Secretin of the Enteropathogenic Escherichia coli Type III Secretion System Requires Components of the Type III Apparatus for Assembly and Localization

Bacterial strains.The wild-type EPEC strain E2348/69 (streptomycin resistant) was used in this study. Strains were grown in Luria-Bertani broth supplemented with appropriate antibiotics as standing overnight cultures at 37°C.

Construction of nonpolar mutants. (i) escC deletion mutant.As detailed by de Grado et al. (9), oligonucleotides ESCC-01F (XhoI restriction site) and ESCC-02R (SacI restriction site) were used to amplify escC with chromosomal DNA from EPEC E2348/69. The amplification product was cloned into pCR2.1 TOPO (Invitrogen), generating pCR-escC. Primers ESCC-03R and ESCC-04F were used to create an in-frame deletion of the escC gene in pCR-escC by using inverse PCR amplification. Both oligonucleotides ESCC-03R and ESCC-04F introduced an MluI restriction site. The 2,049-bp SacI-XhoI escC deletion fragment was cloned into the positive-selection suicide vector pCVD442 (13), which had been digested with SacI-SalI. The resulting plasmid, pCVD442-ΔescC, was used to construct the escC deletion mutant in EPEC E2348/69 (streptomycin resistant) by allelic exchange as described previously (13), generating the EPEC ΔescC strain (9).

(ii) escV deletion mutant.The EPEC ΔescV strain was constructed as described above by using primers ESCV-05F, ESCV-06R, ESCV-07R, and ESCV-08F.

(iii) escN deletion mutant.The EPEC ΔescN strain was constructed as described above by using primers ESCN-09F, ESCN-10R, ESCN-13R, and ESCN-14F, except that a BstEII site was introduced.

Construction of plasmids expressing escC, escV, escN, and their HSV-tagged versions.By using the appropriate primer pairs (Table 1), esc genes were amplified from EPEC E2348/69, introducing BamHI and SalI restriction sites. The amplified products were cloned into the BamHI-SalI sites in pACYC184 (New England Biolabs), leaving the genes under the control of the tetracycline resistance gene promoter, thus generating pEscC, pEscV, and pEscN. By using the appropriate primer pairs (Table 1), esc genes were amplified from EPEC E2348/60, introducing BamHI and NheI restriction sites. The amplified products were cloned into the BamHI and NheI sites in pET27b(+) (Novagen) in order to be tagged with herpes simplex virus (HSV), creating an in-frame translational fusion on the 3′ end of the gene (corresponding to the C terminus of the protein). Esc-HSV-tagged genes were amplified from the pET27b derivatives and cloned into the BamHI-SalI sites in pACYC184, leaving the genes under the control of the tetracycline resistance gene promoter, thus generating pEscC-HSV, pEscV-HSV, and pEscN-HSV.

