The Serine Protease EspC from Enteropathogenic Escherichia coli Regulates Pore Formation and Cytotoxicity Mediated by the Type III Secretion System

Type III secretion systems (T3SSs) are specialized macromolecular machines critical for bacterial virulence, and allowing the injection of bacterial effectors into host cells. The T3SS-dependent injection process requires the prior insertion of a protein complex, the translocon, into host cell membranes consisting of two-T3SS hydrophobic proteins, associated with pore-forming activity. In all described T3SS to date, a hydrophilic protein connects one hydrophobic component to the T3SS needle, presumably insuring the continuum between the hollow needle and the translocon. In the case of Enteropathogenic Escherichia coli (EPEC), the hydrophilic component EspA polymerizes into a filament connecting the T3SS needle to the translocon composed of the EspB and EspD hydrophobic proteins. Here, we identify EspA and EspD as targets of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE). We found that in vitro, EspC preferentially targets EspA associated with EspD, but was less efficient at proteolyzing EspA alone. Consistently, we found that EspC did not regulate EspA filaments at the surface of primed bacteria that was devoid of EspD, but controlled the levels of EspD and EspA secreted in vitro or upon cell contact. While still proficient for T3SS-mediated injection of bacterial effectors and cytoskeletal reorganization, an espC mutant showed increased levels of cell-associated EspA and EspD, as well as increased pore formation activity associated with cytotoxicity. EspP from enterohaemorrhagic E. coli (EHEC) also targeted translocator components and its activity was interchangeable with that of EspC, suggesting a common and important function of these SPATEs. These findings reveal a novel regulatory mechanism of T3SS-mediated pore formation and cytotoxicity control during EPEC/EHEC infection.


Bacterial culture and Priming
The bacterial strains and plasmids used in this study are listed in Table 1. All strains were grown in Luria-Bertani broth (LB) (17 hr at 37°C, 200 rpm). To activate the type III secretion system, EPEC strains were primed by diluting overnight LB culture (1:100 dilution) into DMEM containing 1g/L glucose and incubated at 37°C in a 5% CO 2 incubator for the indicating times [9]. When required, ampicillin was added to the medium at 100 µg/mL. When strains carrying pJLM174 were used, LB broth or DMEM were supplemented with 0.2% arabinose to induce or 0.2% glucose to repress EspC expression.

Antibodies and reagents
Rabbit polyclonal antibody directed against EspC was produced by Genescript Corporation (USA) using a KLH conjugate peptide (CNQVSSFEQPDWENR). Mouse monoclonal anti-EspB (clone B292) and anti-EspD (clone A84), kindly provided by F. Ebel, were described previously [10]. Rabbit polyclonal anti-Tir and anti-EspA have been described previously [11,12]. Anti-caspase 3 antibodies were from Cell signalling. Alexa-conjugated secondaries antibodies as well as Alexa-conjugated Phalloïdin were from Invitrogen (Life technology) and used at a dilution of 1:200 for immunofluorescence. The horseradish peroxidase-coupled antibodies were from Jackson Immunoresearch (West Grove, PA) and used at 1:20000 for Western blot analysis. Anti-actin directly coupled to horseradish peroxidase was from Cell Signaling and used at 1:20,000.
Phenylmethylsulfonyl fluoride (PMSF), Lucifer yellow CH dilithium salt (LY), the caspase inhibitor zVAD, and DAPI were from Sigma. The Complete TM Protease inhibitor cocktail was from Roche diagnostic and used following the manufacturer's instructions.

Immunofluorescent staining
For staining of bacteria grown in DMEM or of eluted samples corresponding to the peak A and A/D of the MonoQ anion exchange chromatography, samples were incubated for 15 min at 21°C with coverslips that were previously coated with poly-L-lysine at a final concentration of 50 µg / ml prior to fixation for 20 min in PBS containing 3.7 % paraformaldehyde (PFA).
For cell challenge studies, following incubation with bacteria at indicated time, samples were washed with PBS and fixed with PFA. Following fixation, samples were blocked for at least 60 min at 21°C in PBS containing 10 % FCS. For infected cells, samples were permeabilized by incubation in PBS containing 0.1 % Triton-X100 for 4 min at 21°C. Samples were processed for immunofluorescent staining as previously described, using anti-EspA antibody at a dilution of 1:400, or anti-EspD antibody at a dilution of 1:300.

Cloning and generation of deletion mutants of bfpA, espC, and espP genes in EPEC and EHEC
The EHEC espP gene was amplified with its RBS sequence using the synthetic

Translocation assay
Cells were infected for 45 min at 37°C with primed bacteria. Monolayers were washed 3 times with ice-cold PBS containing 0.1 mM CaCl 2 , 1 mM MgCl 2 , and solubilized in 300 µL ice-cold PBS containing protease inhibitors and 0.1% Triton X-100 (TX100). All subsequent steps were performed on ice. Samples were scraped with a rubber policeman, transferred to a Dounce homogenizer, and subjected to 20 strokes. An aliquot of total cell lysates was analysed by Western blotting, while the rest were subjected to two successive centrifugations for 5 min at 20 000 g to remove cell debris and bacteria. Supernatants corresponding to the Triton-X100 soluble fraction containing the translocated proteins were analyzed by Western blotting using the highly sensitive detection reagent (SuperSignal West Femto Chemiluminescent Substrate, Thermo Scientific).

Proteinase K digestion
Insoluble forms of EspA, EspB and EspD released during overnight bacterial culture in DMEM were analyzed by proteinase K digestion. Bacterial pellets were incubated in DMEM containing 1 µg / mL proteinase K for 20 min at 20°C. Proteinase K was inactivated by addition of protease inhibitors (Complete TM , Roche Pharmaceuticals) followed by immediate addition of Laemmli sample buffer and incubation for 10 min at 100 °C. Samples were subjected to Western blot analysis.

Caspase-3 activation
HeLa cells were infected with primed EPEC strains for 45 min at 37°C. The medium was removed and samples were further incubated for 1 additional hour in fresh medium.
Gentamicin was added to kill bacteria and samples were incubated for 17h. When mentioned, zVAD was added and maintained during all the duration of the experiment. Cells were scrapped in Laemmli sample buffer, transferred to an eppendorf tube and incubated for 10 min at 100 °C. Samples were subjected to Western blot analysis using antibodies as mentioned. Staurosporine was used at a final concentration of 1µM as a positive control for caspase-activation.