YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal^*

Bacterial Strains, Plasmids, and Growth Conditions

Bacterial strains and plasmids used in this study are listed in supplemental Table S1. Standard molecular biology methods were used to generate the different plasmid constructs used in this study. The PCR primers used in the different cloning strategy are listed in supplemental Table S2. For the complementation assay of YopE secretion, the divergent yerA-yopE operon was cloned into pBADmycHis A. The expression of yopE and yerA is under control of their native promoter and co-regulated with the T3S machinery. The different yopE variants were cloned similarly. The sequences of all the constructs were systematically checked (Eurofins MWG Operon). The pET-yscN plasmid was generated by GenScript Corporation. Escherichia coli strains were grown in Luria-Bertani broth (LB) or on Luria agar plates at 37 °C. Y. pseudotuberculosis strains were grown at 26 °C in LB or on Luria agar plates. Antibiotics were added to the medium for selection according to the resistance markers carried by the plasmid. The following concentrations were used: kanamycin, 50 μg/ml and carbenicillin, 100 μg/ml.

Yop Secretion Analysis

To induce Yops secretion, Yersinia pseudotuberculosis strains were first grown at 26 °C for 2 h in Ca²⁺-depleted LB medium (medium containing 5 mm EGTA and 20 mm MgCl₂) and shifted at 37 °C for 3 h. Cultures were started at an A₆₀₀ of 0.1. Samples were treated as described previously (26) and separated on Tris-Tricine polyacrylamid gels. Proteins were either stained with Coomassie R250 or, alternatively, transferred onto a PVDF membrane for immunoblotting. Anti-Yop, anti-YscU, anti-YscP, anti-DnaJ, and anti-YscF antibodies were diluted 1:5,000. Horseradish peroxidase-conjugated anti-rabbit antibody was diluted at 1:10,000 (GE Healthcare). Proteins were detected with a chemiluminescence detection kit (GE Healthcare). Quantification by densitometry was made using Multi Gauge software (Fuji film). The bands to be quantified were selected and quantitated after background subtraction.

HeLa Cell Cytotoxicity Assay

Yersinia cultures were started at an A₆₀₀ of 0.1 in LB medium containing 1 mm Ca²⁺. After 1 h of growth at 26 °C, cultures were shifted at 37 °C for 2 h. HeLa cells were infected for 45 min at multiplicity of infection of 10 and 20. The cytotoxicity was assayed as previously described by Rosqvist and coworkers with some modifications (3). For immuno-staining samples were subsequently fixed with 4% PFA and permeabilized with 0.5% Triton X-100 (Sigma Aldrich). Nonspecific binding were prevented by treatment with PBS, 0.1 m glycine, and PBS, 1% bovine serum albumin. Alexa Fluor 488 phalloidin (Life Technologies) and DAPI were used to, respectively, stain cells actin cytoskeleton and nucleic acids. Samples were mounted in mounting medium (Dako) and examined with a fluorescence microscope (Nikon Eclipse C1 plus).

Co-purification Assay

For YscN/YopE interaction analysis, the plasmid encoding His₆-YscN was introduced into BL21(DE3) strain. Cells were grown at 37 °C. At A₆₀₀ = 0.7 protein production was induced by addition of 1 mm IPTG for 2 h. Cells were pelleted 10 min at 6,000 × g. Cell pellets were resuspended in PBS, 1% Triton X-100, and sonicated on ice. Broken cells were centrifuged 20 min at 20,000 × g at 4 °C, and the supernatant was incubated with HIS-Select Nickel Affinity Gel (Sigma Aldrich) for 1 h at 4 °C. Resin was washed, resuspended in PBS, 0.1% Triton X-100, and used in an interaction assay. Yersinia strains bearing plasmids encoding different YopE constructs were grown in conditions that allow induction of the T3SS. Cells were lysed as described above, and supernatants were incubated with gel-bound YscN for interaction. After 2 h of gentle rocking at 4 °C, samples were washed with PBS, 0.1% Triton X-100. Proteins that remained bound to the gel were eluted using SDS-PAGE sample buffer. Sample were separated on SDS-PAGE and transferred onto PVDF membrane for immuno-detection. Similar protocol was used for GST-YscU_CC/YscN interaction with some modifications. GST constructs were bound to glutathione-Sepharose resin (GE Healthcare), and His6-YscN was in solution for the interaction assay.

