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J Bacteriol. Jul 2009; 191(13): 4259–4267.
Published online Apr 24, 2009. doi:  10.1128/JB.01730-08
PMCID: PMC2698497

Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins [down-pointing small open triangle]

Abstract

YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscUCC. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca2+ conditions) and calcium-depleted medium (−Ca2+ conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca2+-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca2+ conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca2+ conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.

The type III secretion system (T3SS) occurs in many gram-negative pathogenic or symbiotic bacteria (6, 16, 19). The T3SS is evolutionarily related to the bacterial flagellum (19, 24), but while the flagellar apparatus is dedicated to bacterial motion, the T3SS specifically allows bacterial targeting of effector proteins across eukaryotic cell membranes into the lumen of the target cell (19). The main function of the effectors is to reprogram the cell to the benefit of the bacterium (28). The two organelles are superficially similar in form and can be divided into two physical substructures; a basal body is connected to a multimeric filamentous protein structure protruding from the bacterial surface. The basal body is embedded in the cell wall and spans from the cytosol to the surface of the bacterium with a cytosolic extension called the C-ring. The proximal center of the basal body is likely involved in the actual export of nonfolded substrates, which are thought to pass through the cell wall through this hollow structure (6, 16, 41). Early and elegant work by Macnab's group showed that morphogenesis of the flagella is ordered such that first the cell-proximal hook structure is polymerized and then the flagellar filament is assembled on top of the hook structure (43). Thus, there is ordered switching from secretion of hook proteins to flagellin, which was called substrate specificity switching by Macnab et al. (15, 27). Mutants expressing extraordinarily long hooks have been isolated and connected to regulation and determination of hook buildup and subsequent substrate specificity switching (18, 29, 43). A central factor in this process is the integral 42-kDa cytoplasmic membrane protein FlhB, which has four putative transmembrane helices in its N-terminal domain, which is designated FlhBTM. The hydrophilic C-terminal domain (FlhBC) is predicted to protrude into the cytosol. In addition, FlhBC can be further divided into two subdomains, FlhBCN (amino acids 211 to 269) and FlhBCC (amino acis 270 to 383), that are connected via a proposed flexible hinge region (27). The hinge region contains a highly conserved NPTH motif, which is found in all T3SSs. Interestingly, FlhBC is specifically cleaved within this NPTH sequence (N269↓P270) (27). Site-specific mutagenesis of the NPTH site has a significant effect on the substrate switching, and the ability of flhB(N269A) and flhB(P270A) mutants to cleave FlhB is impaired, indicating that autoproteolysis is important (13, 15). Interestingly, the proteolysis is most likely the outcome of an autochemical process rather than an effect of external proteolytic enzymes (13). The FlhB homolog in the Yersinia pseudotuberculosis plasmid-encoded T3SS is the YscU protein, which has been shown to be essential for proper function of the T3SS since a yscU-null mutant is unable to secrete Yop proteins (Yops) into the culture supernatant (1, 21). YscU has been coupled to needle and Yop secretion regulation, as second-site suppressor mutations introduced into YscUCC restore the yscP-null mutant phenotype. A yscP mutant is unable to exhibit substrate specificity switching and carries excess amounts of the needle protein YscF on the bacterial surface compared to the wild type. (11) Furthermore, YscP has been implicated in regulation of the T3SS needle length as a molecular ruler, where the size and helical content of YscP determine the length of the needle (20, 42). Together, these findings suggest that YscP and YscU interact and that this interaction is important for regulation of needle length, as well as for Yop secretion. As in FlhB, four predicted transmembrane helices followed by a cytoplasmic tail can be identified in YscU (1). In addition, the cytoplasmic part (YscUC) can be divided into the YscUCN and YscUCC subdomains (Fig. (Fig.1A).1A). Variants of YscU with a single substitution in the conserved NPTH sequence (N263A) have been found to be unable to generate YscUCC, suggesting that YscU of Yersinia also is autoproteolysed (21, 33, 38). The T3SS of Y. pseudotuberculosis secretes about 11 proteins, which collectively are called Yops (Yersinia outer proteins). These Yops have different functions during infection. Some are directly involved as effector proteins, attacking host cells to prevent phagocytosis and inflammation, while others have regulatory functions. Although the pathogen is extracellularly located, the Yop effectors are found solely in the cytosol of the target cell, and secretion of Yops occurs only at the zone of contact between the pathogen and the eukaryotic target cell (7, 36). Close contact between the pathogen and the eukaryotic cell also results in elevated expression and secretion of Yops (12, 30). Hence, cell contact induces the substrate switching; therefore, here we studied the connection between YscU autoproteolysis and expression, as well as secretion and translocation of Yops. Previous studies of YscU function were conducted mainly with in trans constructs instead of introduced YscU mutations in cis. Such studies reported loss of T3SS regulation (21). To avoid potential in trans problems, we introduced all mutations in cis with the aim of elucidating the function of YscU in type III secretion (T3S). Our results suggest that YscU autoproteolysis is not an absolute requirement either for Yop/LcrV secretion or for Yop translocation but is important for accurate regulation of Yop expression and secretion.

