Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier

Abstract

BackgroundListeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown.MethodsAmounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells.ResultsWestern blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates.ConclusionThis study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate.

Identification of genes essential for anaerobic growth of Listeria monocytogenes

“…It has already been shown that anaerobic pre-culture of Listeria monocytogenes enhances adhesion in in vitro cell culture assays and virulence in vivo in the guinea pig model (Bo Andersen et al, 2007). Anaerobic induction of InlB (inlB, lmo0433, log 2 RTL 1.3), involved in adherence to (Lindén et al, 2008) and invasion in (Pentecost et al, 2010) intestinal tissue, and LAP (a bifunctional enzyme, also with metabolic capability, lmo1634, described previously in the text as adh, log 2 RTL 1.4), involved in adherence (Burkholder et al, 2009) and paracellular translocation (Burkholder & Bhunia, 2010;Kim & Bhunia, 2013), was already described previously (Burkholder et al, 2009;Stritzker et al, 2004Stritzker et al, , 2005. In addition, we observed anaerobically an upregulation of lmo0971-lmo0973, encoding the Dlt proteins involved in Dalanine esterification of lipoteichoic acid and wall teichoic acid (dltD, dltC and dltB, log 2 1.2, 1.3 and 1.5).…”

Virulence Gene-Associated Mutant Bacterial Colonies Generate Differentiating Two-Dimensional Laser Scatter Fingerprints

“…In our previous study, it was demonstrated that three L. monocytogenes strains (i.e., H4, F4244, and F4262) of serotype 4b express various levels of LAP and exhibit variable adhesion and pathogenesis in a cell culture model (49). In the present study, these strains also generated differentiating scatter patterns on both BHIA and LBA media.…”

“…Western blotting and ELISA confirmed the high LAP production of the H4 strain (Fig. 8d) (49). This variation in the amount of protein synthesis may be one of the factors that contributed to the differences observed in the scatter patterns of the serotype 4b strains.…”

“…Previously, we demonstrated that strains expressing various levels of LAP exhibited variable adhesion and virulence properties in a cell culture model (49). Here we examined the colony scatter patterns of three L. monocytogenes strains, i.e., H4, F4244, and F4262, with differential LAP expression.…”

Listeria monocytogenes remodels the cell surface in the blood‐stage

“…These proteins were mostly cytosolic proteins previously documented as ‘moonlighting’ proteins (Henderson and Martin, ). Among these, we identified metabolic enzymes that do not have a typical signal peptide and proteins lacking a cell wall anchoring motif: pyruvate dehydrogenase (Lmo1053); 6‐phosphogluconate dehydrogenase (Lmo1376); alcohol‐acetaldehyde dehydrogenase (Lmo1634), also known as Lap for Listeria adhesion protein (Kim and Bhunia, ); glucose‐6‐phosphate isomerase (Lmo2367); triose phosphate isomerase (Lmo2457); a putative phosphoglycerate kinase (Lmo2458); and, fructose‐1,6‐bisphosphate aldolase (Lmo2556) (Table ). Some of these enzymes have been proposed to have more than one biological function in other pathogens, including virulence implications (Henderson and Martin, ; Henderson, ).…”