Pseudomonas aeruginosa Expresses a Lethal Virulence Determinant, the PA-I Lectin/Adhesin, in the Intestinal Tract of a Stressed Host: The Role of Epithelia Cell Contact and Molecules of the Quorum Sensing Signaling System

Gut-derived sepsis is a term used to describe a state of systemic inflammation with organ dysfunction hypothesized to be initiated and perpetuated by the intestinal tract microflora. This process is observed to be a life-threatening problem following clinical disorders such as burn injury,1 neonatal enterocolitis,2 severe neutropenia,3 inflammatory bowel disease, and following acute rejection of intestinal graft transplantation.4 We have developed a unique model of gut-derived sepsis whereby Pseudomonas aeruginosa is introduced directly into the cecum of surgically stressed mice.5 P. aeruginosa was chosen to model gut-derived sepsis based on the observation that hospitalized patients have a high prevalence of this organism in their stool following exposure to antibiotics and by the observation that the persistence of P. aeruginosa in the feces of catabolically stressed patients is associated with a high mortality rate.6 We induced catabolic stress in mice by performing a 30% surgical hepatectomy. At the time of the hepatectomy, P. aeruginosa was introduced into the intestinal tract by direct puncture of the cecum. This model resulted in a mortality rate of nearly 100%, whereas sham-operated controls injected with identical strains of P. aeruginosa completely recovered. In this model there was equal translocation and bacteremia between control and hepatectomy mice. This finding, coupled with the observation that systemic injection of P. aeruginosa resulted in no mortality, suggested that the lethal effect of this organism in the intestinal tract might require in vivo virulence activation locally to effectuate a systemic response.5,7,8 Continuous work in our laboratory with P. aeruginosa has identified that a key virulence determinant, the PA-I lectin/adhesin, facilitates the adherence of P. aeruginosa to the intestinal epithelium and causes a major defect in the intestinal epithelial barrier to 1 of its potent cytotoxins, exotoxin A.5,9 The PA-I lectin/adhesin may be up-regulated in response to local environmental factors that are unique to the intestinal tract of a stressed host, thereby explaining why control animals survive whereas stressed animal do not. Local factors within the intestinal tract of a stressed host, which affect bacterial growth rates, colony pattern formation, exposure to the intestinal epithelium, etc, might play an important role in virulence transformation among opportunistic pathogens such as P. aeruginosa. Virulence gene expression in bacteria is a complex process that is dictated by multiple factors present within the local microenvironment.10 Bacterial contact with the intestinal epithelium, a result of stress-induced mucosal immune dysfunction, itself could induce virulence gene expression in intestinal bacteria. In addition, the expression of multiple virulence genes in P. aeruginosa could be further enhanced by activation of its quorum sensing signaling system, a system by which individual bacteria sense changes in their population density and respond when a critical mass is present, presumably that amount necessary to overcome the host.11 Therefore, understanding the response of certain virulence genes to changes in bacterial growth rate, colony formation patterns, exposure to quorum sensing molecules, and contact to their target cells, such as the intestinal epithelium, should provide important insight into aspects of in vivo virulence transformation for the PA-I lectin of P. aeruginosa that have not been previously addressed. Therefore, the aims of the present study were to build on our previous work on the effect of P. aeruginosa on the intestinal epithelial barrier by first determining whether bacterial-epithelial responses could be reproduced in additional human cultured intestinal epithelial cells not previously reported. We next sought to characterize the interaction between P. aeruginosa and the intestinal epithelium by determining the effects of bacterial growth phase, epithelial cell contact, and the exposure of butanoyl homoserine lactone (C4-HSL), a quorum sensing signaling molecule known to affect various extracellular virulence factors in P. aeruginosa, on PA-I expression in P. aeruginosa. In vivo experiments were then designed to determine whether additional soluble virulence factors in P. aeruginosa could be identified that might also mediate the lethal effect of P. aeruginosa in the intestinal tract of mice. We also determined whether surgical stress itself up-regulated the virulence of intestinal P. aeruginosa by analyzing isolated strains from the cecum of stressed mice for up-regulated PA-I mRNA. Finally, we analyzed various environmental and clinical strains of P. aeruginosa for the presence of the PA-I gene to determine the clinical relevance of PA-I in critically ill patients.