Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2016 Jul 29.
Published in final edited form as: Environ Microbiol. 2010 Apr 7;12(9):2426–2435. doi: 10.1111/j.1462-2920.2010.02216.x

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

Stephanie Schüller 1,*, Alan D Phillips 1
PMCID: PMC4966633  EMSID: EMS69259  PMID: 20406285

Summary

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.

Introduction

Enterohaemorrhagic Escherichia coli (EHEC) is a human intestinal pathogen that causes diarrhoea and can induce severe complications such as haemorrhagic colitis and haemolytic uraemic syndrome associated with the production of Shiga toxin (Tarr et al., 2005). After ingestion via contaminated food or water, EHEC colonizes the intestinal mucosa by forming attaching/effacing lesions (A/E), which are characterized by intimate bacterial adherence and localized microvillous destruction (Knutton et al., 1987). The A/E lesion formation has been attributed to the presence of a type III secretion (T3S) system that is encoded on the locus of enterocyte effacement (LEE) pathogenicity island and enables the bacterium to inject effector proteins into the host cell (reviewed by Frankel et al., 1998). After establishment of initial contact via EspA containing filaments, two further effector proteins, EspB and D, are translocated into the host cell membrane where they form a pore structure (Ebel et al., 1998; Knutton et al., 1998; Ide et al., 2001). Delivery of the translocated intimin receptor (Tir) into the host cell membrane is followed by dissolution of the EspA filament and intimate bacterial attachment via binding of Tir to the bacterial adhesin intimin (Kenny et al., 1997a; Deibel et al., 1998). In vitro cell culture models have been useful in elucidating the mechanisms of A/E lesion formation; however, they generally do not consider the altered environment that EHEC encounters in vivo. Apart from high osmolarity, 37°C temperature, neutral to alkaline pH and different concentrations of ions and nutrients than in the ecological environment, the milieu in the intestinal tract is characterized by low oxygen levels (He et al., 1999). Studies on enteropathogenic Salmonella ssp. have shown that anaerobic conditions increase bacterial adherence, invasion and virulence (Ernst et al. 1990; Lee and Falkow, 1990; Schiemann and Shope, 1991; Singh et al., 2000). Whereas most of these studies have used anaerobic bacterial cultures to infect cells in an aerobic environment, we have employed a system that allows apical infection under microaerobic conditions (oxygen concentrations of 1–2% atmospheric pressure) while basolateral cell compartments are maintained under oxygenated conditions. Using this system, we show increased EHEC adherence and T3S under apical microaerobic conditions.

Results

Establishment of a vertical diffusion chamber system for microaerobic EHEC infection

A vertical diffusion chamber system was used to generate an environment that mimics the microaerobic milieu at the luminal side of the intestinal mucosa. Polarized T84 cells were mounted between half chambers resulting in separation of apical and basolateral sides of the monolayer. Apical chambers were perfused with either anaerobic or oxygenated gas mixtures whereas basolateral compartments were maintained under oxygenated conditions to ensure optimal cell survival (Fig. 1). Cells were infected apically with EHEC for 6 h and apical media were exchanged every 2 h to maintain bacteria in early to midlogarithmic growth phase.

Fig. 1.

Fig. 1

Open in a new tab

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.

Oxygen concentrations in apical chambers were monitored during the experiment and were found to be 90–92% (percentage of atmospheric pressure) under oxygenated and 1–1.7% under apical anaerobic perfusion, thus generating a microaerobic environment. Oxygen concentrations of standing bacterial overnight cultures used for infection were close to 0% at the bottom of the tube and around 4% at the top. Determination of transepithelial electrical resistance (TER) of cell monolayers before and after the experiment showed that neither microaerobic apical conditions nor EHEC infection affected epithelial barrier function and monolayer integrity as TER remained stable under all conditions (data not shown). This was corroborated by occludin staining that showed intact tight junctions after 6 h of infection under microaerobic or oxygenated conditions (Fig. 2A). Scanning electron microscopy (SEM) of membrane filters showed a distinctive brush border of short microvilli and loose bacterial microcolonies on infected cells (Fig. 2B). This was similar under high and low oxygen levels.

Fig. 2.

Fig. 2

Open in a new tab

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.

Apical microaerobiosis enhances EHEC colonization

Bacterial adherence to polarized T84 monolayers was quantified by plating out serial dilutions of cell lysates and determining colony-forming units (cfu). As shown in Fig. 3A, numbers of adherent EHEC were significantly increased under apical microaerobic conditions compared with an oxygenated environment (P = 0.0004). This effect was specific as it was not observed with the nonpathogenic E. coli strain HB101 that adhered poorly independent of oxygen levels applied [483.5 ± 47.4 cfu/filter oxygenated (OX) versus 489.8 ± 40.1 cfu/filter microaerobic (MA); mean ± SEM, n = 4, P > 0.05]. Despite increased adherence under microaerobiosis, growth of non-adherent EHEC in the apical medium (as assessed by OD600) was slower under microaerobic than under oxygenated conditions (Fig. 3B, P = 0.0001).

