Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies.

Proteomic approach and expression analysis revealed the differential expression of predicted leptospiral proteases capable of ECM degradation.

Leptospira, the causative agent of leptospirosis is known to have many proteases with potential to degrade extracellular matrix. However, a multipronged approach to identify, classify, characterize and elucidate their role has not been attempted. Our proteomic approach using high-resolution LC-MS/MS analysis of Triton X-114 fractions of Leptospira interrogans resulted in the identification of 104 proteases out of 130 proteases predicted by MEROPS. In Leptospira approximately 3.5% of the genome complements for proteases, which include catalytic types of metallo-, serine-, cysteine-, aspartic-, threonine- and asparagine- peptidases. Comparison of proteases from different serovars revealed that M04, M09B, M14A, M75, M28A, A01 and U73 protease families are exclusively present in pathogenic form. The M23 and S33 protease families are represented with >14 members in Leptospira. The differential expression under physiological temperature (37 °C) and osmolarity (300 mOsM) showed that proteases belonging to the catalytic type of Metallo-peptidases are upregulated significantly in pathogenic conditions. In silico prediction and characterization of the proteases revealed that several proteases are membrane anchored and secretory, classical as well as non-classical system. The study demonstrates the diversity and complexity of proteases, while maintaining conservation across the serovars in Leptospira and their differential expression under pathogenic conditions.

Extracellular Proteome Analysis Shows the Abundance of Histidine Kinase Sensor Protein, DNA Helicase, Putative Lipoprotein Containing Peptidase M75 Domain and Peptidase C39 Domain Protein in Leptospira interrogans Grown in EMJH Medium.

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.