FIG. 1.
Autoproteolysis of YscU. (A) Schematic diagram of YscU in the bacterial inner membrane. The diagram shows the NPTH motif and the different parts of YscU after autoproteolysis and is the result of a prediction of transmembrane helices in proteins performed ...

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Bacterial strains and plasmids used in this study are listed in Table Table1.1. Escherichia coli strains were grown in Luria-Bertani broth or on Luria agar plates at 37°C. Y. pseudotuberculosis was grown either at 26°C or at 37°C on Luria agar plates or in TMH (39), a defined rich medium, with antibiotics corresponding to resistance markers carried by the strains. EGTA at a final concentration of 5 mM was added to TMH to create −Ca2+ conditions, and addition of 2.5 mM CaCl2 created +Ca2+ conditions. Antibiotics were used at the following concentrations: kanamycin, 50 μg/ml; chloramphenicol, 25 μg/ml; ampicillin, 50 μg/ml; carbenicillin, 100 μg/ml; and streptomycin, 5 μg/ml in liquid cultures and 30 μg/ml in plates.

TABLE 1.
Bacterial strains and plasmids used in this study

DNA methods.

Standard methods (37) were used for plasmid DNA preparation, restriction enzyme digestion, separation by gel electrophoresis, ligation, preparation of competent cells, and transformation of E. coli.

Construction of vectors and introduction of yscU site mutants in cis.

Suicide vectors pML40 to pML44 carrying fragments corresponding to 456 to 468 bp of different yscU site mutant variants were generated as follows. PCR was performed with primers 5′-GCTCACGAGCTCATAGCCGACTATGCCTTTGAATA-3′ (SacI site underlined) and 5′-TCTAGATTATAACATTTCGGAATGTTGTTTCT-3′ (XbaI site underlined) [bold type indicate bases 5049 to 5071 and 5491 to 5516, respectively, in the Y. pseudotuberculosis YPIII(pIB1) yscU sequence (accession no. L25667)]. Plasmids pIB102, pPE40, pPE41, pPE42, and pPE43 (Table (Table1)1) were used as templates to generate fragments of wild-type yscU, ΔNPTH263-266, N263A, P264A, and T264A, respectively. The fragments were introduced into SacI/XbaI-digested pNQ705 (26), generating pML40 to pML44 (Table (Table1).1). These plasmids were transferred into the recipient Yersinia sp. strain YPIII(pIB102) by conjugation. Plasmid pML40 was also transferred into strain YPIII(pIB69). To confirm insertion of the correct sequences of the yscU variants, a PCR fragment was generated using primers 5′-GAGCTCATGAGCGGAGAAAAGACAGAG-3′ (bold type indicate bases 4452 to 4472 of yscU [accession no. L25667]) and 5′-TCTAGATTATAACATTTCGGAATGTTGTTTCT-3′ (see above) and sequenced (MWG-Biotech, Ebersberg, Germany).