Bacterial fractionation.Bacterial cell fractionation was based on previously described procedures (17, 28, 47). Briefly, EPEC from an overnight standing culture was subcultured 1/50 in 100 ml of minimal essential medium for 3 h at 37°C under 5% CO₂. The culture was harvested, washed in phosphate-buffered saline, and resuspended in 1 ml of 50 mM Tris (pH 7)-20% sucrose with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 μg of leupeptin/ml, 1 μM pepstatin, 10 μg of aprotinin/ml). Cells were treated with 40 μl of 0.25 M EDTA-0.25 mg of lysozyme/ml (final concentrations, 10 mM and 10 μg/ml, respectively) for 10 min at room temperature. All subsequent steps were carried out at 4°C. The periplasmic fraction was isolated from the rest of the cells by centrifugation (at 8,000 × g for 10 min). The pellet was resuspended in 1 ml of sonication buffer (10 mM Tris-HCl [pH 7] and protease inhibitors) and sonicated three times for 15 s each time (Fisher Sonicator, amplitude 2.5). Unbroken bacteria were removed by centrifugation (at 16,000 × g for 2 min), and the cleared supernatant containing cytoplasmic proteins and inner and outer membranes was removed and centrifuged (in a Beckman TLA 100 Ultracentrifuge with a TLA100.3 rotor) for 1 h at 50,000 × g to pellet the membranes. The supernatant containing the cytoplasmic fraction was removed; the membrane pellet was washed with sonication buffer, resuspended in 0.1 ml sonication buffer with 0.5% N-lauroylsarcosine, which selectively solubilizes the inner membrane, and centrifuged (at 50,000 × g for 1 h); the supernatant containing the inner membrane fraction was removed; and the outer membrane pellet was washed in sonication buffer with 0.5% N-lauroylsarcosine. The final pellet was resuspended in 0.1 ml of sonication buffer with 0.5% N-lauroylsarcosine and 0.1% sodium dodecyl sulfate (SDS). The protein content of all samples was determined by using the Bio-Rad protein assay (periplasmic fraction) or the bicinchoninic acid (BCA) protein assay (Sigma) (other fractions) before addition of SDS sample buffer with β-mercaptoethanol. The above fractionation protocol yielded approximately 0.33 mg of protein in the periplasmic fraction, 3.3 mg of protein in the cytoplasmic fraction, and 0.1 mg of protein in each of the inner membrane and outer membrane fractions.

Phenol treatment.To reduce the EscC complex seen in the outer membrane fraction to the monomer, outer membrane pellets obtained by the procedure described above were resuspended in 0.1 ml of sonication buffer and treated with phenol as described previously (21). Briefly, outer membrane samples were extracted with 0.1 ml of buffer-saturated phenol (Gibco BRL) at 70°C for 10 min, cooled to 4°C, and centrifuged at 5,000 × g for 10 min. The upper aqueous layer (which had no detectable protein) was discarded. The interface and the phenol layer were treated with 0.1 ml of distilled water at 70°C again, and the aqueous phase was discarded after centrifugation as described above. The proteins in the organic phase were precipitated with 2 volumes of acetone at 4°C overnight and then pelleted by centrifugation (at 16,000 × g for 20 min). The pellet was washed with acetone, and once with 2 volumes of ether, and was collected by centrifugation. The pellet was air dried before being resuspended in 0.1 ml of sonication buffer with 0.5% N-lauroylsarcosine and 0.1% SDS. The protein content of all samples was determined by using a BCA protein assay before addition of SDS sample buffer with β-mercaptoethanol.

Immunoblotting.Samples (10 μg) were subjected to SDS-PAGE and transferred to nitrocellulose filters (pure nitrocellulose; pore size, 0.45 μm; Bio-Rad). Blots were blocked overnight at 4°C in 5% (wt/vol) skim milk-TTBS (0.1% Tween 20-Tris-buffered saline), incubated first with the primary antibody diluted in 1% skim milk-TTBS for 1 h at room temperature and then with the secondary antibody diluted in 1% skim milk-TTBS for 1 h at room temperature, and detected with the ECL reagent (Amersham). Primary antibodies were a mouse anti-Tir monoclonal antibody, clone 2A8 (8), diluted 1/1,000 and preabsorbed with a fixed EPEC tir mutant; a mouse anti-EspB monclonal antibody, clone 2A11, diluted 1/100; mouse anti-DnaK (Stressgen), diluted 1/1,000; rabbit anti-maltose binding protein (anti-MBP) (New England Biolabs), diluted 1/3,000; rabbit anti-intimin, diluted 1/3,000, and rabbit anti-Etk (diluted 1/3,000) (kind gifts from I. Rosenshine); and mouse anti-HSV (Novagen), diluted 1/1,000. Secondary antibodies were a horseradish peroxidase-conjugated goat anti-mouse antibody (Jackson ImmunoResearch), diluted 1/5,000, and a horseradish peroxidase-conjugated goat anti-rabbit antibody (Sigma), diluted 1/5,000.

