Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells

Abstract

Advances in the understanding of the pathogenesis of enterohaemorrhagic Escherichia coli (EHEC) have greatly benefited from the use of human epithelial cell lines under aerobic conditions. However, in the target site of EHEC infection, the human intestine, conditions are microaerobic. In our study we used polarized human colon carcinoma cells in a vertical diffusion chamber system to investigate the influence of reduced apical oxygen levels on EHEC colonization. While apical microaerobiosis did not affect cell integrity and barrier function, numbers of adherent bacteria were significantly increased under low compared with high apical oxygen concentrations. In addition, expression and translocation of EHEC type III secreted (T3S) effector proteins was considerably enhanced under microaerobic conditions and dependent on the presence of host cells. Increased colonization was mainly mediated via EspA as adherence levels of an isogenic deletion mutant were not influenced by low oxygen levels. Other potential adherence factors (E. coli common pilus and flagella) were only minimally expressed under high and low oxygen levels. Addition of nitrate and trimethylamine N-oxide as terminal electron acceptors for anaerobic respiration failed to further increase bacterial colonization or T3S under microaerobiosis. This study indicates that EHEC T3S and colonization are enhanced by the microaerobic environment in the gut and therefore might be underestimated in conventional aerobic cell culture systems.

Fig. 1

Infection in a vertical diffusion chamber system. Polarized intestinal epithelial cells grown on Snapwell filters were inserted between two half chambers and infected apically with EHEC. Apical chambers were perfused with 95% O₂, 5% CO₂ (oxygenated) or 90% N₂, 5% H₂, 5% CO₂ (anaerobic) whereas basolateral compartments were maintained under oxygenated conditions.

Fig. 2

Cell morphology and bacterial adherence patterns of polarized T84 cells infected with EHEC for 6 h under apical microaerobic or oxygenated conditions. Shown are representative images from three independent experiments. A. Confocal micrographs of non-infected (NI) and infected monolayers (EHEC) stained for occludin (green). Bacteria and cell nuclei were stained with propidium iodide (magenta). Bars = 10 µm. B. Scanning electron micrographs of non-infected and infected T84 cells. Bars = 1 µm.

Fig. 3

Microaerobic apical conditions enhance EHEC colonization (A), but impair growth of non-adherent bacteria (B). T84 cells were infected with EHEC for 6 h and maintained under apical oxygenated (OX) or microaerobic conditions (MA). Adherent bacteria were quantified as cfu/filter (A) whereas growth of non-adherent bacteria was assessed by OD₆₀₀ (B). Data are shown as means ± SEM from four independent experiments performed in triplicate. ***P < 0.001.

Fig. 4

Apical microaerobic conditions enhance expression and secretion of EHEC T3S proteins. A. Stained SDS-PAGE of EHEC-secreted proteins under oxygenated (OX) or microaerobic conditions (MA). Differentially secreted proteins are marked by asterisks and molecular weights are indicated in kDa. B. Western blot of EHEC-secreted proteins probed with Tir, EspA and EspB specific antisera. C. Western blot of EHEC and T84 cell lysates showing Tir and EspA expression by non-adherent (NA) and adherent (A) bacteria respectively. Cell lysates of non-infected (NI) T84 monolayers were included to confirm specificity of antisera. Relative amounts of loaded bacterial protein were assessed by GroEL immunoblotting. D. Immunofluorescence staining of EHEC adhering to T84 cells after 6 h of infection showing enhanced EspA filament formation (green) under microaerobic compared with oxygenated conditions (first two panels on the left). In contrast, expression of ECP (green, second panel from the right) and flagella (red, panel on far right) was not affected by oxygen levels. EHEC were labelled with anti-O157:H7 (red) and cellular/bacterial DNA was counterstained with DAPI (blue, far right panel only). Shown are representative images from three independent experiments.

Fig. 5

A. EHEC adherence to polarized Caco-2 cells is elevated under microaerobic conditions. Data are shown as means ± SEM from four independent experiments performed in triplicate. **P < 0.01. B. Western blot of EHEC and Caco-2 cell lysates showing Tir and EspA expression by non-adherent (NA) and adherent (A) bacteria respectively. Translocated Tir is indicated by an asterisk. Relative amounts of loaded bacterial protein were assessed by GroEL immunoblotting. C. Immunofluorescence staining of polarized Caco-2 cells infected with EHEC for 6 h. Translocated Tir (green) is evident beneath adherent bacteria (propidium iodide, red).

Fig. 6

EHEC T3S and growth of non-adherent bacteria are host cell-dependent. (A) Western blot of bacterial supernatant proteins probed with Tir, EspA and EspB specific antisera and (B) OD₆₀₀ of apical media. Bacteria were grown under microaerobic (MA) or oxygenated conditions (OX) in the absence (−T84) or presence of T84 cells (+T84). A. Protein samples were run on two separate gels that were processed simultaneously. B. Data are shown as means ± SEM from two independent experiments performed in duplicate. **P < 0.01, ***P < 0.001.

Fig. 7

Enhanced microaerobic adherence of EHEC to polarized T84 cells is mainly dependent on EspA. T84 cells were infected with wt EHEC, an isogenic ΔespA mutant, and its complemented strain [Δ(pespA)] for 6 h while maintained under apical oxygenated (OX) or microaerobic conditions (MA). Adherent bacteria were quantified as cfu/filter (A) whereas growth of non-adherent bacteria was assessed by OD₆₀₀ (B). Data are shown as means ± SEM from four independent experiments performed in triplicate. **P < 0.01, ***P < 0.001.

Fig. 8

Addition of nitrate or TMAO as terminal electron acceptors does not further enhance EHEC T3S or colonization under microaerobic conditions. T84 cells were infected with EHEC for 6 h under apical oxygenated (OX) or microaerobic (MA) conditions. Nitrate (N) or TMAO (T) was added to apical chambers. A. Western blot of bacterial supernatant proteins probed with anti-Tir, EspA and EspB. B. Colonization of T84 cells by EHEC. Data are shown as means ± SEM from two independent experiments performed in triplicate. *P < 0.05, **P < 0.01.