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.

Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

“…These results revealed that the three selected MAbs reacted more strongly to lineage I isolates than lineage II isolates grown in enrichment cultures, according to methods described in Health Canada's Compendium of Analytical Methods. Unlike previous studies of potential diagnostic surface proteins which reported reduced protein expression under specific conditions (10,11,36,37), the stable expression of LMOf2365_0639 in enrichment culture makes it a suitable candidate biomarker.…”

“…Geng et al previously developed PAbs against four L. monocytogenes proteins, one of which was Lmo0610, the homologue of LMOf2365_0639 (36). Their study, which utilized Western blotting, revealed weak expression of Lmo0610 in L. monocytogenes V7 (serotype 1/2a) cultured in Fraser, UVM, and BHI broths.…”

Actin Polymerization Drives Septation of Listeria monocytogenes namA Hydrolase Mutants, Demonstrating Host Correction of a Bacterial Defect

“…Several previously reported studies classified NamA with primary functions closely associated with the bacterial surface based on the presence of its LysM peptidoglycan binding domains (3,6,7,10), its detection at the bacterial surface (8,52), and its ability to serve as an immunoreactive epitope in L. monocytogenes detection studies (19,20,44). Other studies, however, have detected NamA in supernatant fractions derived from L. monocytogenes strains containing a mutationally activated form of the virulence regulator PrfA [PrfA(L140F)] as well as in wild-type supernatants in comparison to secA2 mutants (33,50), although those studies did not determine if secreted NamA was functionally active.…”

“…Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes at 1.0 A (constant) for 1 h. All membranes were blocked for 1 h in PBS (Gibco, Carlsbad, CA) containing 0.05% Tween (PBST) and 5% milk. Membranes were incubated with a 1:2,500 dilution of monoclonal antibody C11E9 in PBST (a kind gift of Arun Bhunia), which is known to specifically recognize NamA (19), or a 1:500 dilution of monoclonal antibody against internalin A (Toxin Technologies, Sarasota, FL) at 4°C overnight with shaking, followed by three additional washes with 30 ml of PBST for 10 min each. A 1:2,500 dilution of secondary goat anti-mouse antibody conjugated to alkaline phosphatase (Southern Biotech, Birmingham, AL) was incubated with shaking for 2 h at room temperature, followed by three additional 10-min washes in PBST.…”

“…NamA was initially identified as a variably expressed immunogenic epitope of L. monocytogenes recognized by monoclonal antibodies at the bacterial surface (19,20,44). NamA contributes to cell division, as mutants lacking namA form bacterial chains during exponential-phase growth in broth culture (10).…”