GST-YscU_CC Is Secreted and Interferes with Yops Secretion

Earlier results have shown that YscU_CC is secreted by the T3SS (26), and we were therefore interested to localize the minimal T3S signal within YscU_CC. A classical approach to identify and characterize secretion signals is to generate hybrid proteins with reporter proteins such as GFP, adenylate cyclase, or GST (glutathione S-transferase) and follow the secretion pattern of these hybrids. We therefore generated GST-tagged YscU_CC variants and introduced the plasmids expressing these constructs into the Yersinia pseudotuberculosis strain, YPIII/pIB29 (MEK). To induce the T3SS, bacteria were grown in Ca²⁺-depleted medium and shifted from 26 °C to 37 °C. YscU_CC-GST (Ucc-GST), in contrast to GST and GST-YscU_CC (GST-U_CC), could not be detected in the cell lysates of both Yersinia and E. coli Bl21 (DE3) strains after induction. This hybrid protein was therefore not further studied. The level of Yops detected in the supernatant of the strain expressing gst-U_CC was around 70% lower than the level observed for the strain expressing only gst (Fig. 1A, top panel compare lanes 1 and 2). Next we tested whether GST-U_CC was secreted by the T3SS. For this, anti-GST antibodies were used to detect GST in the supernatants and pellets from strains expressing gst and gst-U_CC. Two bands were detected in the supernatant of the strain expressing gst-U_CC (Fig. 1A, middle panel). The high molecular weight band (36 kDa) corresponds to GST-U_CC while the lower band (25 kDa) corresponds to GST, most likely generated by degradation of the hybrid protein. No band was detected in the supernatant of the strain expressing gst only (Fig. 1A, middle panel). These results show that YscU_CC fused to the C terminus of GST promotes secretion of the GST-U_CC hybrid protein. Furthermore, GST-U_CC was not detected in the supernatant when the bacteria were grown in medium supplemented with Ca²⁺ (data not shown), demonstrating that GST-U_CC secretion is dependent of the T3SS. Similar amounts of GST and GST-U_CC were detected in the cell pellets showing that the absence of GST in the supernatant was not linked to expression defect or protein instability (Fig. 1A, lower panel).

YscU_CC Contains a C-terminal Secretion Signal

The data presented above indicated that unlike Yops, YscU_CC does not need to be located at the N terminus to promote secretion of GST through the T3SS. In contrast, our results suggested that YscU_CC harbors a C-terminal T3S signal. To define this putative C-terminal secretion signal, truncated variants of GST-U_CC were generated by deleting the three and six last residues of YscU_CC (GST-U_CCΔ3 and GST-U_CCΔ6). GST-U_CCΔ3 and GST-U_CCΔ6 also interfered with Yops secretion but at a lower extent than GST-U_CC (Fig. 1A, upper panel). Interference in Yop secretion by GST-YscU_C has been observed before by Riordan and Schneewind (30). The authors proposed that interaction sites within YscU_C mediate GST-YscU_C interaction with some components of the machinery (e.g. YscL, YscQ, and YscK), which may interfere in Yop secretion. It is possible that GST-YscU_CC, GST-U_CCΔ3, and GST-U_CCΔ6 interfere in Yop secretion in a similar manner. However, YscU is a critical secretion regulator; it is then most plausible that the interference in Yop secretion is due to increased level of YscU_C that may hamper the effectors secretion regulation. Nevertheless, neither GST-U_CCΔ3 nor GST-U_CCΔ6 was detected in the culture supernatant indicating that those two construct are not secreted by the T3SS (Fig. 1A, middle panel). Similar amounts of GST and GST-U_CC proteins were detected in the cell pellets demonstrating that GST-U_CCΔ3 and GST-U_CCΔ6 are produced and stable (Fig. 1A, lower panel). Altogether, these data show that the absolute C terminus of YscU_CC harbors a T3S signal.

