Testing the efficacy and toxicity of adenylyl cyclase inhibitors against enteric pathogens using in vitro and in vivo models of infection

Abstract

Enterotoxigenic Escherichia coli (ETEC) produces the ADP-ribosyltransferase toxin known as heat-labile enterotoxin (LT). In addition to the toxic effect of LT resulting in increases of cyclic AMP (cAMP) and disturbance of cellular metabolic processes, this toxin promotes bacterial adherence to intestinal epithelial cells (A. M. Johnson, R. S. Kaushik, D. H. Francis, J. M. Fleckenstein, and P. R. Hardwidge, J. Bacteriol. 191:178-186, 2009). Therefore, we hypothesized that the identification of a compound that inhibits the activity of the toxin would have a suppressive effect on the ETEC colonization capabilities. Using in vivo and in vitro approaches, we present evidence demonstrating that a fluorenone-based compound, DC5, which inhibits the accumulation of cAMP in intoxicated cultured cells, significantly decreases the colonization abilities of adenylyl cyclase toxin-producing bacteria, such as ETEC. These findings established that DC5 is a potent inhibitor both of toxin-induced cAMP accumulation and of ETEC adherence to epithelial cells. Thus, DC5 may be a promising compound for treatment of diarrhea caused by ETEC and other adenylyl cyclase toxin-producing bacteria.

FIG. 1.

In vitro cell-based cAMP assay. (A) Bioassay with different concentrations of PGE₂-imidazole and DC5 demonstrating that these compounds inhibited cAMP production induced by edema toxin (EdTx) on RAW 264.7 cells, with IC₅₀s of 14.1 and 8.99, respectively. Controls were medium alone or with the addition of 100 μM each individual compound tested (protective antigen [PA] or edema factor [EF]). (B) DC5 effect on cAMP production by cells treated with CT (equivalent to LT, IC₅₀ of 2.04).

FIG. 2.

Bacterial growth in LB medium in the presence or absence of DC5. The different bacterial cultures were monitored spectrophotometrically for a period of 6 h. (A) ETEC H10407, EPEC E2348/69, EHEC 86-24. (B) ETEC DEC7A, EPEC E2348/69, S. Typhimurium, EAEC O42.

FIG. 3.

Establishing the optimal DC5 dose. (A) Increase in fluid (estimated by weight of the intestine) was reduced in the presence of DC5. P values (t test): *, <0.05 (comparing DMSO plus PBS control with ETEC treatment); **, <0.05 (ETEC treated with ETEC plus 1 mM DC5). (B) The DC5 treatment had an impact on the amount of bacteria (ETEC) recovered from the intestinal lumen at 72 h, particularly at 0.1 mM DC5. P values (t test): *, <0.01 (comparing ETEC treatment with ETEC plus 0.1 mM DC5 or ETEC plus 1 mM DC5). Each group included eight mice.

FIG. 4.

Comparing the effects of DC5 and PGE₂-imidazole. (A) Reduction in the weight of the intestine (indicative of fluid accumulation) was observed in DC5- and PGE₂-imidazole-treated mice. P values (t test): *, <0.05 (comparing ETEC treatment with ETEC plus 0.1 mM DC5). (B) Both compounds had a significant effect on bacterial recovery from the intestinal lumen at 72 h postinfection. P values (t test): *, <0.01 (comparing ETEC treatment with ETEC plus 0.1 mM DC5 or ETEC plus 0.1 mM PGE₂-imidazole). Each group included eight mice.

FIG. 5.

EM images of infected intestine. Ultrastructural studies of the intestine infected with ETEC only (A to C), ETEC treated with PGE₂-imidazole (D to F), and ETEC with DC5 (G to I). ETEC-infected intestines demonstrated destruction of the microvilli and cell death, while integrity of the microvilli in the compound-treated animals remained intact. Control tissue (noninfected) was used for comparison (not shown). Magnification: panels A, D, and G, ×16,000; panels B, E, and H, ×32,000; panels C, F, and I, ×64,000.

FIG. 6.

Comparing effects of DC5 routes of administration on bacterial survival. ETEC-infected animals received DC5 treatment using the oral or the intraperitoneal routes at 24, 48, and 72 h postinfection, and the CFU in the intestine were calculated. P values (t test): *, <0.01 (comparing ETEC treatment with ETEC plus 0.1 mM DC5 or ETEC plus 0.1 mM PGE₂-imidazole). P values (t test): *, <0.01 (comparing treatments at 72 h, ETEC treatment versus ETEC plus 0.1 mM DC5 IP or ETEC plus 0.1 mM DC5 orally administered). Each group included eight mice.

FIG. 7.

Effect of DC5 and LT on bacterial adhesion to HeLa cells. Bacteria were inoculated at a multiplicity of infection of 10:1 onto semiconfluent cultured epithelial cell monolayers in the presence or absence of DC5 or exogenous LT toxin. Bacteria and cells were incubated for 3 h, and the adherent bacteria were quantified by serial dilutions. (A) ETEC H10407, EAEC O42, V. cholerae 596B; (B) EPEC E2348/69, EHEC O157:H7, ETEC DEC7A; (C) S. enterica serovar Typhimurium, S. flexneri. Each panel represents duplicate independent experiments performed in triplicate. *, P = 0.05; **, P = 0.01 (t test; comparing treated samples to their corresponding wild-type strains).