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

Abstract

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.

Quercetin and Apigenin of Cymbopogon citratus mediate inhibition of HCT-116 and PC-3 cell cycle progression and ameliorate Doxorubicin-induced testicular dysfunction in male rats

“…Taken together, our investigation supports the idea that Apigenin may confer a wide range of natural and chemopreventive effects on cells by influencing various cell cycle progression pathways or apoptotic cascades [44]. Hung et al [45] stated that Quercetin has a synergetic chemopreventive efficacy, when combined with Kaempferol, via the arrest of HCT-116 cells due to inhibition of DNA synthesis. As for the cascade pathway, Quercetin induces loss of HCT-116 viability and subsequently supports the structure/activity of 3'-OH moiety and/or 4'-OH moiety.…”

Immunoproteome profiling of Listeria monocytogenes under mild acid and salt stress conditions

“…Only in C2, a specific histidine kinase with a MW of 100 kDa was found [36]. The flagellar motor switch (FliM) protein (MW 38 kDa) was identified only in C3, together with lmo0723 (MW 66 kDa), codified by the gene lmo0723 [37, 38]. In C4, the proteins identified were: PdhB (MW 35 kDa), very similar to pyruvate dehydrogenase (PDH, E1 b‐subunit), thioredoxin reductase (MW 34 kDa), and lmo0132 (MW 55 kDa), which is involved in the process of DNA repair and maintenance (Table 1) [39–41].…”

A comprehensive investigation of protein expression profiles in L. monocytogenes exposed to thermal abuse, mild acid, and salt stress conditions

“…Under the optimal growth condition (C1-opt), surface proteins that contribute to L. monocytogenes host cell adhesion clustered together. Lmo1068 is involved in attachment and biofilm formation (Tiong and Muriana, 2016 ), Lmo0723 in chemotaxis, and Lmo2713 is upregulated by the σ B factor during host cell invasion (Neuhaus et al, 2013 ; Piercey et al, 2016 ). These proteins are also visualized in cluster 3 (C3), while Iap protein was identified in all studied conditions along with autolysin protein (Lmo1076), muramidase protein MurA ( lmo2691 ) (Tiong and Muriana, 2016 ), and Lmo0129, which is involved in the host gastrointestinal tract colonization (Desvaux et al, 2010 ).…”