EscC, EscV, and EscN are required for functional type III translocation by EPEC.As an initial examination of the type III secretion pathway, and to investigate the function of three type III apparatus proteins that are predicted to be located in different compartments of EPEC and that are highly conserved in other TTSSs, nonpolar deletion mutants were constructed for the genes encoding EscN, EscV, and EscC. We assayed for functional type III translocation by EPEC by examining whether pedestals formed in HeLa cells infected with wild-type EPEC or the mutants by immunofluorescence microscopy. While wild-type EPEC formed microcolonies and accumulated phosphotyrosine-containing proteins and actin under the bacteria, the escC, escV, and escN mutants did not form pedestals (data not shown). Lack of secretion of Esps and Tir, and lack of Tir translocation into HeLa cells, also confirmed the essential role of these three proteins in the functionality of the type III apparatus (18) (data not shown).

To demonstrate that the mutations were nonpolar, the three mutants were rescued with the appropriate esc gene. escC, escV, and escN were amplified and cloned either untagged or HSV tagged on the 3′ end (corresponding to the C terminus) into pACYC184. The resulting plasmids carrying either the untagged or the HSV-tagged esc genes (Table 1) were transformed into the corresponding mutants, which were then able to form pedestals on HeLa cells, as demonstrated by immunofluorescence microscopy (data not shown). Therefore, the mutations of the escC, escV, and escN genes were nonpolar and could be complemented, and the epitope tags did not interfere with the function of these three proteins.

Tir and EspB accumulate in the cytoplasm of type III mutants.In order to investigate the pathway of secretion through the type III apparatus, Tir and EspB distribution was examined in wild-type EPEC and type III mutants. Bacteria were fractionated into periplasmic, cytoplasmic, and inner and outer membrane fractions by lysozyme treatment, ultracentrifugation, and selective detergent extraction with sarcosyl, respectively, as detailed in Materials and Methods. While multiple methods exist for fractionating bacteria, this method was chosen because it is well established (17, 47) and, perhaps more importantly, has previously been used successfully to fractionate EPEC (28). The sarcosyl method takes advantage of the differing lipid properties of the inner and outer membranes. Sarcosyl has the same charge density as lipopolysaccharide (LPS), and it is thought that because it mimics LPS, it does not solubilize the LPS-based outer membrane (47). As a control for clean bacterial fractionation, samples (10 μg of protein of each fraction) were probed with anti-DnaK, anti-Etk, anti-intimin, and anti-MBP (Fig. 1B). DnaK is an abundant heat shock protein that functions as a chaperone inside the bacterial cytoplasm (40) and was found in the cytoplasmic fraction. Etk is an E. coli inner membrane tyrosine kinase (28) and was found in the inner membrane fraction. Intimin is the outer membrane ligand in EPEC (29) and is subject to N-terminal processing. As expected, the processed form was found in the outer membrane and the preprocessed form and a small amount of the processed form were found in the inner membrane. MBP is a periplasmic protein (30) and was found primarily in the periplasmic fraction but was also detected in the cytoplasmic fraction. Other methods for isolating clean periplasm, including freeze-thawing, various concentrations of lysozyme, and osmotic shock, were explored, but all methods resulted in significant contamination of the periplasmic fraction with cytoplasmic proteins (data not shown). Because our method resulted in a noncontaminated periplasmic fraction, it was appropriate for the purposes of this study. While the marker proteins were localized correctly by using this bacterial fractionation technique, this does not necessarily indicate that the type III apparatus and associated proteins would fractionate as cleanly, since it potentially spans both membranes. Sucrose density and flotation gradients were not successful at separating the inner and outer membranes (data not shown). The sarcosyl treatment released predicted inner membrane components of the TTSS (see the next section) and was thus used extensively for this study.

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FIG. 1.