Fig. 3.

Fig. 3

Open in a new tab

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.

EHEC T3S protein expression and secretion are increased under microaerobic conditions

Previous studies have shown that the T3S effector protein EspA is involved in initial contact and essential for EHEC adherence to epithelial cells (Ebel et al., 1998). Therefore, we investigated whether increased bacterial adherence under microaerobic conditions was associated with enhanced T3S. Media from apical diffusion chambers were sampled after 6 h of infection and supernatant proteins were analysed by SDS-PAGE and Western blotting. Staining of polyacrylamide gels showed that four protein bands were induced under microaerobiosis whereas two bands were evident under oxygenated conditions only (Fig. 4A). Western blotting revealed that three out of the four proteins induced by low oxygen levels were Tir, EspA and EspB (Fig. 4B). Analysis of lysates from non-adherent bacteria in apical media revealed that increased amounts of T3S proteins in supernatants were associated with enhanced protein expression in the bacterial cell (Fig. 4C). We also examined T3S protein expression by adherent EHEC by analysing cell lysates of infected T84 monolayers and found that EspA expression was enhanced under microaerobic conditions (Fig. 4C). This was confirmed by immunofluorescence staining that showed pronounced EspA filament formation on adherent EHEC under microaerobic conditions whereas fewer and shorter filaments were observed under oxygenated conditions (Fig. 4D). In addition to EspA, we also examined the influence of oxygen levels on expression of other potential EHEC adherence factors, i.e. E. coli common pilus (ECP) and flagella. Bacterial and cell lysates were analysed by SDS-PAGE and Western blotting, but no specific signal was observed under either oxygenated or microaerobic conditions (data not shown). Immunofluorescence staining of infected T84 monolayers showed that only few adherent bacteria expressed ECP (12.5 ± 1.9% OX versus 11.9 ± 2.3 MA; mean ± SEM, n = 4, P > 0.05) or flagella (1.8 ± 0.2% OX versus 1.5 ± 0.3 MA; mean ± SEM, n = 4, P > 0.05) independent of oxygen levels employed (Fig. 4D).

Fig. 4.

Fig. 4

Open in a new tab

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.

Microaerobiosis promotes Tir translocation into the host cell

Having demonstrated increased EspA filament formation under microaerobic conditions we wanted to determine whether this was associated with increased Tir translocation into the host cell. Immunofluorescence staining of infected T84 cells demonstrated that very few bacteria showed Tir translocation after 6 h of infection (data not shown). For this reason we performed infections of polarized Caco-2 cells where EHEC A/E lesion formation was observed at higher frequency. As shown for T84 cells, Caco-2 cell viability and barrier function was unaffected by apical microaerobiosis or EHEC infection for 6 h (data not shown) and EHEC adherence was significantly enhanced by microaerobic compared with oxygenated conditions (Fig. 5A, P = 0.0045). SDS-PAGE and Western blot analysis of bacterial and cell lysates showed increased EspA and Tir expression on non-adherent and adherent bacteria under microaerobiosis respectively (Fig. 5B). An additional Tir band of higher molecular weight was observed in lysates from cells infected under microaerobic conditions (Fig. 5B). This represents translocated Tir that has been serine- and/or threonine-phosphorylated by host cell kinases resulting in an increase in apparent molecular weight from 72 to 88 kDa (Devinney et al., 1999). Enhanced Tir translocation under microaerobic conditions was confirmed by immunofluorescence staining, which showed translocated Tir associated with 49.3 ± 3.6% of adherent EHEC under microaerobiosis compared with 15.0 ± 2.3% under oxygenated conditions (mean ± SEM, n = 4, P= 0.0022; Fig. 5C).

Fig. 5.

Fig. 5

Open in a new tab

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).

Type III secretion in the vertical diffusion chamber system is host cell-dependent

To examine whether the presence of host cells was required for T3S, half chambers were assembled containing either Snapwell filters with T84 monolayers or empty Snapwell support rings without cell monolayers present. Chambers without filters were perfused with oxygenated or anaerobic gas mixtures on both sides whereas chambers with cell monolayers were maintained as described above. Chambers were inoculated with lower bacterial numbers than in previous experiments to prevent medium exchange, and incubations were continued until bacteria reached early to mid-logarithmic phase (OD600 = 0.2−0.3) at approximately 3 h after inoculation. This was equivalent to the bacterial density obtained at 6 h post infection with medium replacement every 2 h in previous experiments. Whereas apical media from T84-containing chambers showed a similar pattern of secreted bacterial proteins as observed after 6 h of infection, no protein bands were observed in supernatants from chambers with EHEC only on stained protein gels (data not shown). Western blotting showed no secretion of Tir, EspA and EspB in supernatants from chambers with EHEC only whereas supernatants from T84-containing chambers showed enhanced T3S under microaerobic conditions as observed before (Fig. 6A). Interestingly, growth of non-adherent bacteria was significantly enhanced in the presence of T84 cells under both microaerobic and oxygenated conditions (P < 0.0001 and P = 0.0036 respectively; Fig. 6B). Enhanced bacterial growth and T3S appeared to be dependent on bacteria–host cell contact as no effect was observed on bacteria incubated in T84 cell-conditioned medium (apical medium obtained from non-infected T84 cells after 6 h incubation in the diffusion chamber system, data not shown).