Yop secretion and production assay.

Yersinia strains were grown in TMH under +Ca2+ and −Ca2+ conditions for 1 h at 26°C and then for 3 h at 37°C with appropriate antibiotics. The optical densities at 600 nm (OD600) of the cultures were measured, and total samples and supernatant samples were collected in sodium dodecyl sulfate (SDS) sample buffer and boiled. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis with anti-YopB monoclonal antibodies and anti-YopE and anti-YopD polyclonal antibodies. The membrane was incubated with horseradish peroxidase-labeled anti-rabbit and anti-mouse antibodies (Amersham Pharmacia Biotech), and the proteins were detected with a chemiluminescence detection kit (Millipore).

Western blot quantification.

YopE, YopB, and YopD total protein expression and supernatant protein levels were quantified in three sequential experiments. Wild-type YPIII(pIB102)/pNQ and YPIII(N263A)/pNQ were grown in TMH under −Ca2+ conditions for 1 h at 26°C and then for 3 h at 37°C. Total samples and supernatants were collected in SDS sample buffer, boiled, and 16-fold serially diluted to avoid saturated signals, and this was followed by OD600-regulated loading and separation by 12% SDS-PAGE. Western blotting was carried out with anti-YopE and anti-YopD polyclonal antibodies and anti-YopB monoclonal antibodies. The membranes were probed with horseradish peroxidase-labeled anti-rabbit and anti-mouse antibodies (Amersham Pharmacia Biotech), and the proteins were detected with a chemiluminescence detection kit (Millipore). The signals were quantitatively measured by use of the Bio-Rad Imager ChemiDoc XRS system.

LcrV expression and secretion analysis.

Triplicate cultures of Y. pseudotuberculosis wild-type strain YPIII(pIB102)/pNQ, YPIII(N263A)/pNQ, YPIII(P264A)/pNQ, and YPIII(pIB19) were grown in TMH supplemented with 0.1% Triton X-100 under +Ca2+ and −Ca2+ conditions for 1 h at 26°C and then for 3 h at 37°C. The OD600 of the cultures were measured, and supernatant and pellet samples of each culture were collected in SDS sample buffer and boiled. Samples were separated by 12% SDS-PAGE, and Western blot analysis was performed with anti-LcrV polyclonal antibodies. The membrane was probed with horseradish peroxidase-labeled anti-rabbit antibodies, and protein detection was carried out with a chemiluminescence detection kit (Millipore). The signals were quantitatively measured with the Bio-Rad Imager ChemiDoc XRS system.

LcrQ secretion detection.

Y. pseudotuberculosis wild-type strain YPIII(pIB102)/pNQ, YPIII(N263A)/pNQ, YPIII(P264A)/pNQ, YPIII(T265A)/pNQ, and YPIII(pIB26) were grown in TMH under +Ca2+ and −Ca2+ conditions for 1 h at 26°C and then for 3 h at 37°C. The OD600 of the cultures were measured, and supernatant and pellet samples of each culture were collected in SDS sample buffer and boiled. Samples were separated by 15% SDS-PAGE, and Western blot analysis was performed with anti-LcrQ polyclonal antibodies. The membrane was probed with horseradish peroxidase-labeled anti-rabbit antibodies, and protein detection was carried out with a chemiluminescence detection kit (Millipore).

YscU protein expression assay.