Tir and EspB accumulate in the cytoplasm of type III mutants. Bacteria were fractionated into periplasmic (P), cytoplasmic (C), and inner membrane (IM) and outer membrane (OM) fractions by lysozyme treatment, ultracentrifugation, and selective detergent extraction, respectively. Samples (10 μg of each fraction) were resolved by SDS-10% PAGE and transferred to a nitrocellulose filter. (A) Western blots were probed with an anti-Tir or anti-EspB antiserum. (B) Western blots were probed with an anti-DnaK, anti-MBP, anti-Etk, or anti-intimin antiserum to monitor for contamination of fractions.

In wild type EPEC, Tir and small amounts of EspB were detected in the cytoplasmic and inner membrane fractions (Fig. 1A), but most was secreted into the culture medium (data not shown). All three type III mutants had greater amounts of Tir and EspB detected in the cytoplasmic and inner membrane fractions than wild-type EPEC. All three mutants showed the same pattern of Tir and EspB distribution. The three mutants were rescued to give approximately a wild-type EPEC distribution of Tir and EspB when complemented in trans with the appropriate untagged (data not shown) or HSV-tagged (Fig. 1A) esc gene. The differences are likely due to the unbalanced expression of the genes that is driven by the tetracycline promoter of the plasmid, which uncouples the production of these proteins from the rest of the type III apparatus.

Localization of EscC, EscV, and EscN.We next examined the location of EscC, EscV, and EscN by using the bacterial fractionation method and detecting the C-terminal HSV tag. EscC is predicted to be a 56-kDa preprotein with a putative signal sequence that has a cleavage site at amino acid 19, which would result in a 54-kDa outer membrane protein upon export by the sec-dependent secretion pathway. EscC-HSV was detected as a 60-kDa protein in the cytoplasmic and inner membrane fractions (Fig. 2A). A faint band was detectable above the 60-kDa band in the inner membrane fraction and the cytoplasm (at longer exposures); this most likely corresponds to the preprocessed form. Notably, we did not detect EscC in the outer membrane fraction. We hypothesized that boiling of the sample may have led to aggregation and precipitation of EscC from this fraction, as has been observed for other membrane proteins (66). When the samples were not boiled prior to electrophoresis, a high-molecular-weight form of EscC was detectable by Western blotting in the stacking gel in the inner membrane and outer membrane fractions. The high-molecular-weight complex in the outer membrane can be dissociated by phenol treatment to yield monomeric EscC (Fig. 2B). These results are analogous to those obtained with other EscC homologues (12, 22, 35, 54).

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FIG. 2.

Localization of EscC, EscV, and EscN. Bacteria were fractionated into periplasmic (P), cytoplasmic (C), and inner membrane (IM) and outer membrane (OM) fractions by lysozyme treatment, ultracentrifugation, and selective detergent extraction, respectively. Samples (10 μg of each fraction) were resolved by SDS-10% PAGE, transferred to a nitrocellulose filter, and probed with anti-HSV antisera. (A) Asterisks indicate samples that were not boiled prior to electrophoresis. (B) The outer membrane fraction was treated with phenol prior to addition of SDS sample buffer.

EscV is predicted to be a 75-kDa preprotein with a putative sec-dependent signal sequence, cleavable at residue 28, yielding a 72-kDa inner membrane protein with 7 predicted transmembrane domains. Virtually all of the EscV-HSV was found in the inner membrane fraction (Fig. 2A). The major band of EscV-HSV had an apparent molecular mass of 64 kDa in the inner membrane, although a smear of protein was detected at higher molecular weights, suggesting that EscV forms SDS-resistant complexes. Smearing was reduced by not boiling the samples prior to electrophoresis (Fig. 2A).

EscN is predicted to be a 49-kDa cytoplasmic protein, with no signal sequence. EscN-HSV was detected at the predicted molecular mass in both the cytoplasmic and inner membrane fractions (Fig. 2A). Since EscN does not have any predicted transmembrane domains, it is unlikely to be a membrane protein. Furthermore, EscN is not found in the periplasmic fraction, suggesting an association with the cytoplasmic side of the inner membrane or with other inner membrane components of the TTSS (Fig. 2A). A less-abundant 60-kDa band was also detected in the inner membrane.

