RT-qPCR Analysis of 15 Genes Encoding Putative Surface Proteins Involved in Adherence of Listeria monocytogenes

摘要

L. monocytogenes adherence to food-associated abiotic surfaces and the development of biofilms as one of the underlying reasons for the contamination of ready-to-eat products is well known. The over-expression of internalins that improves adherence has been noted in cells growing as attached cells or at elevated incubation temperatures. However, the role of other internalin-independent surface proteins as adhesins has been uncharacterized to date. Using two strains each of weakly- and strongly-adherent L. monocytogenes as platforms for temperature-dependent adherence assays and targeted mRNA analyses, these observations (i.e., sessile- and/or temperature-dependent gene expression) were further investigated. Microplate fluorescence assays of both surface-adherent strains exhibited significant ( P < 0.05) adherence at higher incubation temperature (42 °C). Of the 15 genes selected for RT-qPCR, at least ten gene transcripts recovered from cells (weakly-adherent strain CW35, strongly-adherent strain 99-38) subject to various growth conditions were over expressed [planktonic/30 °C (10), sessile/30 °C (12), planktonic/42 °C (10)] compared to their internal control (16SrRNA transcripts). Of four genes overexpressed in all three conditions tested, three and one were implicated as virulence factors and unknown function, respectively. PCR analysis of six unexpressed genes revealed that CW35 possessed an altered genome. The results suggest the presence of other internalin-independent adhesins (induced by growth temperature and/or substratum) and that a group of suspect protein members are worthy of further analysis for their potential role as surface adhesins. Analysis of the molecular basis of adherence properties of isolates of L. monocytogenes from food-associated facilities may help identify sanitation regimens to prevent cell attachment and biofilm formation on abiotic surfaces that could play a role in reducing foodborne illness resulting from Listeria biofilms.