Fig. 6.

Fig. 6

Open in a new tab

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.

Enhanced microaerobic colonization is mainly mediated via EspA

We further investigated whether bacterial adherence factors other than EspA contributed to increased bacterial colonization under microaerobic conditions. Therefore, infections were performed with an isogenic espA deletion mutant (EHEC ΔespA) and its complemented strain [EHEC ΔespA (pespA)] and numbers of adherent bacteria were determined. As shown in Fig. 7A, colonization levels of EHEC ΔespA were not significantly enhanced by low oxygen levels and were similar to those of wild-type (wt) EHEC under oxygenated conditions (Fig. 7A, P > 0.05). In contrast, wt EHEC and EHEC ΔespA (pespA) showed significantly increased adherence under microaerobiosis (Fig. 7A, P = 0.0002 and P = 0.0044 respectively). Growth of non-adherent EHEC was significantly enhanced under oxygenated conditions in all strains tested (Fig. 7B, wt: P = 0.0048, ΔespA: P = 0.0026 and ΔespA (pespA): P = 0.0012).

Fig. 7.

Fig. 7

Open in a new tab

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.

Influence of electron acceptors on T3S and colonization

Previous studies have shown that maturation of the EHEC T3S system under anaerobiosis requires the presence of terminal electron acceptors (Ando et al., 2007). To investigate whether T3S under microaerobic conditions could be further enhanced by the presence of electron acceptors, diffusion chamber experiments were conducted in the presence of trimethylamine N-oxide (TMAO) or nitrate that have been shown to act as electron acceptors for anaerobic respiration in EHEC (Ando et al., 2007). As shown in Fig. 8A, secretion of Tir, EspA and EspB was comparable under microaerobic conditions with or without electron acceptors. Similarly, colonization levels under microaerobic conditions were not significantly affected by the addition of TMAO or nitrate (P > 0.05) whereas a significant reduction in colonization levels was observed under oxygenated conditions (P < 0.05 relative to microaerobic conditions without electron acceptor or with nitrate and P < 0.01 relative to microaerobiosis with TMAO, Fig. 8B).

Fig. 8.

Fig. 8

Open in a new tab

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.

Discussion

In this study we have investigated EHEC colonization in an environment simulating the microaerobic conditions in the human gut. Although accurate data about oxygen concentrations in the human intestine are still lacking, non-invasive imaging of oxygen levels in the gastrointestinal tract of living mice indicate that the milieu in the mid-small intestine and mid-colon is microaerobic (1.4% of atmospheric pressure) (He et al., 1999). This is very similar to the apical oxygen concentrations generated in the vertical diffusion chamber system (1–1.7%). Polarized T84 and Caco-2 colon carcinoma cells have been chosen as model cell lines as they have been extensively used to study EHEC pathogenesis and develop high TER after polarization. Morphology and integrity of cell monolayers in the diffusion chamber was not influenced by apical microaerobic conditions during the 6 h experiment, which is consistent with Cottet and colleagues (2002) who have used a similar system with polarized Caco-2 cells to investigate Helicobacter pylori infection under microaerobiosis. Tight junction integrity was not affected by EHEC infection under the conditions employed [infection with stationary culture at a multiplicity of infection (moi) of 200 bacteria per cell for 6 h], which agrees with previous studies showing that EHEC causes a much slower and less pronounced decrease in TER than EPEC (Philpott et al., 1998; Viswanathan et al., 2004). Whereas cell morphology and EHEC colonization patterns did not differ under apical microaerobic or oxygenated conditions, adherence levels were approximately 2× higher when apical chambers were maintained under microaerobiosis. Similar diffusion chamber studies have been performed with H. pylori and more than 20× increased adherence levels after 4 h of infection have been reported under microaerobic conditions (Cottet et al., 2002). This more dramatic effect of microaerobiosis on adherence levels is likely due to the high oxygen sensitivity of H. pylori that resulted in poor survival of bacteria under aerobic conditions (Cottet et al., 2002). In contrast, EHEC is a facultative anaerobe and non-adherent bacteria showed better growth at high than at low oxygen levels. Escherichia coli can utilize two respiratory oxidases for reduction of oxygen. Whereas the low-affinity cytochrome bo3 oxidase is active under oxygen-rich conditions, the high-affinity cytochrome bd oxidase can still bind oxygen at low concentrations but is energetically less efficient (Gunsalus and Park, 1994; Jones et al., 2007). Interestingly, an earlier study (James and Keevil, 1999) investigated growth of EHEC in steady-state chemostat cultures and yielded similar results to our diffusion chamber study: whereas growth of anaerobic cultures was impaired relative to aerobic ones, anaerobic cultures were more adhesive for HEp-2 cells.

