Mutual enhancement of virulence by enterotoxigenic and enteropathogenic Escherichia coli

Abstract

Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) are common causes of diarrhea in children in developing countries. Dual infections with both pathogens have been noted fairly frequently in studies of diarrhea around the world. In previous laboratory work, we noted that cholera toxin and forskolin markedly potentiated EPEC-induced ATP release from the host cell, and this potentiated release was found to be mediated by the cystic fibrosis transmembrane conductance regulator. In this study, we examined whether the ETEC heat-labile toxin (LT) or the heat-stable toxin (STa, also known as ST) potentiated EPEC-induced ATP release. We found that crude ETEC culture filtrates, as well as purified ETEC toxins, did potentiate EPEC-induced ATP release in cultured T84 cells. Coinfection of T84 cells with live ETEC plus EPEC bacteria also resulted in enhanced ATP release compared to EPEC alone. In Ussing chamber studies of chloride secretion, adenine nucleotides released from the host by EPEC also significantly enhanced the chloride secretory responses that were triggered by crude ETEC filtrates, purified STa, and the peptide hormone guanylin. In addition, adenosine and LT had additive or synergistic effects in inducing vacuole formation in T84 cells. Therefore, ETEC toxins and EPEC-induced damage to the host cell both enhance the virulence of the other type of E. coli. Our in vitro data demonstrate a molecular basis for a microbial interaction, which could result in increased severity of disease in vivo in individuals who are coinfected with ETEC and EPEC.

FIG. 1.

Effect of ETEC culture filtrates on EPEC-induced ATP release from host cells. Culture filtrates were prepared from overnight cultures of ETEC strain H10407 or H10407ΔLT (D) in CAYE medium as described in Materials and Methods. ETEC sterile filtrates were applied to T84 cells in 96-well plates for 18 to 20 h (60 μl per well unless otherwise indicated). The medium was changed to serum-free medium without antibiotics, and cells were infected with EPEC at an MOI of 100:1. After 45 min to allow EPEC adherence, the medium was changed again to include an ATP-regenerating system (creatine kinase plus phosphocreatine) and the nucleotidase inhibitor α,β-methylene-ADP; incubation was continued for 2 to 3 h as shown on the figures. Then, the plates were swirled to allow mixing to occur; aliquots were collected, subjected to filtration via a 0.45-μm filter to remove bacteria or detached host cells, and assayed for ATP. Abbreviations: forsk, 20 μM forskolin added the day of the experiment; ETEC SF, ETEC sterile filtrate; CT, 200-ng/ml cholera toxin; E2348, EPEC strain E2348/69; B171, EPEC strain B171-8; JCP, EPEC strain JCP88; wt, wild type; ΔLT SF, sterile filtrate prepared from strain H10407ΔLT.

FIG. 2.

Effect of purified E. coli enterotoxins on EPEC-induced ATP release. T84 cells were grown in 48-well (A) or 96-well (B) plates. (A) Purified, pyrogen-free LT-IIa toxin at 720 ng/ml was applied overnight; on the next day, the medium was changed as described in the legend to Fig. 1 and in Materials and Methods. After EPEC infection, supernatant medium was collected 2 h after the medium change. (B) Purified STa was applied to some wells just before infection with EPEC strain E2348/69, and then STa was added again to the same 1 μM concentration after the medium change. The phosphodiesterase inhibitor Zaprinast (10 μM) was added to some wells after the medium change (B, right). Supernatants were collected 3 h after the medium change shown in panel B.

FIG. 3.

Effect of infection with live ETEC on EPEC-induced ATP release from T84 cells. ETEC strain H10407 was grown overnight in CAYE medium, and then T84 cells were infected with 2.5 μl or 5 μl of overnight culture, resulting in an ETEC MOI of 60 or 120, respectively. Overnight cultures of the EPEC strains, in contrast, were subcultured for 2 h in serum-free Dulbecco's modified Eagle's medium and then used to infect T84 cells in 48-well plates. The MOIs were 140, 260, and 120 for EPEC strains B171-8, JCP88, and E851/71, respectively. In this experimental design, therefore, the ETEC had a 2-h “head start” relative to the EPEC strains. Supernatants were collected at 2 and 3 h for the ATP assay as shown in the graphs.

FIG. 4.

Additive effects of AMP and ETEC toxins on short-circuit current in T84 cell monolayers in the Ussing chamber. T84 cells were grown on collagen-coated Snap-Well culture inserts as described in Materials and Methods for 7 to 9 days until they reached confluence and a high transepithelial resistance. AMP, ETEC toxins, and PI-PLC were applied on the apical (i.e., mucosal or luminal) side of the monolayer, while carbachol was applied on the basolateral side. U73122 is an inhibitor of PI-PLC. Raw data (current, voltage, and resistance) were collected in a digital file and then imported into GraphPad Prism for the creation and labeling of the graphs shown. All experiments shown were repeated at least three times, and representative tracings are shown. Using this cell line, preparation, and electrode configuration, a positive short-circuit current (shown as an upward deflection on the graph) represents chloride secretion toward the apical side of the tissue.

FIG. 5.

Additive effects of AMP and STa toxin and guanylin on short-circuit current in T84 cell monolayers in the Ussing chamber. T84 cells were grown on collagen-coated Snap-Well culture inserts, as described in Materials and Methods and in the legend to Fig. 4. (A to C) Secretory effect of 10 μM AMP alone, STa alone, and STa followed by AMP. (D to F) Parallel series of tracings, except that the AMP concentration was reduced to 2 μM and guanylin was used instead of STa.

FIG. 6.

Additive effects of adenosine and cyclic AMP-elevating toxins on vacuole formation in T84 cells. T84 cells were grown to confluence on Permanox plastic Lab-Tek chamber slides and then treated with cholera toxin, ETEC culture filtrates, adenosine, or combinations of stimuli. Vacuoles were allowed to form for 3 or 16 h. Slides were fixed, stained with Giemsa, and photographed at a magnification of ×200 magnification for all panels. The size bar shown in panel A represents 30 μm, as determined with latex sizing beads. (A) Control T84 cells; (B) cells treated with 20 μM adenosine for 3 h; (C) cells treated with 25-ng/ml CT for 3 h; (D) cells treated with 20 μM adenosine and 25-ng/ml CT for 3 h, showing enhanced formation of vacuoles; (E) cells treated with 100 μl of a sterile filtrate of culture supernatant of ETEC strain H10407 for 19 h; (F) cells treated with 100 μl of ETEC sterile filtrate for 19 h with 20 μM adenosine added for the last 3 h; (G) cells treated for 19 h with a sterile filtrate of ETEC mutant H10407ΔLT.

FIG. 7.

Effect of ion channel inhibitors on cholera toxin-induced vacuole formation. T84 cells were grown as described in the legend to Fig. 6, treated with CT at 50 ng/ml for 3 h, fixed, stained, and photographed as before. Size bars, 30 μm. (A) Cells treated with CT alone; (B) cells treated with CT plus 10 μM CFTR_inh-172, a thiazolidinone CFTR inhibitor; (C) cells treated with CT plus 100 μM glyburide, a sulfonylurea which is a CFTR and potassium channel inhibitor; (D) sketch depicting the proposed mechanism for the formation of giant vacuoles in T84 cells in response to CT and LT (adenosine receptors are omitted from the diagram for the sake of simplicity).