Gut-derived sepsis occurs when the right pathogen with the right virulence genes meets the right host: evidence for in vivo virulence expression in Pseudomonas aeruginosa - PubMed (original) (raw)

Gut-derived sepsis occurs when the right pathogen with the right virulence genes meets the right host: evidence for in vivo virulence expression in Pseudomonas aeruginosa

J Alverdy et al. Ann Surg. 2000 Oct.

Abstract

Objective: To define the putative role of the PA-I lectin/adhesin, a binding protein of Pseudomonas aeruginosa, on lethal gut-derived sepsis after surgical stress, and to determine if this protein is expressed in vivo in response to physical and chemical changes in the local microenvironment of the intestinal tract after surgical stress.

Summary background data: Previous work from the authors' laboratory has established that lethal gut-derived sepsis can be induced after the introduction of P. aeruginosa into the cecum of mice after a 30% hepatectomy. This effect does not occur when P. aeruginosa is introduced into the cecum of sham operated control mice. Previous experiments further established that the mechanism of this effect is due to the presence of the PA-I lectin/adhesin of P. aeruginosa, which induces a permeability defect to a lethal cytotoxin of P. aeruginosa, exotoxin A.

Methods: Three strains of P. aeruginosa, one lacking functional PA-I, were tested in two complementary systems to assess virulence. Strains were tested for their ability to adhere to and alter the permeability of cultured human colon epithelial cells, and for their ability to induce mortality when injected into the cecum of mice after a 30% hepatectomy. To determine if PA-I is "in vivo expressed" when present in the cecal environment after hepatectomy, strains were retrieved from the cecum of sham-operated and hepatectomy-treated mice 24 and 48 hours after their introduction into the cecum and their PA-I expression was assessed.

Results: Results indicated that PA-I plays a putative role in lethal gut-derived sepsis in the mouse, because strains lacking functional PA-I had an attenuated effect on cultured human epithelial cells, and were nonlethal when injected into the cecum of mice after 30% surgical hepatectomy. Furthermore, surgical stress in the form of hepatectomy significantly altered the intestinal microenvironment, resulting in an increase in luminal norepinephrine associated with an increase in PA-I expression in retrieved strains of P. aeruginosa. Co-incubation of P. aeruginosa with norepinephrine increased PA-I expression in vitro, suggesting that norepinephrine plays a role in the observed response in vivo.

Conclusions: Lethal gut-derived sepsis may occur when intestinal pathogens express virulence determinants in response to environmental signals indicating host stress. In this regard, the PA-I lectin/adhesin of P. aeruginosa appears to be a specific example of in vivo virulence expression in colonizing pathogens in the intestinal tract in response to surgical stress.

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Figures

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Figure 1. Effect of hepatectomy and starvation on pH, redox state, and norepinephrine concentration in the cecum of mice. Mice in the control group underwent sham laparotomy and were allowed access to chow and water ad libitum (Control/fed). Mice in the hepatectomy group underwent a 30% surgical hepatectomy and were allowed water ad libitum only (Hepatectomy/starved). Results demonstrate that a statistically significant increase in pH, redox state, and norepinephrine concentration was observed in cecal contents of mice after hepatectomy and 48 hours of starvation (*P < .001). Norepinephrine concentration was increased in the cecal contents only, while cecal tissue levels were not statistically different between groups (data not shown). The small graph represents the cecal tissue levels of dopamine, the immediate precursor of norepinephrine, between groups. A statistically significant decrease (*P < .001) in dopamine was observed in mice after hepatectomy, suggesting that the elevated luminal concentration of norepinephrine is derived from the cecal tissues.

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Figure 2. Characterization of strains of Pseudomonas aeruginosa for PA-I concentration and their effects on the barrier function of Caco-2 cells and mouse mortality after introduction into mouse cecum after a 30% hepatectomy. Strain 33347 demonstrated a high degree of PA-I expression by both Western blot analysis and erythrocyte agglutination activity. None of the strains had surface expressed PA-I, as assessed by erythrocyte agglutination activity using whole bacterial cells. PA-I mRNA was not detectable in 33347–66. PA-I mRNA correlated to protein expression in 33347 and 27853. The PA-I negative strain ATCC 33347–66 (mutant) had an attenuated effect on the adherence to Caco-2 cells and did not decrease transepithelial electrical resistance to the same degree as PA-I positive strains (*P < .001). The PA-I negative strain (33347–66) was completely nonlethal when introduced into the cecum of mice after hepatectomy.

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Figure 3. Extracellular virulence factor profile of strains of Pseudomonas aeruginosa. Extracellular virulence factor concentration is expressed in relative scale from + to +++++. Strains differed significantly in extracellular virulence factor concentrations. While strain 33347–66 had a considerable increase in the amount of exotoxin A compared to 33347, it did not induce mortality in mice (see Fig. 2).

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Figure 4. Erythrocyte agglutination assays of the various strains of Pseudomonas aeruginosa exposed to norepinephrine in vitro or harvested from the cecum of sham-operated control or 30% surgical hepatectomy mice. Results demonstrate that while neither 27853 nor 33347 had functionally (surface) expressed PA-I, both strains expressed PA-I when coincubated with 0.01% norepinephrine. This effect was GalNAc-inhibitable. Strains were harvested and assayed for PA-I 24 and 48 hours after cecal injection of strain 27853 into the cecum of sham operated control and 30% surgical hepatectomy. Only strains harvested from the cecum of mice undergoing 30% surgical hepatectomy displayed evidence of functionally expressed PA-I. The agglutination titer of PA-I after 48 hours in the mouse cecum after hepatectomy (1:80) was higher than its total PA-I titer at baseline in vitro (1:20; see Fig. 2), suggesting that the cecal environment after 30% hepatectomy may increases the synthesis as well as the surface expression of PA-I as a result of the in vivo condition.

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