The Adherent/Invasive Escherichia coli Strain LF82 Invades and Persists in Human Prostate Cell Line RWPE-1, Activating a Strong Inflammatory Response

Abstract

Adherent/invasive Escherichia coli (AIEC) strains have recently been receiving increased attention because they are more prevalent and persistent in the intestine of Crohn's disease (CD) patients than in healthy subjects. Since AIEC strains show a high percentage of similarity to extraintestinal pathogenic E. coli (ExPEC), neonatal meningitis-associated E. coli (NMEC), and uropathogenic E. coli (UPEC) strains, here we compared AIEC strain LF82 with a UPEC isolate (strain EC73) to assess whether LF82 would be able to infect prostate cells as an extraintestinal target. The virulence phenotypes of both strains were determined by using the RWPE-1 prostate cell line. The results obtained indicated that LF82 and EC73 are able to adhere to, invade, and survive within prostate epithelial cells. Invasion was confirmed by immunofluorescence and electron microscopy. Moreover, cytochalasin D and colchicine strongly inhibited bacterial uptake of both strains, indicating the involvement of actin microfilaments and microtubules in host cell invasion. Moreover, both strains belong to phylogenetic group B2 and are strong biofilm producers. In silico analysis reveals that LF82 shares with UPEC strains several virulence factors: namely, type 1 pili, the group II capsule, the vacuolating autotransporter toxin, four iron uptake systems, and the pathogenic island (PAI). Furthermore, compared to EC73, LF82 induces in RWPE-1 cells a marked increase of phosphorylation of mitogen-activated protein kinases (MAPKs) and of NF-κB already by 5 min postinfection, thus inducing a strong inflammatory response. Our in vitro data support the hypothesis that AIEC strains might play a role in prostatitis, and, by exploiting host-cell signaling pathways controlling the innate immune response, likely facilitate bacterial multiplication and dissemination within the male genitourinary tract.

FIG 1

LF82 and EC73 adhesion (A), invasion (B), and ability to survive within infected cells (C) and role of host cell actin polymerization and microtubule in the invasion process (D). Data are expressed as means ± standard deviations from at least three independent experiments performed in triplicate. Asterisks indicate statistically significant differences at P ≤ 0.05 by ANOVA with post hoc unpaired (*) or paired (**) Student's t test. ND, not determined.

FIG 2

Immunofluorescence staining of RWPE-1 cell monolayers 24 h postinfection. (A) LF82; (B) EC73; (C) noninvasive control strain MG1655. Intracellular bacteria stained green, while extracellular bacteria appear orange/yellow. Magnification, 400×.

FIG 3

Light and electron microscopy of Epon-embedded RWPE-1 cells infected with LF82 (C and D) or EC73 (A and B) for 24 h. Groups of bacteria are frequently observed to occupy cytoplasmic vacuoles, suggesting active proliferation and/or clustering. (A and C) Light micrograph of toluidine blue-stained thin sections. Arrows indicate cells with groups of bacteria within cytoplasmic compartments. (B and D) Electron micrographs of ultrathin sections. Bars: A and C, 8 μm; B and D, 0.9 μm.

FIG 4

Phosphorylation of MAPKs and of the NF-κB p65 subunit induced by LF82 and EC73 in RWPE-1 cells. Shown are representative Western blots of cells infected for various lengths of time with different E. coli strains, as indicated (A, B, C and D). CC, uninfected cells. Stripped membranes were reprobed using antibodies anti-ERK1/2, anti-p38, anti-JNK1/2, and anti-p65 to control protein loading. The levels of phosphorylated proteins were quantified by densitometry (ImageJ software) and calculated as the ratios of phosphorylated to total kinases and phosphorylated to total p65. Data are expressed as arbitrary units (A.U.) and are means ± standard deviations from at least three independent experiments, in duplicate. Asterisks indicate statistically significant difference at P ≤ 0.05 by one-way ANOVA with post hoc unpaired Student's t test.

FIG 5

Levels of release of cytokines IL-6 and IL-8 in RWPE-1 cell monolayers infected with the E. coli LF82, EC73, and MG1655 strains (MOI of 10). Cytokines were measured 3 and 24 h postinfection by ELISA. Ctrl, control. Data are expressed as means ± standard deviations from at least three independent experiments. **, statistically significant difference at P ≤ 0.05 by repeated-measures ANOVA with post hoc paired Student's t test.