Enhanced EHEC adherence under microaerobic conditions was associated with increased expression and secretion of T3S proteins by both adherent and non-adherent bacteria. Environmental conditions influencing EHEC or EPEC T3S have been investigated in the past and it has been shown that maximal T3S and A/E lesion formation occurs at 37°C, pH 7 and physiological osmolarity, i.e. conditions that prevail in the human intestinal tract (Rosenshine et al., 1996; Kenny et al., 1997b). The T3S is also enhanced in the presence of sodium bicarbonate, calcium and millimolar concentrations of Fe(NO3)3 (Kenny et al., 1997b; Abe et al., 2002) and is affected by acid stress (House et al., 2009). Interestingly, only one report has investigated the impact of low oxygen concentrations on EHEC T3S so far. In the study (Ando et al., 2007), EHEC were grown to early stationary phase in LB medium and supernatant proteins were examined. While good T3S was observed in cultures grown under aerobic conditions, only very low amounts of T3S proteins were detected under anaerobiosis. However, addition of alternative terminal electron acceptors (nitrate or TMAO) restored anaerobic T3S to aerobic levels and resulted in enhanced EspA filament formation and adherence to Caco-2 cells. Further experiments demonstrated that growth without electron acceptor resulted more frequently in a premature T3S apparatus lacking EspA filament and EscF needle complex (Ando et al., 2007). This study prompted us to investigate the influence of terminal electron acceptors in our system. Neither addition of nitrate nor of TMAO resulted in further enhancement of T3S or colonization. In contrast to Ando and colleagues (2007), oxygen concentrations in our diffusion chamber system were microaerobic rather than anaerobic. Under these conditions, E. coli can still perform aerobic respiration via the high-affinity cytochrome bd oxidase, whereas other electron acceptors than oxygen (nitrate, nitrite, DMSO/TMAO, or fumarate) are required for respiration under anaerobic conditions (Gunsalus and Park, 1994; Jones et al., 2007). Although Ando and colleagues (2007) observed good EHEC T3S under aerobic conditions, we did not detect secretion of Tir, EspA or EspB without the presence of host cells. This discrepancy may arise from differences in growth media (LB versus DMEM/F-12), bacterial growth phase (early stationary versus early to mid-logarithmic phase), or the presence of 5% CO2 in our diffusion chamber system. While there are no further reports regarding T3S in EPEC or EHEC, a study of diffusely adhering E. coli (Diard et al., 2006) demonstrated increased secretion of virulence associated Dr fimbriae under anaerobiosis.

In addition to environmental conditions, T3S and A/E lesion formation are dependent on growth phase, with maximal activity occurring at early to mid-logarithmic phase (OD600 = 0.2−0.4) (Rosenshine et al., 1996; Kenny et al., 1997b). The T3S is also activated by quorum sensing during transition from late exponential to stationary phases (Sperandio et al., 1999). In our diffusion chamber system, we aimed to maintain bacterial growth within early to mid-logarithmic phase to avoid the effect of quorum sensing. This was achieved by exchanging apical media every 2 h, which also helped to level out bacterial growth differences in oxygenated and microaerobic apical chambers.

Another important factor contributing to T3S and bacterial growth is the presence of host cells. This has also been observed by Cottet and colleagues (2002) where growth of H. pylori in the microaerobic diffusion chamber system was only sustained in the presence of Caco-2 cells. As T84-conditioned medium failed to promote EHEC T3S and bacterial growth, this effect appears to be dependent on direct host cell contact. In the infection protocol, apical media were exchanged every 2 h so that the majority of non-adherent bacteria are probably derived from adhering and detaching bacteria, which continue to secrete effector proteins into the medium. Microarray studies have investigated EHEC gene transcription in response to host cell adherence. Whereas Dahan and colleagues (2004) showed downregulation of most of the LEE mRNAs at 5 h of infection of red blood cells compared with incubation in medium alone, other studies (Kim et al., 2009) reported no significant difference between LEE mRNA expression at 3 h of infection of intestinal HT-29 cells and the medium control. While both of these studies focus on a particular time point of infection, kinetic real-time PCR analysis of EPEC LEE gene expression during HEp-2 cell infection showed induced tir and espA mRNA expression at 3 h, which remained constant throughout 5 h of infection (Leverton and Kaper, 2005). Inconsistent results in virulence gene expression during infection are likely to be caused by differences in host cell type but may also vary according to the infection protocol. In particular, it is difficult to achieve synchronized infection at later stages where bacteria will consist of a mixed population of attached, released and newly attaching bacteria.

