Extracellular Signals of a Human Epithelial Colorectal Adenocarcinoma (Caco-2) Cell Line Facilitate the Penetration of Pseudomonas aeruginosa PAO1 Strain through the Mucin Layer

Abstract

Pseudomonas aeruginosa can penetrate the layer of mucus formed by host intestinal epithelial cells, often resulting in sepsis in immunocompromised patients. We have previously demonstrated that P. aeruginosa can penetrate the mucin layer by flagellar motility and the degradation of the mucin layer. However, it remains unclear how P. aeruginosa initially recognizes epithelial cells. Using the human epithelial colorectal adenocarcinoma (Caco-2) cell line, we investigated extracellular signaling that could facilitate the penetration of P. aeruginosa through the mucin layer. The supernatant from Caco-2 cell cultures increased penetration of P. aeruginosa through an artificial mucin layer. The Caco-2 cell supernatant increased bacterial flagella-dependent swarming motility, but it did not influence P. aeruginosa growth or protease activity. Filtering of the Caco-2 cell supernatant indicated that proteins weighing <10 kDa increased mucin penetration, swarming motility, and, based on a tethered cell assay, induced acceleration of the flagellar filament rotational rate. Furthermore, a capillary assay showed that <10 kDa proteins in the Caco-2 cell supernatant attracted P. aeruginosa cells. Finally, we identified that growth-regulated oncogene-α (GRO-α) secreted by Caco-2 cells was a factor facilitating flagellar filament rotation and swarming motility, although it did not attract the bacteria. We conclude that penetration of the mucin layer by P. aeruginosa is facilitated by small proteins (<10 kDa) secreted by Caco-2 cells, both by inducing acceleration of flagellar motility and increasing chemotaxis.

Sulfated vizantin suppresses mucin layer penetration dependent on the flagella motility of Pseudomonas aeruginosa PAO1

“…aeruginosa colonies in the bottom chamber exposed to sulfated vizantin (1 and 10 μM) as compared with untreated cells ( Fig 1A ; P > 0.05). As previously reported [ 8 , 22 ], deletion of the fliC gene significantly decreased the number of P . aeruginosa colonies in the bottom chamber ( Fig 1A ; P < 0.05).…”

“…We previously demonstrated that P . aeruginosa PAO1 penetrates the mucin layer by flagellar motility and a mucin degrading protease [ 8 , 22 ]. In the present study, sulfated vizantin (100 μM, 3 h incubation) did not significantly decrease bacterial protease activity ( Fig 2A ; P > 0.05), although protease inhibitors significantly decreased bacterial protease activity in P .…”

“…We performed a mucin penetration assay, as described previously [ 8 , 22 ]. Mucin chambers were prepared by adding 50 μL of 5% bovine submaxillary mucin (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco’s modified Eagle medium (DMEM) with high glucose (Sigma-Aldrich) to 0.143-cm 2 membranes of Transwell inserts (3.0-μm pore filter; Corning, Corning, NY, USA).…”

“…Furthermore, we previously demonstrated that P . aeruginosa PAO1 can penetrate the mucin layer through flagellar motility and a mucin degrading protease [ 8 , 22 ]. These findings indicate that compounds attenuating P .…”

Evolutionary conservation of the antimicrobial function of mucus: a first defence against infection

“… 122 A recent in vitro study, using the human colorectal adenocarcinoma cell line Caco-2, demonstrated that unidentified low molecular weight proteins present in the supernatant of Caco-2 cultures, also attracted P. aeruginosa in capillary chemotaxis assays and increased bacterial motility and mucin penetration (bovine submaxillary mucin). 123 Thus, there is growing evidence to suggest there is a link between production of chemoattractant molecules, motility of pathogens and their ability to colonise mucosal layers.…”