Y. pseudotuberculosis wild-type strain YPIII(pIB102) was grown in TMH under either +Ca2+ or −Ca2+ conditions with 50 μg/ml kanamycin for 1 h at 26°C and then shifted to 37°C. Samples were withdrawn after the 1 h of incubation at 26°C (zero time) and 30, 60, and 120 min after the shift to 37°C. At the same time YPIII(pIB102) was grown in TMH supplemented with 50 μg/ml kanamycin under +Ca2+ conditions for 1 h at 26°C and for 30 min at 37°C. Thereafter, calcium was chelated by addition of 5 mM EGTA. Samples were withdrawn from this culture after 30, 60, and 120 min of induction after addition of EGTA to the culture. The sample obtained after 1 h of incubation at 26°C plus 30 min of incubation at 37°C under +Ca2+ conditions was designated the zero-time sample for this experimental setup. At each time point samples were pelleted, taken up in SDS sample buffer, and boiled. The protein samples were loaded onto an 11% Tris-Tricine SDS-PAGE gel based on OD600 values and subjected to Western blot analysis with peptide anti-YscUCC polyclonal antibodies (this study). The membrane was incubated with horseradish peroxidase-labeled anti-rabbit antibodies (Amersham Pharmacia Biotech), and the proteins were detected with a chemiluminescence detection kit (Amersham Pharmacia Biotech).

YscU autoproteolysis assay.

An overnight culture of BL21 carrying plasmid pPE5 [His6-yscU(202-354)] was inoculated to obtain an OD600of ~0.1. The culture was grown at 37°C to an OD600 of 0.5, and then it was induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h. The culture was pelleted, washed, and taken up in binding buffer (8 M urea, 10 mM imidazole). After sonication and another centrifugation step, the overexpressed protein was found to be in the soluble fraction. The lysate was flushed over a calibrated nickel column (GE Healthcare), and the flowthrough was kept. The column was washed five times with binding buffer, and the wash fractions were saved. Next, the column-bound proteins were eluted with elution buffer (8 M urea, 300 mM imidazole). The eluate was diluted 1:30 in 10 mM Tris (pH 7.4) to obtain final concentrations of urea and imidazole of 0.2 M and 10 mM, respectively. The diluted eluate was left at room temperature overnight. The next day the diluted eluate was precipitated on ice with trichloroacetic acid (TCA) at a final concentration of 10%. The precipitate was washed with acetone, and the resulting pellet was taken up in binding buffer. All of the fractions collected were separated by 15% Tris-Tricine SDS-PAGE, followed by Coomassie blue staining. All fraction volumes were adjusted to the lysate volume. The diluted and TCA-precipitated sample was also separated by 15% Tris-Tricine SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. N-terminal sequencing analysis, carried out at the Protein Analysis Center, Karolinska Institute, was performed for the three bands on the membrane.

Surface localization of YscF.

The analysis of surface-localized YscF was performed essentially as previously described (11), but with the changes described below. Yersinia strains were grown in TMH under +Ca2+ and −Ca2+ conditions for 1 h at 26°C and induced by incubation for 3 h at 37°C. The OD600 of the cultures were measured, and samples of each culture were gently pelleted by centrifugation and dissolved in TMH under +Ca2+ and −Ca2+ conditions, resulting in 10-fold concentration. The concentrated pellets were sheared by five passages through a hypodermic needle (23Gx1; 0.6 by 25 mm; B. Braun) to release surface proteins and organelles from the bacterial surface. After a centrifugation step, sheared supernatants and pellets were obtained after shearing. The samples were separated by 15% Tris-Tricine SDS-PAGE and transferred to a polyvinylidene difluoride membrane. YscF was detected with an anti-YscF polyclonal antibody and a horseradish peroxidase-labeled anti-rabbit antibody (Amersham Pharmacia Biotech). Protein detection was carried out with a chemiluminescence detection kit from Amersham Pharmacia Biotech. The signals were quantitatively measured by use of the Bio-Rad Imager ChemiDoc XRS system. The data are presented separately below for +Ca2+ and −Ca2+ conditions, and all results are expressed as a ratio compared to the wild type.

HeLa cell cytotoxicity assay.

Yersinia cultures were grown overnight in TMH under −Ca2+ conditions in the presence of the appropriate antibiotics. The cultures were diluted and grown for 1 h at 26°C and then at 37°C for 1 h. HeLa cells were infected for 45, 90, or 135 min. The cytotoxicity was later assayed as previously described (34). Pictures were taken with a microscope (Zeiss Axioskop) at a magnification of ×20.

