Differential inlA and inlB expression and interaction with human intestinal and liver cells by Listeria monocytogenes strains of different origins

Abstract

In this study, a number of Listeria monocytogenes strains of different origins were evaluated for in vitro invasion capacity for various human cell types (monocytic THP-1, enterocytic Caco-2, and hepatocytic HepG2 cells) and for expression levels of specific virulence genes. For THP-1 cells, no differences between clinical and nonclinical L. monocytogenes strains in invasion capacity or in production of the proinflammatory cytokine interleukin-8 (IL-8) were observed, whereas for the Caco-2 and HepG2 cells, significant differences in invasion capacity were noticed. On average, the clinical strains showed a significantly lower invasion capacity than the nonclinical L. monocytogenes strains. Furthermore, it was shown that the clinical strains induce lower IL-8 levels in HepG2 cells than do the nonclinical strains. This observation led us to study the mRNA expression levels of inlA, inlB, and ami, important virulence genes mediating adhesion and invasion of eukaryotic cells, by real-time reverse transcription-PCR for 27 clinical and 37 nonclinical L. monocytogenes strains. Significant differences in inlA and inlB expression were observed, with clinical strains showing a lower expression level than nonclinical strains. These observations were in accordance with in vitro invasion of Caco-2 and HepG2 cells, respectively. The results of this study indicate that differential expression levels of inlA and inlB possibly play a role in the virulence capacities of L. monocytogenes strains. The lower capacity of clinical strains to invade HepG2 cells and to induce IL-8 is possibly a mechanism of immune evasion used by specific L. monocytogenes strains.

FIG. 1.

Assays of invasion by L. monocytogenes strains of different origins (clinical and nonclinical strains) into three human cell lines, monocytic THP-1 cells (upper panel), enterocytic Caco-2 cells (middle panel), and hepatocytic HepG2 cells (lower panel). Results are expressed as mean log₁₀ CFU/ml (plus standard deviations) for three independent experiments analyzed in triplicate. Numbers of intracellular bacteria were determined after 1 h. •, strains with a truncated, nonfunctional InlA protein; C, clinical strains; NC, nonclinical strains.

FIG. 2.

IL-8 production after infection of human monocytic THP-1 cells (upper panel) and human hepatocytic HepG2 cells (lower panel) with L. monocytogenes strains of different origins (clinical and nonclinical strains). IL-8 was detected by ELISA. Mean values (plus standard deviations) for three independent experiments carried out in triplicate are shown. C, clinical strains; NC, nonclinical strains; −, negative control; LPS, lipopolysaccharide (positive control).

FIG. 3.

inlA, inlB, and ami mRNA expression levels in the late logarithmic growth phase of clinical and nonclinical L. monocytogenes strains. The bars represent inlA, inlB, and ami mRNA expression levels (plus standard deviations) relative to tufA (for inlA) or to three housekeeping genes (rpoD, tufA, and the 16S rRNA gene) (for inlB and ami). n, number of experiments; ▴, strains that are underestimated in relative inlA expression level because of the presence of additional polymorphisms in the primer-binding site of the reverse primer.