Apart from EspA, which has been shown to be essential for EHEC adherence to human epithelial cells (Ebel et al., 1998), no other major adherence factors appear to be involved in increased microaerobic colonization as adherence levels of an isogenic espA deletion mutant were not affected by low oxygen levels and were similar to those of wt EHEC under oxygenated conditions. Although previous studies have shown that EHEC flagella are involved in adherence to bovine intestinal epithelial cells (Erdem et al., 2007; Mahajan et al., 2009), they do not play a role in colonization of pigs (Best et al., 2006) and were only expressed on very few bacteria in the diffusion chamber system independent of oxygen levels. Similar results were observed for the expression of ECP, which has been demonstrated to promote adherence to human epithelial cells (Rendón et al., 2007). Interestingly, a study on sorbitol-fermenting EHEC O157:NM described induced expression of Sfp fimbriae under anaerobiosis, which resulted in increased adherence to intestinal epithelial cells (Müsken et al., 2008). However, genes for Sfp fimbriae are unique for sorbitol-fermenting EHEC O157:NM and absent in O157:H7 strains (Friedrich et al., 2004).

In E. coli, changes from aerobic to anaerobic growth and vice versa are regulated by the two global regulators Fnr (anaerobiosis) and ArcA (microaerobiosis) (Gunsalus and Park, 1994; Alexeeva et al., 2003; Jones et al., 2007). Whereas it has been shown that many Salmonella virulence genes are regulated by Fnr and thus are affected by anaerobic conditions (Fink et al., 2007; Van Immerseel et al., 2008), no such link has been reported for pathogenic E. coli so far. Our study has shown that EHEC T3S and colonization are enhanced by microaerobiosis and it will be important to elucidate the underlying pathways involved in this phenomenon to gain a better understanding of how EHEC adapts to the microaerobic environment and successfully colonizes the human gut.

Experimental procedures

Cell culture

Human colon carcinoma T84 cells (ATCC CCL-248) were cultured in DMEM/F-12 mixture supplemented with 10% foetal bovine serum (Sigma) and used between passage 42 and 55. Human colon carcinoma Caco-2 cells (ECACC 86010202) were grown in DMEM medium supplemented with 10% foetal bovine serum and non-essential amino acids (Sigma). For diffusion chamber experiments, 5 × 105 T84 cells cm−2 or 2 × 105 Caco-2 cells cm−2 were seeded on collagen-coated polyester Snapwell filter inserts (12 mm diameter, 0.4 mm pore; Corning Costar). The TER was monitored using an EndOhm chamber and EVOM resistance meter (WPI) and values of around 1500 Ω × cm2 (T84) or 500 Ω × cm2 (Caco-2) after 10–14 days of differentiation indicated establishment of epithelial barrier function. Cells were grown at 37°C in a 5% CO2 atmosphere.

Bacterial strains and culture conditions

The EHEC strain TUV 93-0 (Shiga toxin-negative O157:H7 EDL933), TUV 93-0 ΔespA (ICC288, Shaw et al., 2008), TUV 93-0 ΔespA (pespA) (ICC288 complemented with pBAD-EspA EHEC O157:H7, Crepin et al., 2005) and the non-pathogenic E. coli strain HB101 were obtained from G Frankel (Imperial College London). For infections, bacteria were grown standing in Brain Heart Infusion broth overnight at 37°C. Ampicillin (100 µg ml−1) and kanamycin (50 µg ml−1) were added as required.

Infection in a vertical diffusion chamber system

Snapwell filter inserts with polarized cells were mounted between two half chambers of a vertical diffusion chamber system (Harvard Apparatus). Both compartments were filled with serum-free culture medium and 108 bacteria (moi of 200) were added to the apical side (Fig. 1). Apical chambers were perfused with 95% O2, 5% CO2 (oxygenated) or 90% N2, 5% H2, 5% CO2 (anaerobic) whereas basolateral compartments were kept under oxygenated conditions. Oxygen concentrations in apical compartments were monitored during the experiment using an ISO2 dissolved oxygen meter (WPI). Apical media were exchanged every 2 h to maintain bacteria at early exponential growth phase and incubations were continued for 6 h. For electron acceptor studies, 10 mM sodium nitrate or TMAO (Sigma) was added to the apical chamber. To investigate the influence of host cells on T3S, parallel diffusion chamber experiments were performed with or without cell monolayers. Bacteria were inoculated at a lower concentration (2.5 × 107, moi of 50) to prevent medium exchange. Incubations were carried out for approximately 3 h when bacteria had reached early exponential phase (OD600 = 0.2–0.3), which was similar to bacterial densities at 6 h post infection with 2 h medium exchange. At the end of the experiment, apical media were collected and OD600 values were determined to assess growth of non-adherent bacteria. Bacteria were pelleted by centrifugation and supernatants and pellets were stored at −20°C for SDS-PAGE analysis. Membrane filters were cut out from supports, washed twice in cold PBS to remove non-adherent bacteria and processed according to further analysis.

SEM

Filters were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, post-fixed in 1% aqueous osmium tetroxide and dehydrated in 2,2 dimethoxy-propane. Specimens were transferred to absolute ethanol, critically point-dried using liquid carbon dioxide (Emitech K850 apparatus), mounted on aluminium stubs, sputter-coated with gold-palladium (Polaron E5100 sputter coater) and viewed in a JEOL 5300 SEM.

