Triton X‐114 Fractionated Subcellular Proteome of Leptospira interrogans Shows Selective Enrichment of Pathogenic and Outer Membrane Proteins in the Detergent Fraction

Abstract

The Triton X-114-based solubilization and temperature-dependent phase separation of proteins is used for subcellular fractionation where, aqueous, detergent, and pellet fractions represents cytoplasmic, outer membrane (OM), and inner membrane proteins, respectively. Mass spectrometry-based proteomic analysis of Triton X-114 fractions of proteomic analysis of Leptospira interrogans identified 2957 unique proteins distributed across the fractions. The results are compared with bioinformatics predictions on their subcellular localization and pathogenic nature. Analysis of the distribution of proteins across the Triton X-114 fractions with the predicted characteristics is performed based on "number" of unique type of proteins, and "quantity" which represents the amount of unique protein. The highest number of predicted outer membrane proteins (OMPs) and pathogenic proteins are found in aqueous and pellet fractions, whereas detergent fraction representing the OM has the highest quantity of OMPs and pathogenic proteins though lower in number than the aqueous and pellet fractions. This leaves the possibility of an upsurge in pathogenic proteins and OMPs on the OM under pathogenic conditions suggesting their potential use to combat leptospirosis. Further, the Triton X-114 subcellular fractions are more correlated to enrichment of pathogenic proteins predicted by MP3 software than predicted localization.

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

“…LC-MS/MS analysis of TritonX-114 fractions of L. interrogans serovar Copenhageni strain Fiorcruz L1-130 and subsequent identification yielded 2425 proteins in aqueous fractions, 2646 protein in detergents, and 1684 proteins in a pellet that comprised 2957 unique proteins reported under the ProteomeXchange dataset identifiers PXD009050 and PXD016204 [ 22 ]. The analysis of wash and supernatant identified 837 proteins in wash 851 proteins in the supernatant comprising 1176 unique proteins ( Figure 1 A).…”

“…The surface proteins were extracted in PBS containing 5 mM MgCl2 that was easily detachable and also had the unique presence of six proteins without shedding out into the supernatant, which showed its sufficiently good binding on the surface of Leptospira . It is also worth noting that 57% of the extracellular proteins found were pathogenic in nature, as was predicted by MP3 tool with a 27.9% rate (826 pathogenic proteins out of the 2957 proteins identified) in the whole proteome of Leptospira [ 22 ]. Even though some of these proteins were found in lower quantities, the possibility of upregulation of the proteins under pathogenic conditions cannot be ignored.…”

“…The Leptospira pellet obtained after wash was used for Triton X-114 fractionation as described earlier [ 22 ]. The extraction buffer containing 10 mM Tris (pH 8) carrying 1% Triton X-114 and 150 mM NaCl at 4 °C at the rate of 1 mL of extraction buffer per amount of pellet derived from a 25 mL mid-log phase culture was used for extraction.…”

“…In this context, a multipronged approach coupled with the enrichment of secretory proteins from culture supernatant, its analysis using high throughput Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) based proteomics, and the use of bioinformatics tools for the selection of candidate proteins from the supernatant of Leptospira culture in complete EMJH medium was attempted. Previously, Triton X-114 fractionation coupled with LC-MS/MS-based proteomics to determine OMPs of L. interrogans [ 22 ] was used to compare the cellular portion with the extracellular proteins from the wash and supernatant of the culture identified through LC-MS/MS in this study. This article describes the enrichment of secretory proteins from the EMJH culture supernatant and surface proteins from the wash fraction and the proteomic and bioinformatics analysis of the results.…”

Surface proteomics and label-free quantification of Leptospira interrogans serovar Pomona

“…The conventional proteomics approach using two-dimensional gel electrophoresis (2DE) coupled with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) [ 10 , 43 ], mainly identifies abundant proteins but inefficiently identifies highly hydrophobic or membrane proteins. In more recent studies, LC-MS/MS, a high-throughput and high-resolution method, was used to identify leptospiral membrane proteins from the samples of subcellular fractionation using Triton X-114 [ 44 , 45 ]. Moreover, LC-MS/MS-based studies and isotope labeling couple revealed protein abundance of leptospires [ 46 ].…”