Selective Enrichment Media Affect the Antibody-Based Detection of Stress-Exposed Listeria monocytogenes due to Differential Expression of Antibody-Reactive Antigens Identified by Protein Sequencing

Abstract

Selective enrichment broths are frequently used to recover stressed Listeria cells to detectable levels, but the ability of antibodies to detect these cells from various commonly used enrichment media is unknown. In this study, a polyclonal (PAb) and monoclonal (MAb) antibody were used to examine the variation in antigen expression on healthy or stress-recovered Listeria monocytogenes cells grown in brain heart infusion broth, buffered Listeria enrichment broth (BLEB), Listeria repair broth (LRB), University of Vermont medium (UVM), and Fraser broth (FB) for immunodetection. Indirect enzyme-linked immunosorbent assay (ELISA) data showed that L. monocytogenes subjected to stresses (acid, cold, heat, and salt) and then grown in BLEB gave the highest reaction with the anti-Listeria PAb while those grown in LRB gave the highest reaction with the MAb C11E9. Cells grown in UVM and FB gave poor ELISA values with both antibodies. Western blotting with PAb revealed differential expression of surface proteins of 62, 58, 50, 43, and 30 kDa on L. monocytogenes cells, with most proteins displaying elevated expression in BLEB and LRB but reduced or no expression in UVM or FB. Similar differential expressions were noticed for C11E9. PAb-reactive proteins were identified as putative LPXTG-motif cell-wall anchor-domain protein (62 kDa; lmo0610), flavocytochrome C fumarate reductase chain A homolog protein (58 kDa; lmo0355), enolase (50 kDa; lmo2455), glyceraldehyde 3-phosphate dehydrogenase (43 kDa; lmo2459), and hypothetical phospho-sugar binding protein (30 kDa; lmo0041), respectively, and the MAb-reactive 66-kDa protein was confirmed to be N-acetylmuramidase (lmo2691). In conclusion, BLEB and LRB favorably supported increased expression of antigens and proved to be superior to UVM and FB for immunodetection of stressed L. monocytogenes cells.