Immunofluorescence staining and microscopic quantification

Filters were fixed in formalin for 10 min and blocked/permeabilized with 0.1% Triton X-100 (Tx-100), 0.5% BSA in PBS for 20 min. For occludin staining, cells were pre-extracted with 0.2% Tx-100 in PBS for 2 min on ice, fixed in formalin and permeabilized with 0.05% Tx-100 in PBS for 5 min on ice as recommended by the manufacturer. Cells were subsequently incubated in primary antibodies (goat anti-O157:H7 from Fitzgerald Industries; rabbit anti-occludin from Invitrogen; rabbit anti-Tir and anti-EspA from G Frankel, Imperial College London; rabbit anti-ECP from JA Girón, University of Florida, USA) for 60 min, washed and incubated in Alexa Fluor-conjugated anti-goat or anti-rabbit IgG (Invitrogen) for 30 min. Cell nuclei/bacterial DNA were labelled with propidium iodide (Sigma) or DAPI (Roche). Filters were mounted in Vectashield (Vector Laboratories) and analysed using a fluorescence light microscope (Axio Imager, Zeiss) or confocal laser scanning microscope (LSM 510 Meta, Zeiss). To quantify ECP and flagella expression and Tir translocation, 50 randomly chosen infected cells were examined and at least 200 adherent bacteria were counted in three separate experiments.

Quantification of adherent bacteria

Cell monolayers on filters were lysed in 1% Tx-100 in PBS for 30 min on ice. Serial dilutions were plated out on LB agar plates and cfu were determined after overnight incubation at 37°C.

SDS-PAGE and Western blot analysis of T3S proteins

The EHEC lysates were prepared by suspending bacterial pellets in reducing SDS-PAGE sample buffer. Supernatant proteins were precipitated by addition of 10% trichloroacetic acid (v/v) for 1 h at 4°C. Precipitated proteins were pelleted by centrifugation at 4°C (16 000 g, 15 min) and suspended in reducing sample buffer. For cell lysates, polarized monolayers were lysed in ice-cold RIPA buffer containing 1 mM PMSF, 1 mM Na3VO4 and protease inhibitor cocktail (1:200, Sigma). Sample volumes of bacterial lysates and precipitated proteins were adjusted according to bacterial density (OD600) and resolved on 12% SDS-polyacrylamide gels. Proteins were visualized by Instant Blue staining (Triple Red). For Western blotting, proteins were transferred to nitrocellulose (Hybond, GE Healthcare). Membranes were blocked with 3% BSA in TBS/0.05% Tween-20 for 60 min and incubated with primary antibodies (rabbit anti-Tir, EspA and EspB from G Frankel, Imperial College London; rabbit anti-GroEL from Sigma) overnight at 4°C. After washing, blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) and developed using enhanced chemiluminescence (Immobilon Western, Millipore).

Statistics

Statistical analysis was performed using GraphPad prism software. Student’s paired t-test was used to determine differences between two groups; one-way ANOVA with Tukey-Kramer’s multiple comparison post test was used for multiple groups. A P-value of < 0.05 was considered significant.

Acknowledgements

We thank Gadi Frankel (Imperial College London, UK) and Jorge Girón (University of Florida, USA) for bacterial strains and antisera. This project was supported by the Wellcome Trust and the Peter Samuel Royal Free Fund.

