88. Low Intestinal Phosphate Following Surgical Injury Directly Activates the Virulence of P. Aeruginosa Causing Lethal Gut Derived Sepsis

Abstract

Introduction: Severe hypophosphatemia often develops following surgical injury and independently predicts postoperative sepsis-induced mortality. Here we explored the possibility that opportunistic pathogens directly sense low phosphate (Pi) conditions as a signal of host injury and activate their virulence circuitry to cause lethal gut-derived sepsis. Methods: Mice underwent a 30% hepatectomy (Hep) and simultaneous injection of P. aeruginosa (P.a) into the cecum to create phosphate depletion and lethal gut-derived sepsis. Groups of these mice were supplemented with high dose phosphate either orally or systemically (IV, IP). To determine if phosphate concentration directly activates the virulence of P. aeruginosa, we examined the expression of a key determinant that causes gut-derived sepsis in mice, the PA-I lectin, in low phosphate media as well as other key virulence factors including pyocyanin, a redox active compound, PstS a phosphate scavenging protein, and biofilm. Finally to determine if the phosphate acquiring protein PstS is expressed in vivo in this model indicating that P. a is actively “sensing” low phosphate, P. a was recovered 24 hrs post-cecum injection and PstS expression measured by RT-PCR from freshly isolated bacterial RNA. Results: A 30% surgical hepatectomy resulted in low intestinal phosphate concentration despite normal serum phosphate (control = 0.61 ± 0.12 vs. Hep + P.a.= 0.21 ± 0.11 mg/g, p=.006). As previously shown, a 30% hepatectomy + injection of P.a. Into the cecum resulted in high mortality (60%) that was PA-I dependent since the mutant strain Δ PA-I lacking PA-I failed to induce mortality in this model. Oral phosphate repletion restored and exceeded baseline intestinal phosphate levels (Pi=0.89±0.18) and significantly protected mice against mortality (figure 1A) while systemic administration (IV or IP) neither restored intestinal phosphate (not shown) nor protected against mortality. Complementary in vitro experiments demonstrated that low phosphate conditions, similar to those observed in vivo, resulted in a significant (p=0.001) increase in the expression of key virulence products of P. aeruginosa including the PA-I lectin, biofilm, pyocyanin, and PstS (figure 1B). Conversely, high phosphate conditions in vitro suppressed P. aeruginosa virulence expression even when stimulated by host stress compounds known to activate its virulence (data not shown). Finally PstS gene expression, the phosphate scavenging protein, was 4.5 fold higher in P.a in the cecum following hepatectomy vs. control (n=3/group p=0.005) demonstrating that P.a “senses” local phosphate depletion during surgical injury.