An anti-pyruvate kinase monoclonal antibody and translocated intimin receptor (tir) for specific detection of listeria species and shiga-toxigenic escherichia coli
Abstract
Foodborne illnesses pose a significant health concern and economic impact worldwide. In this study, we aimed at developing alternate and improved methods for Shiga toxigenic E. coli (STEC) and Listeria species detection. In Listeria monocytogenes, an auxiliary secretory system, SecA2, plays an important role in translocating virulence and housekeeping proteins to cell surface to aid bacteria to maintain saprophytic and intracellular life styles. Here we investigated if pyruvate kinase (PyK), present in both pathogenic and nonpathogenic Listeria, is translocated by SecA2 system and determined its potential application in immunologic detection of these bacteria. Additionally, cell surface localization and enzymatic activity of PyK were examined. Enzyme immunoassay with anti-PyK antibody, MAb EM-7H10, indicated the presence of PyK in all Listeria species except L. roquortiae. Immunofluorescence assay confirmed surface localization. Analysis of L. monocytogenes ΔsecA2 mutant revealed the absence of PyK in cell wall and the supernatant fractions along with reduced levels in the intracellular fraction indicating that PyK translocation to cell surface is SecA2-dependent. Reverse transcriptase PCR (RT-PCR) confirmed reduced levels of PyK transcript in the ΔsecA2 mutant indicating SecA2-dependent regulation of pyk. Furthermore, PyK expression was found to be 10-fold higher in L. monocytogenes cultured in Brain-Heart Infusion Broth (BHI), Tryptic Soy Broth (TSB) and buffered Listeria enrichment broth (BLEB) than in University of Vermont medium (UVM) or Fraser Broth (FB). In summary, PyK is determined to be a SecA2-dependent surface displayed glycolytic enzyme present in both pathogenic and nonpathogenic Listeria, which could serve as a strong immunologic target for Listeria species detection. Shiga toxigenic E. coli (STEC) has been implicated in several foodborne outbreaks exhibiting severe hemolytic uremic syndrome (HUS) and fatalities. Here, we focus on a novel approach for STEC detection. Translocated Intimin Receptor (TIR) binds exclusively with intimin, a STEC adhesion protein which mediates intimate attachment of the bacteria to the host cell. This receptor-ligand system is unique to STEC and can be used for its detection on biosensor platforms. Collectively, data provide strong evidence for the use of anti-PyK antibody and TIR and for specific detection of Listeria species and STEC, respectively.
Degree
M.S.
Advisors
BHUNIA, Purdue University.
Subject Area
Food Science
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