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

Abstract

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.

Unique Posttranslational Modification Sites of Acetylation, Citrullination, Glutarylation, and Phosphorylation Are Found to Be Specific to the Proteins Partitioned in the Triton X-114 Fractions of Leptospira

“…The LC-MS/MS data of Triton X-114 fractionated proteins of Leptospira submitted to PRIDE PXD016204 7 and PXD026044 17 were analyzed using Proteome Discoverer 2.4. The reassigned spectra are available in the Proteome Xchange Consortium ( ) via the PRIDE partner repository under the dataset identifier PXD030370.…”

“… 23 Our previous results showed a quantitative difference in protein content across subcellular locations in Leptospira . 7 , 17 , 24 The active form of protein can be assessed with the type of modification, subcellular location, coexpressed molecules, interacting molecules, and the environment. Hence, identifying the PTM content of a protein at the subcellular level is more beneficial to finding the role and characteristics of the protein than the PTMs identified from the total protein extract.…”

“…The beads were recovered from the supernatant and repacked in the ProteoMiner column and washed with 100 μL of PBS. Finally, elution was performed using a 2 × 20 μL elution reagent (8 M urea, 2% CHAPS). , …”

“… 7 , 16 − 18 Our earlier study analyzed Triton X-114 fractionated leptospiral proteins, showing a quantitative difference in the distribution across subcellular locations. 7 , 17 This report comprises the analysis of proteomic data and identification of PTMs, their distribution across the subcellular fractions that can lead to a better understanding of the protein function, and their stability in subcellular locations.…”

“…The Triton X-114 fractions aqueous, detergent, pellet, wash, and supernatant represent subcellular locations such as cytoplasmic and periplasmic proteins, outer membrane proteins, inner membrane proteins, surface, and secretory proteins, respectively. ,− Our earlier study analyzed Triton X-114 fractionated leptospiral proteins, showing a quantitative difference in the distribution across subcellular locations. , This report comprises the analysis of proteomic data and identification of PTMs, their distribution across the subcellular fractions that can lead to a better understanding of the protein function, and their stability in subcellular locations.…”