Differential expression of InlB and ActA in Listeria monocytogenes in selective and nonselective enrichment broths

Abstract

Aim: To investigate the effect of selective and nonselective media on the expression of ActA and InlB proteins in Listeria monocytogenes. Methods and Results: Polyclonal antibodies to InlB and ActA were used in western blotting to determine the effect of selective (BLEB, UVM, and FB) or nonselective (BHI and LB) enrichment broths or hotdog exudates. Of the 13 L. monocytogenes serotypes tested, 11 and 12 serotypes showed a strong InlB expression in brain heart infusion (BHI) and Luria‐Bertani (LB), respectively, while only seven and one serotypes showed a strong ActA expression in these two respective broths, and others showed a weaker or no expression. On the contrary, in selective broths, expression of InlB was either very weak or undetectable. However, ActA expression was stronger in 12 serotypes when grown in buffered Listeria enrichment broth (BLEB), 11 in University of Vermont medium (UVM), and 10 in Fraser broth (FB). When tested in hotdog exudates, InlB and ActA were detected in serotypes grown at 37°C but not at 4°C. Transmission electron microscopy, enzyme‐linked immunosorbent assay, and mRNA analysis further supported these observations. Conclusion: Overall, selective enrichment broths promote ActA while nonselective broths promote InlB expression. Significance and Impact of the study: As commonly recommended enrichment broths show differential InlB and ActA expression, proper media must be selected to avoid false results during antibody‐based detection of L. monocytogenes.

Association of ActA to Peptidoglycan Revealed by Cell Wall Proteomics of Intracellular Listeria monocytogenes

“…It is therefore possible that a low number of ActA molecules, hardly detectable by Western analysis, may exist in the membrane of bacteria growing in BHI medium. This postulate agrees with observations reported by other authors (37). Likewise, the ⌬actA mutant was highly impaired in the ability to promote invasion of epithelial and fibroblast cells when bacteria were grown in IMM, a condition in which a fraction of ActA molecules associates with peptidoglycan.…”

“…However, we also reasoned that varied interactions of ActA with cell wall components could take place when bacteria grow in markedly different environmental conditions such as the cytosol of eukaryotic cells and laboratory media. Of note, several authors have reported the production in laboratory media of multiple ActA-derived breakdown products resulting from cleavage at defined sites (36,37). Considering this fact, we assessed whether the ActA polypeptide identified by proteomics could display a different envelope association compared with ActA molecules present either in the extracellular milieu or in the membrane but not bound to the peptidoglycan.…”

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.…”

“…Most surface proteins used as targets for antibody-based methods for L. monocytogenes detection are virulence factors regulated by PrfA (15). Although virulence factors are specific to L. monocytogenes, expression of these factors in in vitro culture is highly variable (10,16,17), most likely due to their adapted expression under intracellular conditions (18,19). Antibody-based assays with specificity for L. monocytogenes have been developed (20)(21)(22); however, the antigen targets recognized by these antibodies are unknown and may be shared with other Listeria species and other non-Listeria foodborne bacteria (21,22).…”

“…Finally, the surface protein candidate should be expressed by all L. monocytogenes serotypes under standard enrichment culture conditions. Certain surface antigens previously examined may not have been suitable for L. monocytogenes detection due to the lack of expression under enrichment culture conditions (10)(11)(12), weak association with the cell surface (13), or lack of conservation in some serotypes (12,14). Most surface proteins used as targets for antibody-based methods for L. monocytogenes detection are virulence factors regulated by PrfA (15).…”