References

  1. Abe H, Tatsuno I, Tobe T, Okutani A, Sasakawa C. Bicarbonate ion stimulates the expression of locus of enterocyte effacement-encoded genes in enterohemorrhagic Escherichia coli O157:H7. Infect Immun. 2002;70:3500–3509. doi: 10.1128/IAI.70.7.3500-3509.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexeeva S, Hellingwerf KJ, Teixeira de Mattos MJ. Requirement of ArcA for redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic conditions. J Bacteriol. 2003;185:204–209. doi: 10.1128/JB.185.1.204-209.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ando H, Abe H, Sugimoto N, Tobe T. Maturation of functional type III secretion machinery by activation of anaerobic respiration in enterohaemorrhagic Escherichia coli. Microbiology. 2007;153:464–473. doi: 10.1099/mic.0.2006/000893-0. [DOI] [PubMed] [Google Scholar]
  4. Best A, La Ragione RM, Clifford D, Cooley WA, Sayers AR, Woodward MJ. A comparison of Shiga-toxin negative Escherichia coli O157 aflagellate and intimin deficient mutants in porcine in vitro and in vivo models of infection. Vet Microbiol. 2006;113:63–72. doi: 10.1016/j.vetmic.2005.10.033. [DOI] [PubMed] [Google Scholar]
  5. Cottet S, Corthesy-Theulaz l, Spertini F, Corthesy B. Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling. J Biol Chem. 2002;277:33978–33986. doi: 10.1074/jbc.M201726200. [DOI] [PubMed] [Google Scholar]
  6. Crepin VF, Shaw R, Abe CM, Knutton S, Frankel G. Polarity of enteropathogenic Escherichia coli EspA filament assembly and protein secretion. J Bacteriol. 2005;187:2881–2889. doi: 10.1128/JB.187.8.2881-2889.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahan S, Knutton S, Shaw RK, Crepin VF, Dougan G, Frankel G. Transcriptome of Enterohemorrhagic Escherichia coli O157 Adhering to Eukaryotic Plasma Membranes. Infect Immun. 2004;72:5452–5459. doi: 10.1128/IAI.72.9.5452-5459.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deibel C, Kramer S, Chakraborty T, Ebel F. EspE, a novel secreted protein of attaching effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine-phosphorylated 90 kDa protein. Mol Microbiol. 1998;28:463–474. doi: 10.1046/j.1365-2958.1998.00798.x. [DOI] [PubMed] [Google Scholar]
  9. DeVinney R, Stein M, Reinscheid D, Abe A, Rusch-kowski S, Finlay BB. Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated. Infect Immun. 1999;67:2389–2398. doi: 10.1128/iai.67.5.2389-2398.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Diard S, Toribio AL, Boum Y, Vigier F, Kansau I, Bouvet O, Servin A. Environmental signals implicated in Dr fimbriae release by pathogenic Escherichia coli. Microbes infect. 2006;8:1851–1858. doi: 10.1016/j.micinf.2006.02.023. [DOI] [PubMed] [Google Scholar]
  11. Ebel F, Podzadel T, Rohde M, Kresse AU, Kramer S, Deibel C, et al. Initial binding of Shiga toxin-producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA-containing surface appendages. Mol Microbiol. 1998;30:147–161. doi: 10.1046/j.1365-2958.1998.01046.x. [DOI] [PubMed] [Google Scholar]
  12. Erdem AL, Avelino F, Xicohtencatl-Cortes J, Girón JA. Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli. J Bacteriol. 2007;189:7426–7435. doi: 10.1128/JB.00464-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ernst RK, Dombroski DM, Merrick JM. Anaerobiosis, type 1 fimbriae, and growth phase are factors that affect invasion of HEp-2 cells by Salmonella typhimurium. Infect Immun. 1990;58:2014–2016. doi: 10.1128/iai.58.6.2014-2016.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fink RC, Evans MR, Porwollik S, Vazquez-Torres A, Jones-Carson J, Troxell B, et al. FNR is a global regulator of virulence and anaerobic metabolism in Salmonella enterica serovar Typhimurium (ATCC 14028s) J Bacteriol. 2007;189:2262–2273. doi: 10.1128/JB.00726-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frankel G, Phillips AD, Rosenshine I, Dougan G, Kaper JB, Knutton S. Enteropathogenic and enterohemorrhagic Escherichia coli: more subversive elements. Mol Microbiol. 1998;30:911–921. doi: 10.1046/j.1365-2958.1998.01144.x. [DOI] [PubMed] [Google Scholar]
  16. Friedrich AW, Nierhoff KV, Bielaszewska M, Mellmann A, Karch H. Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H. J Clin Microbiol. 2004;42:4697–4701. doi: 10.1128/JCM.42.10.4697-4701.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gunsalus RP, Park SJ. Aerobic-anaerobic gene regulation in Escherichia coli: control by the ArcAB and Fnr regulons. Res Microbiol. 1994;145:437–450. doi: 10.1016/0923-2508(94)90092-2. [DOI] [PubMed] [Google Scholar]
  18. He G, Shankar RA, Chzhan M, Samouilov A, Kuppusamy P, Zweier JL. Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging. Proc Natl Acad Sci USA. 1999;96:4586–4591. doi: 10.1073/pnas.96.8.4586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. House B, Kus JV, Prayitno N, Mair R, Que L, Chingcuanco F, et al. Acid Stress Induced Changes in Enterohemorrhagic Escherichia coli O157: H7 Virulence. Microbiology. 2009;155:2907–2918. doi: 10.1099/mic.0.025171-0. [DOI] [PubMed] [Google Scholar]
  20. Ide T, Laarmann S, Greune L, Schillers H, Oberleithner H, Schmidt MA. Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli. Cell Microbiol. 2001;3:669–679. doi: 10.1046/j.1462-5822.2001.00146.x. [DOI] [PubMed] [Google Scholar]