Preparation of YscUCC peptide antibodies.

The antibodies directed to YscUCC were generated by Pacific Immunology, Ramona, CA. Essentially, a synthetic peptide (CLERQNIEKQHSEML) corresponding to amino acids 341 to 354 in the C-terminal sequence of YscU (YscUCC) was generated and used to immunize rabbits to obtain peptide-derived polyclonal antibodies. These antibodies detect full-length YscU, YscUC, and YscUCC.

RESULTS

YscU is autoproteolysed.

The YscU homologs FlhB, Spa40, SpaS, and EscU were recently shown to be autoproteolysed between the asparagine and the proline in the conserved NPTH motif of the proteins (8, 13, 45). Since YscU (40 kDa) also contains this motif, we asked whether YscU had the same autoproteolytic property as the proteins mentioned above. A histidine-tagged construct of the C-terminal cytoplasmic portion (YscUC) of YscU was overexpressed in E. coli and purified on an Ni column under denaturing conditions (8 M urea). The eluate produced three bands corresponding to YscUC-His6 and two additional products (Fig. (Fig.1B).1B). As YscUC-His6 was anticipated to be autoproteolysed at position N263A, we predicted that the following three protein fragments would appear in this fraction following autoproteolysis: YscUCC-His6 (12 kDa), YscUCN (6 kDa), and unprocessed YscUC-His6 (17 kDa). The eluate was diluted, incubated overnight at 21°C, and precipitated with TCA. The eluate fraction and the TCA-precipitated diluted fraction were analyzed by SDS-PAGE followed by Coomassie blue staining, resulting in the predicted tripartite protein pattern (Fig. (Fig.1B).1B). High levels of YscUC-His6 were detected in the eluate containing 8 M urea compared to the diluted fraction, which, in contrast, contained elevated levels of YscUCC-His6 and YscUCN (Fig. (Fig.1B),1B), suggesting that YscU is autoproteolysed at the predicted site. Interestingly, YscUCNcoeluted with YscUC-His6 and YscUCC-His6, indicating that there is a possible interaction between YscUCC-His6 and YscUCN, as suggested previously for FlhB (27) (Fig. (Fig.1B).1B). In addition, the different fragments were analyzed by performing an N-terminal sequence analysis, which confirmed that the autoproteolysis site is between the asparagine and the proline in the NPTH motif of YscU.

YscU protein expression is calcium regulated, whereas autoproteolysis is not calcium regulated.

In all studies conducted so far, the role of YscU in T3S has been studied mainly by expressing YscU variants in trans rather than in cis. We were concerned about this fact, since our experience with Yersinia is that expression of proteins in trans often generates anomalous results that can be misleading or difficult to interpret. Therefore, in this study, all yscU mutants were generated in cis under control of the native promoter. First, the pattern of YscU expression was examined to explore any possible link between YscU expression or autoproteolysis and regulation of the T3SS. Wild-type Y. pseudotuberculosis was grown in TMH supplemented either with 2.5 mM CaCl2 (+Ca2+ conditions) or 5 mM EGTA (−Ca2+ conditions), and YscU protein expression was monitored over time using Western blotting (Fig. (Fig.2).2). The YscU expression levels were low under +Ca2+ conditions, whereas elevated levels of YscU were seen under −Ca2+ conditions (Fig. (Fig.2).2). A temperature shift from 26°C to 37°C did not induce increased YscU expression. In contrast, if the bacterial culture was pregrown at 37°C in the presence of Ca2+, prior to removal of calcium by EGTA upregulation of YscU expression in response to calcium depletion was evident (Fig. (Fig.2).2). Furthermore, appearance of the YscUCC autoproteolysis product was found to be neither calcium regulated nor temperature regulated, since the levels of YscUCC corresponded to the overall levels of YscU expression (Fig. (Fig.2).2). Interestingly, the protein expression profile of YscU/YscUCC is in fact very similar to the expression profile of the Yops; i.e., expression of YscU/YscUCC is calcium regulated. This similarity was further reinforced by the finding that derepressed mutants (such as ΔlcrH, ΔlcrQ, and ΔyopD mutants) expressed elevated levels of YscU/YscUCC under under −Ca2+ conditions as well as under +Ca2+ conditions, whereas repressed mutants (for example, ysc gene mutants) showed lower levels of the two forms of the protein (data not shown) (2, 3, 25, 32). Thus, we conclude that YscU/YscUCC expression is calcium regulated and that the regulation is highly similar to the regulation of Yop expression. These results suggest that since YscU autoproteolysis is equally efficient under conditions that allow Yop secretion and under conditions that do not allow Yop secretion, it is highly unlikely to be a signal for Yop secretion.

