The key role of Pseudomonas aeruginosa PA-I lectin on experimental gut-derived sepsis

Abstract

Objective: To examine the effect of Pseudomonas aeruginosa on intestinal barrier function and its lethal potential when introduced into the intestinal tract of mice.

Summary background data: The mere presence of P. aeruginosa in the intestinal tract of critically ill patients is associated with a threefold increase in death compared with matched cohorts without this pathogen. Whether this effect is a cause or a consequence of the critically ill state has not been previously addressed.

Methods: Transepithelial electrical resistance, a measure of tight junction permeability, was evaluated in Caco-2 intestinal epithelial cells cells apically inoculated with live P. aeruginosa, exotoxin A, or purified PA-I lectin, an adhesin of P. aeruginosa. Lethality studies to P. aeruginosa were carried out in mice undergoing 30% surgical hepatectomy by injecting the bacteria or its various components directly into the cecum.

Results: Only cells exposed to P. aeruginosa or its PA-I lectin developed alterations in barrier function. P. aeruginosa or the combination of PA-I and exotoxin A was lethal to mice when injected into the cecum after partial hepatectomy. Alterations in epithelial barrier function and death in mice were prevented when Pseudomonas was pretreated with N-acetyl D-galactosamine (GalNAc), a binder of PA-I.

Conclusions: P. aeruginosa may act as a pathogen in the gastrointestinal tract, resulting in altered epithelial barrier function and death in a susceptible host. The PA-I lectin of P. aeruginosa may play a key role in its pathogenicity to the intestinal epithelium by inducing a permeability defect to its cytotoxic exoproducts such as exotoxin A.

Figure 1. Percentage change in transepithelial resistance of Caco-2 epithelial monolayers exposed to Pseudomonas aeruginosa or its components at 2 and 4 hours after exposure. Data are means ± standard error of the mean of triplicate cultures (n = 7) at the time point specified. Dose-response data are mean ± standard error of the mean of duplicate cultures (n = 7) of resistance measured at 4 hours after exposure. Data demonstrate that both live and killed P. aeruginosa alter transepithelial resistance in a dose- and time-dependent manner. PA-I altered transepithelial resistance equal to that of whole bacteria; exotoxin A had no effect.

Figure 2. (Small graph) Percentage adherence of Pseudomonas aeruginosa (10⁷ cfu/mL) to dispersed Caco-2 epithelial monolayers after 45 minutes of incubation at 4°C. Data are mean ± standard error of the mean of duplicate cultures (n = 7). (Large graph) Percentage change in transepithelial resistance of Caco-2 epithelial monolayers exposed to P. aeruginosa or its PA-I lectin. Experiments were performed in the absence and presence of GalNAc, a specific binder of PA-I, or dextran, a nonspecific inhibitor of P. aeruginosa adherence to epithelial cells and an epithelial membrane stabilizer. Data are mean ± standard error of the mean of triplicate cultures displayed at the 4-hour time point. As seen with other intestinal pathogens, approximately 1–2% of the initial inoculum of bacteria adhere to dispersed epithelial cells (see the small graph). Experiments with blocking compounds demonstrate that P. aeruginosa adherence to and alteration of the transepithelial resistance of Caco-2 cells are attenuated by the presence of either GalNAc or dextran. However, only GalNAc attenuated the decrease in resistance induced by PA-I. (Right Graph) [³H]-mannitol flux (nmol/h · cm²) across Caco-2 epithelial monolayers at 4 hours after incubation with P. aeruginosa or its PA-I lectin in the absence or presence of GalNAc or dextran. Data are mean ± standard error of the mean of triplicate cultures (n = 7). The apical exposure of either live P. aeruginosa or PA-I resulted in an increase in mannitol flux across the epithelial monolayer. Both GalNAc and dextran attenuated this effect.

Figure 3. Western blot analysis of the effect of live Pseudomonas aeruginosa or PA-I on the tight junctional proteins ZO-1, ZO-2, and occludin. Live P. aeruginosa was pretreated with 2% GalNAc. Dose and time responses of occludin after exposure to live P. aeruginosa was assessed. Heat shock protein (HSP-72) was also measured as a positive control. Results suggest that P. aeruginosa alters both ZO1 and occludin in Caco-2 cells. That this effect is mediated by its PA-I lectin is suggested by experiments using PA-I alone and by blocking experiments with GalNAc, where attenuation of the effect is seen. As with functional experiments, this effect appears to be both dose- and time-dependent

Figure 4. Death rate of mice subjected to sham laparotomy or 30% surgical hepatectomy and receiving direct injection into the cecum of live cultures of Pseudomonas aeruginosa, saline, or virulence components. Death is defined as the percentage of mice dead at 48 hours after bacterial exposure. Results demonstrate that cecal injection of greater than 10⁵ cfu/mL of P. aeruginosa results in a significant (90%) death rate after a 30% surgical hepatectomy. Only the combination of PA-I and exotoxin A induced death in mice; this was greater for mice after hepatectomy (*P < .001). Dose-response experiments and the lack of death with exotoxin A alone suggest that PA-I is a key element in this response.

Figure 5. Death rate of mice after 30% hepatectomy and injection of either live P. aeruginosa or the combination of PA-I and exotoxin A in the presence and absence of 13% GalNAc or 15% dextran. Results demonstrate that although both GalNAc and dextran appeared to protect against death in this model, only GalNAc resulted in a statistically significant and complete prevention of death in mice injected intracecally with either live P. aeruginosa or the combination of PA-I and exotoxin A (*P < .001).