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.

Bacterial Biofilm and its Role in the Pathogenesis of Disease

“…UPEC strains can also invade bladder epithelial cells, proliferate and form matrix-enclosed biofilm-like structures that has been referred to as intracellular bacterial communities (IBC) [210,211]. Interestingly, the invasive capacity of different UPEC strains into the prostate cell line RWPE-1 correlated with both the ability to adhere to epithelial cells and the capacity to form biofilm on a plastic surface, in vitro [208] and similar results were observed with an adherent/invasive E. coli (AIEC) strain, which is highly similar to UPEC, isolated from an ileal lesion in a Crohn's disease patient [212]. It is therefore possible, but to our knowledge has not been demonstrated, that bacteria can persist in the prostate using a similar strategy.…”

A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria

“…To determine if the ICW assay could represent a reliable technique for the quantification of intracellular bacteria, gentamicin protection assays were performed using either the M90 T or the LF82 strains. Upon entering host epithelial cells, strain M90 T displays a high and fast replication rate and the ability to spread into adjacent cells [29] while strain LF82 is well adapted to persist within host cells [30]. Quantification of intracellular bacteria was performed using fixed MOIs in time course infection experiments.…”

“…After invasion, S. flexneri multiplies and spreads rapidly in cell monolayers, irrespective from the MOI used [29]. Conversely, E. coli , as a typical persister, needs at least 24 h to reach an appreciable number of intracellular bacteria [30]. Therefore, the most common MOIs used (MOI 100 for S. flexneri and 10 for E. coli ) were applied for our experiments.…”

“…Hela cell monolayers were infected with strains M90 T and LF82 at MOIs of 100 and 10, respectively, and centrifuged at 2000× g , 10 min, 37 °C. After incubation, 45 min for M90 T and 2 h for strain LF82, monolayers were extensively washed with phosphate-buffered saline (PBS) and further incubated with cell culture medium supplemented with 100 μg/ml gentamicin to kill extracellular bacteria [30, 37]. Three different time points were selected for intracellular bacteria quantification: 1, 2, and 4 HPI for strain M90 T and 24, 48, and 72 HPI for strain LF82.…”

Oligomannose-Rich Membranes of Dying Intestinal Epithelial Cells Promote Host Colonization by Adherent-Invasive E. coli

“…They use the same protein surface appendages, namely, type 1 fimbriae with oligomannose-specific lectin FimH at their tips (Martinez et al, 2000 ; Kline et al, 2009 ), making them highly invasive. For the fimH gene, the prototypic E. coli strains LF82 and UTI89 are classified in the same phylogenetic group (Miquel et al, 2010 ; Conte et al, 2016 ) known as AIEC, which was used in this work.…”