comprising high-resolution
TMT-labeled LC-MS/MS-based proteome analysis coupled with bioinformatics
on leptospiral proteins following Triton X-114 subcellular fractionation
of leptospires treated under physiological temperature and osmolarity
that mimic infection. Although there were significant changes in the
DE proteins at the level of the entire cell, there were notable changes
in proteins at the subcellular level, particularly on the outer membrane
(OM), that show the significance of subcellular proteome analysis.
The detergent-enriched proteins, representing outer membrane proteins
(OMPs), exhibited a dynamic nature and upregulation under various
physiological conditions. It was found that pathogenic proteins showed
a higher proportion of upregulation compared to the nonpathogenic
proteins in the OM. Further analysis identified 17 virulent proteins
exclusively upregulated in the outer membrane during infection that
could be useful for vaccine and diagnostic targets. The DE proteins
may aid in metabolic adaptation and are enriched in pathways related
to signal transduction and antibiotic biosynthesis. Many upregulated
proteins belong to protein export systems such as SEC translocase,
T2SSs, and T1SSs, indicating their sequential participation in protein
transport to the outer leaflet of the OM. Further studies on OM-localized
proteins may shed light on the pathogenesis of leptospirosis and serve
as the basis for effective countermeasures.