Leptospirosis is a re-emerging form of zoonosis that is caused by the spirochete pathogen Leptospira. Extracellular proteins play critical roles in the pathogenicity and survival of this pathogen in the host and environment. Extraction and analysis of extracellular proteins is a difficult task due to the abundance of enrichments like serum and bovine serum albumin in the culture medium, as is distinguishing them from the cellular proteins that may reach the analyte during extraction. In this study, extracellular proteins were separated as secretory proteins from the culture supernatant and surface proteins were separated during the washing of the cell pellet. The proteins identified were sorted based on the proportion of the cellular fractions and the extracellular fractions. The results showed the identification of 56 extracellular proteins, out of which 19 were exclusively extracellular. For those proteins, the difference in quantity with respect to their presence within the cell was found to be up to 1770-fold. Further, bioinformatics analysis elucidated characteristics and functions of the identified proteins. Orthologs of extracellular proteins in various Leptospira species were found to be closely related among different pathogenic forms. In addition to the identification of extracellular proteins, this study put forward a method for the extraction and identification of extracellular proteins.

Posttranslational modifications (PTMs) are decisive factors in the structure, function, and localization of proteins in prokaryotic and eukaryotic organisms. However, prokaryotic organisms lack subcellular organelles, and protein localization based on subcellular locations like cytoplasm, inner membrane, periplasm, and outer membrane can be accounted for functional characterization. We have identified 131 acetylated, 1182 citrullinated, 72 glutarylated, 5 palmitoylated, and 139 phosphorylated proteins from Triton X-114 fractionated proteins of Leptospira, the pathogen of re-emerging zoonotic disease leptospirosis. In total, 74.7% of proteins were found exclusively in different Triton X-114 fractions. Additionally, 21.9% of proteins in multiple fractions had one or more PTM specific to different Triton X-114 fractions. Altogether, 96.6% of proteins showed exclusiveness to different Triton X-114 fractions either due to the presence of the entire protein or with a specific PTM type or position. Further, the PTM distribution within Triton X-114 fractions showed higher acetylation in aqueous, glutarylation in detergent, phosphorylation in pellet, and citrullination in wash fractions representing cytoplasmic, outer membrane, inner membrane, and extracellular locations, respectively. Identification of PTMs in proteins with respect to the subcellular localization will help to characterize candidate proteins before developing novel drugs and vaccines rationally to combat leptospirosis.

Leptospirosis, a re-emerging zoonotic disease, is challenging human and animal health due to the lack of early and rapid diagnostic tools and effective vaccines. The exoproteome of the pathogen expressed under pathogenic conditions possesses a rational diagnostic significance due to its consistent presence in body fluids. were challenged to conditions simulating infection using physiological temperatures and osmolarity. Using state-of-the-art extraction techniques, efficient enrichment of nonabundant proteins, and high-resolution LC-MS/MS, we identified 1575 exoproteins from both the pathogen surface and culture supernatant. The results indicate a significant upregulation of 155 exoproteins, of which 41 were predicted to have moonlighting properties, 35 were identified as adhesins, and several proteins were components of the type 2 secretion system (T2SS). Additionally, 10 proteins showed extracellular matrix (ECM) binding properties, out of which 4 orthologs were found using the T2SS. The overall characteristics of upregulated proteins show that they can help Leptospira establish infection, invasion, and protection from the host defense, thereby providing new insights into the pathogen to confront the host via an increased energy level, secretion system, and host ECM binding molecules. Furthermore, the study suggests potential candidates for efficient antileptospiral countermeasures.