Structure–Function Aspects of PstS in Multi-Drug–Resistant Pseudomonas aeruginosa

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Abstract
The increasing prevalence of multi-drug–resistant (MDR) strains of Pseudomonas aeruginosa among critically ill humans is of significant concern. In the current study, we show that MDR clinical isolates of P. aeruginosa representing three distinct genotypes that display high virulence against intestinal epithelial cells, form novel appendage-like structures on their cell surfaces. These appendages contain PstS, an extracellular phosphate binding protein. Using anti-PstS antibodies, we determined that the PstS-rich appendages in MDR strains are involved in adherence to and disruption of the integrity of cultured intestinal epithelial cell monolayers. The outer surface–expressed PstS protein was also identified to be present in P. aeruginosa MPAO1, although to a lesser degree, and its role in conferring an adhesive and barrier disruptive phenotype against intestinal epithelial cells was confirmed using an isogenic ΔPstS mutant. Formation of the PstS rich appendages was induced during phosphate limitation and completely suppressed in phosphate-rich media. Injection of MDR strains directly into the intestinal tract of surgically injured mice, a known model of phosphate limitation, caused high mortality rates (60%–100%). Repletion of intestinal phosphate in this model completely prevented mortality. Finally, significantly less outer surface PstS was observed in the MPAO1 mutant ΔHxcR thus establishing a role for the alternative type II secretion system Hxc in outer surface PstS expression. Gene expression analysis performed by RT-PCR confirmed this finding and further demonstrated abundant expression of pstS analogous to pa5369, pstS analogous to pa0688/pa14–55410, and hxcX in MDR strains. Taken together, these studies provide evidence that outer surface PstS expression confers a highly virulent phenotype of MDR isolates against the intestinal epithelium that alters their adhesive and barrier disrupting properties against the intestinal epithelium. The resistance of bacteria to multiple antibiotics is a major problem in critically ill patients who often become colonized by highly lethal pathogens such as Pseudomonas aeruginosa. During the course of critical illness, as many as 50% of patients' intestinal tracts become colonized with P. aeruginosa, with as many as 30% of strains being resistant to multiple antibiotics. Concomitantly, critical illness is characterized by acute depletion of phosphate, which itself has been shown to be an independent predictor of infection-related mortality. In the present study we determined that during low phosphate conditions, highly virulent multi-antibiotic–resistant strains of P. aeruginosa isolated from critically ill patients produce an abundance of the phosphate-binding protein, PstS, located on extracellular finger-like structures. These PstS rich appendages participate in the binding of P. aeruginosa to intestinal lining cells and may allow P. aeruginosa to acquire phosphate from its host while remaining at arm's length from the host immune system. This clever tactic may be one example by which successful opportunistic pathogens such as P. aeruginosa survive within complex ecological niches such as the intestinal tract and harm their hosts during the course of critical illness.

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