FIG. 2.
YscU protein expression is calcium regulated, but autoproteolysis to YscUCC is not calcium regulated. Wild-type Y. pseudotuberculosis bacterial pellets were analyzed for YscU and YscUCC protein expression. Bacteria were grown for 1 h at 26°C under ...

Yop regulatory phenotypes of NPTH motif mutants.

We next generated a number of mutants with mutations in the conserved NPTH processing site in cis. These mutants, the N263A, P264A, T265A, and ΔNPTH mutants, were all evaluated with respect to autoproteolysis of YscU and Yop expression and secretion. Bacteria were grown in TMH under +Ca2+ and −Ca2+ conditions at 37°C, and bacterial pellets were analyzed for the presence of YscU and YscUCC by immunoblotting using a YscU polyclonal antiserum specifically designed to recognize the YscUCC part of the protein (Fig. (Fig.3).3). In contrast to recently published data (33, 38), the N263A mutant displayed autoproteolysis, albeit at a much reduced rate compared to the wild-type strain (Fig. (Fig.3).3). On the other hand, no YscUCC could be detected when the P264A mutant was analyzed (Fig. (Fig.3).3). Compared to the wild type, both the N263A and P264A mutants expressed increased amounts of full-length YscU under both growth conditions (Fig. (Fig.3).3). The T265A mutant showed a YscU-YscUCC expression pattern highly similar to that of the wild type (Fig. (Fig.3).3). Deletion of the whole motif (ΔNPTH) resulted in a faster-migrating full-length YscU due to loss of the four amino acids, and this protein was unable to undergo autoproteolysis to generate YscUCC, which further reinforces the importance of the NPTH motif. Unlike the wild type, the P264A and N263A mutants secreted Yops not only under −Ca2+ conditions but also under +Ca2+ conditions. However, the level of Yop secretion by the mutants under −Ca2+ conditions was reduced compared to the level of Yop secretion by the wild type (Fig. (Fig.4A).4A). The increased level of Yop secretion observed for the N263A and P264A mutants paralleled the increased Yop expression when the strains were grown under +Ca2+ conditions (Fig. (Fig.4A),4A), suggesting that these mutants are affected in regulation rather than in secretion or are affected both in regulation and in secretion. The N263A mutant showed elevated levels of Yop expression and secretion compared to the P264A mutant, indicating that YscU autoproteolysis may be important as a regulatory function of YscU (Fig. (Fig.4A).4A). The ΔNPTH mutant had a phenotype identical to the phenotype of a yscU-null mutant and a yscP-null mutant; i.e., the mutant expressed very low levels of Yops, and no Yop secretion could be observed (Fig. (Fig.4A).4A). In contrast, the T265A mutant had a phenotype that corresponded to that of the wild type (data not shown). Interestingly, our analysis showed that the N263A and P264A mutants had a derepressed phenotype for Yop expression and secretion when they were grown under +Ca2+ conditions. While both mutants showed calcium-independent growth, they were still able to express Yops and secrete Yops into the culture supernatant in the presence of Ca2+. Our results show that while autoproteolysis of YscU is not an absolute prerequisite for Yop secretion, it is important for regulation of the T3SS in Yersinia. In a recent publication, the authors presented results suggesting that the N263A and P264A substitutions prevented autoproteolysis of YscU and specifically abolished export of the translocators LcrV, YopB, and YopD, while the levels of secretion of Yop effectors were close to wild-type levels (38). These results are not compatible with our findings, since we detected translocator secretion in the N263A and P264A substitution mutants (Fig. (Fig.4A).4A). We therefore addressed this discrepancy in more detail. The N263A mutant, along with the wild type, was analyzed by quantitative Western blotting for YopE, YopB, and YopD expression and secretion under −Ca2+ conditions. Compared to the wild type, which secretes almost 100% of the YopE, YopD, and YopB, the secretion by the N263A mutant was affected, and this mutant secreted only 40%, 22%, and 19% of the total amounts of YopE, YopD, and YopB, respectively, expressed (Fig. (Fig.4B).4B). Moreover, the N263A mutant expressed smaller total amounts of YopE, YopD, and YopB (40%, 70%, and 30%, respectively) than the wild type (100%) (Fig. (Fig.4C,4C, upper panel). This means that the levels of YopE and YopD secreted by the mutant were only 20% and 15% of the levels secreted by the wild type (Fig. (Fig.4C,4C, lower panel). The amounts of YopB expressed are smaller than the amounts of the other Yops expressed, and the reduced expression in turn also leads to the clearly lower levels of YopB secretion which are shown in Fig. Fig.4C,4C, lower panel. We conclude that the translocators YopB and YopD are secreted. Thus, YscU is not a discriminator that sorts effectors from translocators; rather, it seems to have a general effect on both T3SS regulation and system-connected protein secretion.

