Heterogeneous expression of Pil3 pilus is critical for Streptococcus gallolyticus translocation across polarized colonic epithelial monolayers

ABSTRACT Streptococcus gallolyticus subspecies gallolyticus ( Sgg ) is an opportunistic pathogen responsible for septicaemia and endocarditis in elderly persons. Sgg is also a commensal of the human gastrointestinal tract. Here we demonstrate that Sgg strain UCN34 translocates across tight intestinal barriers in vitro in a Pil3-dependent manner. Confocal images of UCN34 passage across human colonic cells reveals that Sgg utilizes a paracellular pathway. Pil3 was previously shown to be expressed heterogeneously and WT UCN34 consists of about 90% of Pil3 low and 10% of Pil3 high cells. We found that both the Δ pil3 mutant and the Pil3+ overexpressing variant could not translocate across Caco-2 and T84 barriers. Interestingly, combining live Δ pil3 mutant cells with fixed Pil3+ variants in a 10:1 ratio (mimicking UCN34 WT population) allowed efficient translocation of the Δ pil3 mutant. These experiments demonstrate that heterogeneous expression of Pil3 plays a key role in optimal translocation of Sgg across the intestinal barrier. ABSTRACT IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus ( Sgg ) is an opportunistic pathogen responsible for septicemia and infective endocarditis in elderly persons. Sgg is a commensal of the rumen of herbivores and transmission to humans most probably occurs through the oral route. In this work, we have studied how this bacterium crosses the intestinal barrier using well-known in vitro models. Confocal microscopy images revealed that Sgg UCN34 can traverse the epithelial monolayer in between adjacent cells. We next showed that passage of Sgg from the apical to the basolateral compartment is dependent on the heterogenous expression of the Pil3 pilus at the bacterial surface. We hypothesize that Pil3 high cocci adhere firmly to epithelial cells to activate transient opening of tight junctions thereby allowing the traversal of Pil3l ow bacteria.