EscC localization is dependent on EscV and EscN.To determine whether the localization of EscC, EscV, or EscN was affected by other type III apparatus components, a series of trans-complementation experiments were performed. As seen in Fig. 3A, the localization of EscV-HSV was not affected in the escC and escN mutants. Similarly, the localization of EscN-HSV was not affected in escC and escV mutants transformed with pEscN-HSV.

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FIG. 3.

Localization of EscV and EscN does not require other examined type III apparatus components. Bacteria were fractionated into periplasmic (P), cytoplasmic (C), and inner membrane (IM) and outer membrane (OM) fractions by lysozyme treatment, ultracentrifugation, and selective detergent extraction, respectively. Samples (10 μg of each fraction) were resolved by SDS-10% PAGE, transferred to a nitrocellulose filter, and probed with anti-HSV antisera. (A) Localization of EscV-HSV. (B) Localization of EscN-HSV.

Based on its putative cleavable signal sequence, EscC is predicted to be exported across the inner membrane via the sec-dependent pathway and inserted into the outer membrane. The secretin InvG in Salmonella serovar Typhimurium has been shown to have a cleavable sec-dependent signal sequence (37). Wild-type EPEC transformed with pEscC-HSV was fractionated to ensure that any altered distribution of EscC-HSV in the escV and escN mutants was not due to competition with wild-type EscC expressed from the chromosomal copy of the gene (Fig. 4A). ΔescC/pEscC-HSV and wild-type EPEC/pEscC-HSV had the same pattern of EscC-HSV distribution: monomeric EscC was detected in the cytoplasmic and inner membrane fraction, and an EscC complex was detected in the outer membrane fraction when the samples were not boiled prior to electrophoresis. In contrast, when trans-complementation experiments were performed to examine the location of EscC-HSV in escV and escN mutants, they revealed that EscC localization was altered (Fig. 4B). A large quantity of monomeric EscC was detected in the periplasmic fractions of escV and escN mutants, compared to no detectable EscC in the periplasmic fraction of wild-type EPEC or ΔescC/pEscC-HSV. Furthermore, the monomeric EscC was also more abundant in the inner membrane fraction, but much less EscC was observed in the outer membrane fraction of the trans-complemented strains. Equivalent amounts of EscC-HSV were produced by the mutants and wild-type EPEC, suggesting that the altered distribution was not due to altered expression (data not shown). A similar pattern of EscC distribution in the mutants was also seen by using a suboptimal polyclonal antibody directed against EscC (data not shown), indicating that these results are not an artifact due to the presence of the HSV tag. We next investigated if EscC outer membrane insertion occurs before or after type III needle assembly, by examining the location of EscC-HSV in a needle component mutant (escF) (kindly provided by Akio Abe) (58). EscC-HSV was correctly localized in the escF mutant, giving the same pattern of distribution as the wild type and the ΔescC mutant (Fig. 4C). This indicates that EscC localization is independent of needle formation. Collectively, these data strongly suggest that insertion of EscC into the outer membrane requires both the TTSS and the sec-dependent secretion system.

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FIG. 4.

EscC localization is dependent on EscV and EscN but not on EscF. Bacteria were fractionated into periplasmic (P), cytoplasmic (C), and inner membrane (IM) and outer membrane (OM) fractions by lysozyme treatment, ultracentrifugation, and selective detergent extraction, respectively. Samples (10 μg of each fraction) were resolved by SDS-10% PAGE, transferred to a nitrocellulose filter, and probed with anti-HSV antisera. (A) EscC-HSV distribution is the same in wild-type (wt) EPEC and the ΔescC mutant. Asterisks indicate samples that were not boiled prior to electrophoresis. (B) EscC-HSV distribution in ΔescC, ΔescV, and ΔescN mutants. The outer membrane fraction was treated with phenol prior to addition of SDS sample buffer. (C) EscC-HSV distribution in the wild type and the ΔescC and ΔescF mutants. The outer membrane fraction was treated with phenol prior to addition of SDS sample buffer.