To further explore the C-terminal T3S signal of YscU_CC, GST-U_CC constructs with large deletions in the N-terminal region of YscU_CC were generated. According to the atomic structure of YscU_CC residues Leu³²¹ to Ile³²⁶ form a random coil that is followed by an α-helix ranging from Pro³²⁷ to Arg³³⁹ (Fig. 2A) (32). The structural organization of the last 15 residues (Trp³⁴⁰ to Leu³⁵⁴) remains unknown (Fig. 2A). Based on these structural features three variants were produced: GST-U_CC34, GST-U_CC28, and GST-U_CC15 (Fig. 2A). GST-U_CC34 contains the 34 last residues that form a random coil and the α-helix. In the GST-U_CC28 variant, the random coil is no longer present. GST-U_CC15 contains only the last 15 residues of YscU_CC. These three variants were introduced in Y. pseudotuberculosis YPIII/pIB29(MEK) strain and probed for secretion. Immuno-detection of GST in the cell pellets showed that these constructs were stable and expressed at equivalent levels (Fig. 1B, lower panel). While GST remained cytoplasmic, GST-U_CC34, GST-U_CC28, and GST-U_CC15 were detected in the supernatant showing that those three variants were secreted via the T3SS (Fig. 1B, middle panel). These data show that YscU_CC harbors a C-terminal T3S signal and that the 15 last residues are sufficient to promote secretion of GST. After cleavage at the NPTH motif, YscU_CC forms a stable complex with YscU_CN. The crystal structure of the YscU_CN/YscU_CC complex showed that YscU_CC N-terminal residues are fully or partially buried within the structure while the C-terminal residues are most likely exposed and unstructured (Fig. 2A) (32). These structural features of YscU_CC make the N-terminal residues not accessible for interaction with the ATPase YscN in order to be secreted. On the other hand, the C-terminal residues of YscU_CC, may constitute an alternative to overcome this structural situation.

C-terminal Targeting of YopE to the T3SS

It is known that most of the late T3S substrates interact with specific T3S-chaperones (35). T3S-chaperones bind to N-terminal region of the effectors (residues 25 to 100) to maintain the effectors in a partially unfolded state that facilitate interaction with the T3S-machinery and subsequent secretion. Here we identified and characterized within YscU_CC, the first C-terminal T3S signal. It is plausible that YscU_CC is an exception to the N-terminal location of the T3S signal. However, it is an opportunity to study the separation of the secretion signal from the chaperone binding site by putting the last 15 residues of YscU_CC (U₁₅) at the C terminus of a classical effector lacking its native secretion signal. For this, we used YopE, which is a well characterized substrate of Yersinia T3SS. YopE harbors a “classical” N-terminal secretion signal corresponding to the 15 first residues and the deletion of this sequence blocks YopE secretion (15, 17, 36). To test whether the last 15 residues of YscU_CC (U₁₅) can promote secretion of YopE_ΔSP (lacking the 15 first residues) we construct different variants of YopE and introduced the corresponding plasmids into the strain YPIII/pIB29 (MEK). As expected YopE was detected in the supernatant of the strain expressing the wild type YopE while no YopE was secreted by the strain producing YopE_ΔSP (Fig. 3A, lines 1 to 3). These data confirmed that the 15 first residues of YopE are critical for secretion. In the constructs U₁₅-YopE_ΔSP and YopE_ΔSP-U₁₅, the 15 last residues of YscU_CC were fused to YopE_ΔSP N or C termini, respectively. None of these two constructs were secreted into the supernatant at levels that allowed their detection with Coomassie staining (Fig. 3A, lines 4 and 5). However, YopE_ΔSP-U₁₅ was detected in the supernatant by immunoblotting while neither YopE_ΔSP nor U₁₅-YopE_ΔSP was detected in the culture supernatant (Fig. 3B, lines 1 to 4). These results showed that C-terminal U₁₅ can promote secretion of YopE_ΔSP albeit at a low level.

