one-step-extraction-and-zymographic-analysis-of-bacterial-gelatinases

One-step Extraction and Zymographic Analysis of Bacterial Gelatinases

Proteinases of pathogenic bacteria contribute to invasion and pathogenesis by actively degrading the host's extracellular matrix (ECM) components and ...

enriching-subcellular-proteins-leptospira-using-triton-x-114-based

Enriching Subcellular Proteins in <em>Leptospira</em> Using a Triton X-114-Based Fractionation Approach

Prokaryotes lack precise subcellular organelles but exhibit distinct regions such as cytoplasm, inner membrane, periplasm, and outer membrane, where most ...

ICMR Regional Medical Research Centre, Port Blair

ICMR - Regional Medical Research Centre

Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira. Early and accurate diagnosis is the prime step in managing the disease. Secretory proteins of Leptospira remain distinguished for diagnosis due to their availability as soluble proteins in the serum and their interaction with the host immune response due to their extracellular presence. This study presents the cloning, expression, purification, and characterization of imelysin or LruB (LIC_10713), a putative leptospiral protein. We report that the localization of imelysin showed its presence in the inner membrane and in the culture supernatant. The imelysin was upregulated under in vitro physiological conditions of infection. The LIC_10713 interacted significantly with laminin, fibronectin, collagen type I, and collagen type IV in a dose-dependent manner. Phylogenetic analysis showed that LIC_10713 is predominately found in the pathogenic species of Leptospira, and the GxHxxE motif of imelysin-like proteins is represented as the amino acid sequence GHE. Also, immunoglobulins in leptospirosis-infected patients recognize recombinant-LIC_10713 with 100% specificity and 90.9% sensitivity. The secretion nature, abundance, upregulation, binding to ECM components, and immunogenicity determine LIC_10713 as an important molecule that can be used as an anti-leptospirosis measure. KEY POINTS: • The imelysin-like protein (LIC_10713) of Leptospira is a secretory protein • The protein LIC_10713 can bind ECM molecules • The LIC_10713 is mainly found in pathogenic leptospires • The anti-LIC_10713 antibody from human serum can detect the r-LIC_10713.

Leptospiral imelysin (LIC_10713) is secretory, immunogenic and binds to laminin, fibronectin, and collagen IV.

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.

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

Leptospirosis is one of the re-emerging zoonotic diseases, especially in tropical regions. Many antibiotics are used to treat leptospirosis, but there are no scientific evidence-based guidelines or systematic clinical trials for using these drugs. A bioinformatics approach was made to shortlist some Food and Drug Administration (FDA) of the United States of America-approved and currently used drugs for leptospirosis. The existing drugs from the Drug Bank database, which are currently not used for leptospirosis, were selected to identify their target proteins and binding sites using bioinformatics methods. Orthologues of these target proteins were selected from the proteome database of . The similar sites and their interactions with the drugs were validated and recommended for use in leptospirosis. Further, the sensitivity of recommended drugs was also validated in vitro. The sequences and structures of these proteins were compared under strictly controlled parameters and shortlisted Gatifloxacin, Imipenem, Latamoxef, Doripenem, Tigecycline, and Lactams as repurposable drugs for leptospirosis. An in vitro validation of the drugs showed significant antileptospiral activity in 12 serovars with low IC concentrations and also showed that the IC values varied across  serovars. Further, suitable proteins under the concept of "One Target, Many Drugs" identified DNA gyrase subunit A (Q72WD1), 30S ribosomal protein S9 (Q72U99), and 30S ribosomal protein S12 (Q72UA6), and these proteins were found across the pathogenic, saprophytic, and intermediate species of . We describe a method to find repurposable drugs from the approved list that are not currently used to treat leptospirosis and validate them to be taken forward for systematic clinical trials specific to leptospirosis for recommendations in clinical use.

ArticleTotal : 2	Year
Publication title	2025
Publication title Cited by 1	2025

Article	Year
Leptospiral imelysin (LIC_10713) is secretory, immunogenic and binds to laminin, fibronectin, and collagen IV. Applied microbiology and biotechnology\| PubMed ID: 37227474	2023
Physiological Temperature and Osmotic Changes Drive Dynamic Proteome Alterations in the Leptospiral Outer Membrane and Enhance Protein Export Systems. Journal of proteome research\| PubMed ID: 37877620	2023
A New Beginning to the Existing Medicines; Repurposing FDA-Approved Drugs for the Neglected Re-Emerging Disease Leptospirosis. ACS omega\| PubMed ID: 39100284	2024

Suneetha Hariharan

Biography

JoVE Journal Publications

Other Publications