  21. James BW, Keevil CW. Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157. J Appl Microbiol. 1999;86:117–124. doi: 10.1046/j.1365-2672.1999.00639.x. [DOI] [PubMed] [Google Scholar]
  22. Jones SA, Chowdhury FZ, Fabich AJ, Anderson A, Schreiner DM, House AL, et al. Respiration of Escherichia coli in the mouse intestine. Infect Immun. 2007;75:4891–4899. doi: 10.1128/IAI.00484-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA, Finlay BB. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell. 1997a;91:511–520. doi: 10.1016/s0092-8674(00)80437-7. [DOI] [PubMed] [Google Scholar]
  24. Kenny B, Abe A, Stein M, Finlay BB. Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract. Infect Immun. 1997b;65:2606–2612. doi: 10.1128/iai.65.7.2606-2612.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim Y, Oh S, Park S, Kim SH. Interactive transcriptome analysis of enterohemorrhagic Escherichia coli (EHEC) O157:H7 and intestinal epithelial HT-29 cells after bacterial attachment. Int J Food Microbiol. 2009;131:224–232. doi: 10.1016/j.ijfoodmicro.2009.03.002. [DOI] [PubMed] [Google Scholar]
  26. Knutton S, Lloyd DR, McNeish AS. Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun. 1987;55:69–77. doi: 10.1128/iai.55.1.69-77.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Knutton S, Rosenshine I, Pallen MJ, Nisan I, Neves BC, Bain C, et al. A novel Esp-A associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. EMBO J. 1998;17:2166–2176. doi: 10.1093/emboj/17.8.2166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee CA, Falkow S. The ability of Salmonella to enter mammalian cells is affected by bacterial growth state. Proc Natl Acad Sci USA. 1990;87:4304–4308. doi: 10.1073/pnas.87.11.4304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Leverton LQ, Kaper JB. Temporal expression of enteropathogenic Escherichia coli virulence genes in an in vitro model of infection. Infect Immun. 2005;73:1034–1043. doi: 10.1128/IAI.73.2.1034-1043.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mahajan A, Currie CG, Mackie S, Tree J, McAteer S, McKendrick I, et al. An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157 : H7 with bovine intestinal epithelium. Cell Microbiol. 2009;11:121–137. doi: 10.1111/j.1462-5822.2008.01244.x. [DOI] [PubMed] [Google Scholar]
  31. Müsken A, Bielaszewska M, Greune L, Schweppe CH, Müthing J, Schmidt H, et al. Anaerobic conditions promote expression of Sfp fimbriae and adherence of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM to human intestinal epithelial cells. Appl Environ Microbiol. 2008;74:1087–1093. doi: 10.1128/AEM.02496-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Philpott DJ, McKay DM, Mak W, Perdue MH, Sherman PM. Signal transduction pathways involved in enterohemorrhagic Escherichia coli-induced alterations in T84 epithelial permeability. Infect Immun. 1998;66:1680–1687. doi: 10.1128/iai.66.4.1680-1687.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rendón MA, Saldaña Z, Erdem AL, Monteiro-Neto V, Vázquez A, Kaper JB, et al. Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. Proc Natl Acad Sci USA. 2007;104:10637–10642. doi: 10.1073/pnas.0704104104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rosenshine I, Ruschkowski S, Finlay BB. Expression of attaching/effacing activity by enteropathogenic Escherichia coli depends on growth phase, temperature, and protein synthesis upon contact with epithelial cells. Infect Immun. 1996;64:966–973. doi: 10.1128/iai.64.3.966-973.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schiemann DA, Shope SR. Anaerobic growth of Salmonella typhimurium results in increased uptake by Henle 407 epithelial and mouse peritoneal cells in vitro and repression of a major outer membrane protein. Infect Immun. 1991;59:437–440. doi: 10.1128/iai.59.1.437-440.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shaw RK, Berger CN, Feys B, Knutton S, Pallen MJ, Frankel G. Enterohemorrhagic Escherichia coli exploits EspA filaments for attachment to salad leaves. Appl Environ Microbiol. 2008;74:2908–2914. doi: 10.1128/AEM.02704-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Singh RD, Khullar M, Ganguly NK. Role of anaerobiosis in virulence of Salmonella typhimurium. Mol Cell Biochem. 2000;215:39–46. doi: 10.1023/a:1026545630773. [DOI] [PubMed] [Google Scholar]
  38. Sperandio V, Mellies JL, Nguyen W, Shin S, Kaper JB. Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci USA. 1999;96:15196–15201. doi: 10.1073/pnas.96.26.15196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tarr PI, Gordon CA, Chandler WL. Shigatoxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet. 2005;365:1073–1086. doi: 10.1016/S0140-6736(05)71144-2. [DOI] [PubMed] [Google Scholar]
  40. Van Immerseel F, Eeckhaut V, Boyen F, Pasmans F, Haesebrouck F, Ducatelle R. Mutations influencing expression of the Salmonella enterica serovar Enteritidis pathogenicity island I key regulator hilA. Antonie Van Leeuwenhoek. 2008;94:455–461. doi: 10.1007/s10482-008-9262-y. [DOI] [PubMed] [Google Scholar]
  41. Viswanathan VK, Koutsouris A, Lukic S, Pilkinton M, Simonovic I, Simonovic M, Hecht G. Comparative analysis of EspF from enteropathogenic and enterohemorrhagic Escherichia coli in alteration of epithelial barrier function. Infect Immun. 2004;72:3218–3227. doi: 10.1128/IAI.72.6.3218-3227.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES