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. 2012 Oct 2;3(5):e00317-12.
doi: 10.1128/mBio.00317-12. Print 2012.

EspZ of enteropathogenic and enterohemorrhagic Escherichia coli regulates type III secretion system protein translocation

Cedric N Berger  1 , Valerie F Crepin, Kobi Baruch, Aurelie Mousnier, Ilan Rosenshine, Gad Frankel
Affiliations

EspZ of enteropathogenic and enterohemorrhagic Escherichia coli regulates type III secretion system protein translocation

Cedric N Berger et al. mBio. .

Abstract

Translocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli. Consistently, an EPEC espZ mutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal "translocation stop" activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation.

Importance: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

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Figures

FIG 1
FIG 1
EPEC espZ mutant is cytotoxic. HeLa cells were infected with wild-type E2348/69, E2348/69 ΔespZ, and complemented E2348/69 ΔespZ pespZ. Uninfected cells were used as a control. Hematoxylin-and-eosin staining was used to visualize adherent cells 3 h postinfection (A), and Hoechst staining was used to visualize nuclei 1 h postinfection (C). Enumeration of the remaining cells 3 h postinfection revealed extensive cell detachment in monolayers infected with E2348/69 ΔespZ but not in the uninfected cells or cells infected with wild-type E2348/69 or E2348/69 ΔespZ complemented with espZ. Significant differences from uninfected cells are indicated by asterisks (P < 0.01) (B). While deletion of espZ induced nuclear condensation (white arrows), no difference was observed between cells infected with E2348/69 or E2348/69 ΔespZ complemented with espZ and uninfected cells (C). Using AxioVision software to enumerate cells with a condensed nucleus (<100 µm2) (D) revealed a significant increase when cells were infected with E2348/69 ΔespZ compared to uninfected cells or cells infected with wild-type E2348/69 or E2348/69 ΔespZ complemented with espZ. Significant differences from uninfected cells are indicated by asterisks (P < 0.01). Cells were untreated or treated with 50 µM zVAD-FMK 30 min prior to infection with wild-type E2348/69, E2348/69 ΔescN, E2348/69 ΔespZ, and complemented E2348/69 ΔespZ (E). LDH release increased when cells were infected with E2348/69 ΔespZ. Significant differences from uninfected cells are indicated by asterisks (P < 0.01) (see also Fig. S1 and S2 in the supplemental material).
FIG 2
FIG 2
Cells ectopically expressing espZ are resistant to EPEC-induced pedestal and filopodium formation. HeLa-GFP and HeLa-EspZ cells were infected with wild-type E2348/69 (A) or E2348/69 Δmap overexpressing Map (B). Actin was stained with TRITC-phalloidin (red), the Myc-tagged EspZ with mouse anti-Myc (green), and EPEC with rabbit anti-O127 (blue). Transfected cells with pedestals (C) or with microcolony-associated filopodia (D) were quantified by immunofluorescence. One hundred cells were counted from three independent experiments. Results are means and SD. A large proportion of HeLa-GFP cells exhibited either pedestals or microcolony-associated filopodia, whereas neither pedestals nor filopodia were observed in HeLa-EspZ cells. Significant differences are indicated by asterisks (P < 0.01). Images are representative of three replicated experiments.
FIG 3
FIG 3
Ectopic expression of espZ blocks effector translocation. HeLa or HeLa-EspZ cells were infected with E2348/69 Δtir expressing HA-tagged Tir (A), E2348/69 ΔespF expressing Flag-tagged EspF (B), E2348/69 expressing Tir-TEM (C), or E2348/69 expressing EspF-TEM (D). E2348/69 ΔescN bacteria expressing Tir-TEM or EspF-TEM fusions were used as negative controls. Myc-tagged EspZ was stained with mouse anti-Myc-FITC (green), HA-tagged and FLAG-tagged effectors with mouse anti-HA and mouse anti-Flag, respectively (red), and EPEC with rabbit anti-O127 (blue). Tir and EspF were detected in the pedestal and cytoplasm, respectively, in HeLa cells. In contrast, neither effector was detected in HeLa-EspZ cells. Images are representative of three replicated experiments. Translocation assays using TEM fusions showed that Tir and EspF were translocated into HeLa cells. Significantly reduced translocation was seen in HeLa-EspZ cells. No effector translocation was seen from the control strains. Results are the fold increase compared to uninfected cells from three independent experiments and are presented as means and SD (*, P < 0.01) (see also Fig. S5 in the supplemental material).
