Assessment of Listeria monocytogenes Surface Proteins Identified from Proteomics Analysis for Use as Diagnostic Biomarkers

摘要

The Gram-positive bacterium Listeria monocytogenes is an important pathogen that causes a foodborne illness with a high percentage of fatalities. Surface proteins, specifically expressed from a wide range of L. monocytogenes serotypes under selective enrichment culture conditions, can serve as targets for the detection and isolation of this pathogen using antibody-based methods. Among a number of surface proteins identified by mass spectrometry in a previous proteomic study, six candidates (annotated as LMOf2365_0148, LMOf2365_0312, LMOf2365_0546, LMOf2365_1883, LMOf2365_2111, and LMOf2365_2742) were selected here for investigating their expression in the bacterial cells cultured in vitro by raising rabbit polyclonal antibodies (PAbs) against the recombinant form of each candidate. These protein candidates contained regions conserved among various L. monocytogenes isolates but variable in other Listeria species. LMOf2365_0148, an uncharacterized protein with a LPXTG motif accountable for covalent linkage to the cell wall peptidoglycan, exhibited a strong reaction signal from anti-LMOf2365_0148 PAb binding to the cell surface, as detected by immunofluorescence microscopy. Further study, through the generation of a panel of mouse monoclonal antibodies (MAbs) to the recombinant LMOf2365_0148, showed that one of the MAbs, M3686, reacted to bacterial isolates belonging to all three lineages of L. monocytogenes under Health Canada's standard enrichment culture conditions (MFHPB-07 and MFHPB-30). These results demonstrated the potential of using LMOf2365_0148 as a surface biomarker, in conjunction with specific MAbs developed here, for the isolation and detection of L. monocytogenes from foods and food processing environments. IMPORTANCE Strains of Listeria monocytogenes are differentiated serologically into at least 13 serotypes and grouped phylogenetically into 4 distinct lineages (I, II, III, and IV). No single monoclonal antibody (MAb) reported to date is capable of binding to the surface of L. monocytogenes strains representing all the serotypes. This study assessed the expression of six surface proteins selected from a previous proteomic study and demonstrated that surface protein LMOf2365_0148 has the greatest potential as a surface biomarker. A panel of 24 MAbs to LMOf2365_0148 were assessed extensively, revealing that one of the MAbs, M3686, reacted to a wide range of L. monocytogenes isolates (lineage I, II, and III isolates) grown under standard enrichment culture conditions and thus led to the conclusion that LMOf2365_0148 is a useful novel surface biomarker for identifying, detecting, and isolating the pathogen from food and environmental samples. Strains of Listeria monocytogenes are differentiated serologically into at least 13 serotypes and grouped phylogenetically into 4 distinct lineages (I, II, III, and IV). No single monoclonal antibody (MAb) reported to date is capable of binding to the surface of L. monocytogenes strains representing all the serotypes.