YscU_CC Secretion Signal, Orientation Does Matter

To be secreted via the T3SS, proteins have to interact with component of the T3S-machinery, such as the ATPase YscN (24, 25, 37). It is known that protein-protein interactions depend on the spatial arrangement of the side chains from surface-exposed residues (38). Thus the location (N- or C-terminal) as well as the orientation of the signal peptide might affect the secretion efficiency. To test this hypothesis we assayed the secretion of both U_15rev-YopE_ΔSP and YopE_ΔSP-U_15rev. U_15rev corresponds to the peptide that contains the same residues as U₁₅ but in a reverse order. YopE_ΔSP-U_15rev was not secreted whereas U_15rev-YopE_ΔSP was detected in the supernatant after Coomassie Blue staining (Fig. 3A, lines 6 and 7). Thus, U₁₅ promotes secretion when placed at the N terminus but only when the residues are placed in a reverse orientation. These results indicated that the orientation of U₁₅ is important for secretion and most likely for interaction with the machinery. The level of U_15rev-YopE_ΔSP detected in the supernatant is significantly higher than YopE_ΔSP-U₁₅ secreted level but is only about 5% of the amount detected for wt YopE showing that the YopE native secretion signal is more efficient than U_15rev. YopE signal peptide contains only one charged residue (Lys²) while six charged residues are present in U₁₅ (Glu³⁴², Arg³⁴³, Glu³⁴⁷, Lys³⁴⁸, His³⁵⁰, and Glu³⁵²). As demonstrated by a Kyte and Doolityle plot, the charged residues within U₁₅ make this peptide more hydrophilic than YopE signal peptide (Fig. 2B). These hydrodynamic discrepancies between U₁₅ and YopE native signal peptide might explain this difference is secretion level. Indeed, charged residues as well as hydrophobic pockets at the interaction surface are parameters that guide protein-protein interaction (38, 39). It is probable that U_15rev has a lower affinity for the machinery components that recognize the secretion signal.

We also investigated the ability of the native secretion signal of YopE (denoted E₁₅) to promote secretion of YopE_ΔSP when placed at the C terminus. Surprisingly both YopE_ΔSP-E_15rev and YopE_ΔSP-E₁₅ were not detected in the culture supernatant (Fig. 3). Unlike U₁₅, E₁₅ cannot promote C-terminal secretion and that regardless of the orientation, showing that U₁₅ has features that are not shared with classical secretion signal.

The capacity of U₁₅ and U_15rev to promote translocation into eukaryote cells was also evaluated. For this, we followed the development of the cytotoxicity phenotype of HeLa cells after infection with strain expressing different variants of YopE. Thirty minutes after infection a full cytotoxicity phenotype was observed with the strain expressing YopE (Fig. 4B) while no cytotoxicity was observed when HeLa cells were infected with strains carrying an empty plasmid or plasmids expressing YopE_ΔSP or YopE_ΔSP-U₁₅ (Fig. 4, A, C, and D). On the other hand U_15rev-YopE_ΔSP provokes cytotoxicity on around 30% of the HeLa cells (Fig. 4E). The cytotoxicity phenotypes of YopE_ΔSP-U₁₅ and U_15rev-YopE_ΔSP correlate with their secretion efficiency.