FIG 4
FIG 4
EspZ blocks effector translocation from a second EPEC infection wave. Cells were infected with wild-type E2348/69 or E2348/69 ΔespZ that was either untreated or treated with CCCP (carbonyl cyanide m-chlorophenylhydrazone) at the time of infection or 30 or 60 min postinfection. For immunofluorescence (A), actin was stained with TRITC-phalloidin (red) and EPEC with rabbit anti-O127 (green). LDH release (B) was measured after 2 h of infection. Treatment with CCCP, which inhibits the T3SS of EPEC, 30 min postinfection did not block pedestal formation (white arrows) but inhibited LDH release induced by E2348/69 ΔespZ. No inhibition of LDH release was observed in cells treated 60 min postinfection. Results are means and SD from three independent experiments (*, P < 0.01). Cells were either left uninfected or infected with wild-type E2348/69, E2348/69 ΔescV, or E2348/69 ΔespZ and then superinfected with E2348/69 expressing Tir-TEM (C) or EspF-TEM fusions (D). Translocation of effectors by the second wave of bacteria was monitored using a real-time translocation assay on CCF2-preloaded cells. Translocation of Tir and EspF was reduced in cells preinfected with wild-type E2348/69. In contrast, similar translocation kinetics were recorded in uninfected control cells or cells infected with E2348/69 ΔescV or E2348/69 ΔespZ, suggesting that EspZ can block effector translocation of a second wave of infection. Results are means and SD from three independent experiments (*, P < 0.01).
FIG 5
FIG 5
The activity of EspZ is serotype specific. HeLa-GFP, HeLa-EspZEPEC, or HeLa-EspZEHEC cells were infected with EPEC strains E2348/69 (O127:H6) and B171 (O111:H−) (B), EPEC strains ICC219 (O57:H6), E22 (O103:H2), and ICC57 (O55:H7) (C), and EHEC 85-170 (O157:H7). Actin was stained with TRITC-phalloidin (red), Myc-tagged EspZ was detected with mouse anti-Myc (green), and DNA was detected with Hoechst (blue). Transfected cells with pedestals were quantified by immunofluorescence (A). One hundred cells were counted from five independent experiments. Results are means and SD. Pedestals were observed in HeLa-GFP and HeLa-EspZEPEC cells infected with ICC57 or 85-170 (B). No pedestals were observed in HeLa-EspZEHEC cells. Significant differences from HeLa-GFP cells are indicated by asterisks (P < 0.01).
FIG 6
FIG 6
The extracellular loop of EspZ determines strain specificity. HeLa cells transfected with GFP, EspZEHEC, EspZEPEC, HeLa-EspZEHEC with the EPEC loop, or EspZEPEC with the EHEC loop were infected with EHEC EDL933 Δtir expressing HA-tagged Tir (A). Actin was stained with RRX-phalloidin (red), HA-tagged Tir with mouse anti-Myc (green) and EPEC with rabbit anti-O157 (blue). Transfected cells with pedestals were quantified after immunofluorescence (B). One hundred cells were counted from five independent experiments. EHEC formed pedestals associated with Tir staining in HeLa-GFP, HeLa-EspZEPEC or HeLa-EspZEHEC with the loop of EPEC but not in HeLa-EspZEHEC or HeLa-EspZEPEC with the loop of EHEC. Results are presented as means ± SD. Significant differences from HeLa-GFP cells are indicated by asterisks (P < 0.01). (c) LDH release was measured from cells infected for 3 h with wild-type 85-170, 85-170 ΔespZ, or 85-170 ΔespZ expressing EHEC EspZ, EPEC EspZ, EHEC EspZ with the EPEC loop, or EPEC EspZ with the EHEC loop. LDH release increased when cells were infected with 85-170 ΔespZ, 85-170 ΔespZ expressing EPEC EspZ, or EHEC EspZ with the EPEC loop but not when cells were infected with 85-170 ΔespZ expressing EHEC EspZ. Partial complementation was observed with 85-170 ΔespZ expressing EHEC EspZ and EPEC EspZ with the EHEC loop. Significant differences from uninfected cells or from cells infected with 85-170 ΔespZ are indicated (* and #, respectively; P < 0.01) (see also Fig. S7 in the supplemental material).
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