YscU_C Interacts with the ATPase YscN

It was recently suggested that the ATPase YscN interacts with the secretion signal of YopR, and that this interaction is critical for YopR secretion by the T3SS (24). Previous studies showed that surface-exposed residues of some T3S-chaperones in complex with their cargo effector also interact with the ATPase (40–42). The results presented above showed that U₁₅ and U_15rev, respectively, placed at the C-terminal or the N-terminal end of YopE_ΔSP promoted secretion, indicating that both U₁₅ and U_15rev interacted with YscN. To test this hypothesis, His₆-YscN bound on a nickel-agarose resin was incubated with different variants of YopE to evaluate the capacity of these proteins to co-purify with YscN. YerA, the cognate chaperone of YopE, was co-expressed in all constructs to avoid degradation YopE and to maintain YopE chaperone binding domain in a partially unfolded state to facilitate interaction with YscN. YscN was eluted and co-purified YopE variants were detected using anti-YopE antibodies. Like YopR, YopE interacted with YscN, in a secretion signal-dependent manner (Fig. 5A). As expected, YopE_ΔSP did not co-purify with YscN. U_15rev-YopE_ΔSP, the variant with the highest secretion efficiency after wild type YopE, co-purified with YscN (Fig. 5B). This interaction is strictly dependent of the N-terminal U_15rev peptide since YopE_ΔSP and YopE_ΔSP-U_15rev were not co-purified with YscN (Fig. 5B). After YopE, U_15rev-YopE_ΔSP is the variant that co-purified best with YscN (around 60% of YopE amount) correlating with both secretion efficiency and translocation into HeLa cells. Importantly although U₁₅-YopE_ΔSP was not secreted, this variant interacted with YscN albeit at a lower level compared with U_15rev-YopE_ΔSP (Fig. 5B). Surprisingly, we were unable to detect interaction between YopE_ΔSP-U₁₅ and YscN (Fig. 5B), although YopE_ΔSP-U₁₅ was secreted via the T3SS. However, the YopE_ΔSP-U₁₅ level of secretion was relatively lower than YopE level; and it is possible that this low level of secretion is a consequence of a low affinity of YscN for YopE_ΔSP-U₁₅ hybrid protein. Thus, interaction between the secretion signal and the ATPase YscN is critical to allow secretion; however our results suggest that interaction per se may not be sufficient to promote secretion. In line with our findings, we could also show an interaction between YscU_C and YscN (Fig. 5). A similar result has previously been described for Spa40 and Spa47 that are the homologues of YscU and YscN in Shigella flexneri (43).

Deletion of the C Terminus of YscU Affects YscF Secretion

It has been shown earlier that YscU auto-proteolysis followed by YscU_CC dissociation constitute critical steps for regulation of the substrate specificity switch (14, 29, 30). In addition, we recently showed that YscU_CC is secreted by the T3SS when bacteria are grown in conditions allowing Yop secretion (26). These previous published studies with the data presented above in the present article, suggested that the C-terminal end of YscU_CC would be essential for Yops secretion. Surprisingly, when YscU_Δ3, YscU_Δ6, and YscU_Δ9, which correspond to YscU constructs having C-terminal deletions, were introduced into a ΔyscU strain (YPIII/pIB75), Yop secretion was not affected (Fig. 6A). Similarly, these mutants induced a cytotoxic response after infection of HeLa cells that was indistinguishable from the corresponding yscU wt strain (Fig. 6B). Thus, neither Yop secretion into the culture supernatant nor Yop translocation into HeLa cells was affected by the deletion of the last 9 residues of YscU. The fact that these mutants showed secretion and translocation of Yops at wild-type level strongly suggests that both auto-proteolysis and dissociation of YscU_CC were not affected by the C-terminal deletions. As expected, secretion of YscU_CC however, was impaired by YscU C-terminal deletions (Fig. 7A, second panel from the top) suggesting that YscU_CC secretion is not required to trigger dissociation, but it is likely that YscU_CC secretion is a result of dissociation. Interestingly, while similar amounts of intracellular YscF (needle subunit) were detected for the different strains, we observed an increase of YscF secretion for the C-terminal deletions mutants (Fig. 7A, bottom and third panel from the top). These results suggested that the C-terminal signal sequence of YscU is involved in YscF secretion control. Elongated needles have been reported for ΔyscP mutants and yscU point mutants defective in auto-proteolysis (11, 44, 45). However, in contrast to mutants carrying C-terminal deletion of YscU, Yop secretion is impaired in a yscP mutant as well as in the yscU processing mutants. Altogether, the different phenotypes observed for mutants expressing YscU_Δ3, YscU_Δ6 and YscU_Δ9 constructs suggest that the C-terminal end of YscU_CC is directly involved in the secretion regulation of the needle subunit, YscF. Previously, it has been shown in Y. enterocolitica that strains bearing the processing mutants YscU_N263A and YscU_P264A secrete elevated amount of YscF as a consequence of reduced secretion of YscP (12, 45). Interestingly, the secretion of YscP was not affected in any of the YscU mutants with C-terminal deletions (Fig. 7B).