FIG. 3.
Altered and eliminated autoproteolysis to YscUCC for NPTH motif mutants. The wild type and the N263A, P264A, T265A, and ΔNPTH motif mutants were subjected to Western blot analysis with anti-YscUCC antibodies. Bacteria were pregrown for 1 h at ...
FIG. 4.
NPTH motif mutants show alterations in Yop expression and secretion profiles. Bacteria were grown in TMH under +Ca2+ conditions or under −Ca2+ conditions for 3 h at 37°C, and Yop expression and secretion were analyzed. ...

YscU and LcrV secretion.

Since YscU has been described as a unique regulator of translocator protein export, further studies of LcrV were required to connect all translocator proteins to theYscU function (38).

To further analyze LcrV secretion and expression in the autoproteolysis mutants (N263A and P264A), we grew cultures in TMH under +Ca2+ and −Ca2+ conditions in the presence of 0.1% Triton X-100. Addition of Triton X-100 to growing cultures has been shown to increase the solubility of Yops (22). The expression of LcrV was clearly derepressed in the N263A and P264A mutants, but the wild-type exhibited previously described calcium-regulated expression control (Fig. (Fig.5)5) (44). Interestingly, these mutants secrete LcrV under −Ca2+ conditions but not under +Ca2+ conditions (Fig. (Fig.5).5). Moreover, the amount of LcrV secreted by the mutants is reduced (6 to 16%) compared to the amount secreted by the wild type, but the level of secretion of LcrV was the same magnitude as the level of secretion of the Yops (Fig. (Fig.5).5). This indicates that autoproteolysis of YscU is important for retaining calcium-regulated LcrV expression and proper LcrV secretion.

FIG. 5.
YscU N263A and P264A mutants display altered LcrV expression and secretion The wild type (wt) and the N263A, P264A, and ΔlcrV mutants were induced for 3 h at 37°C in TMH supplemented with 0.1% Triton X-100 under +Ca2+ ...

Mutants N263A and P264A secrete LcrQ in calcium-containing medium.

When Y. pseudotuberculosis is grown under conditions allowing maximal expression and secretion of Yops, another protein, the negative regulator LcrQ, is also secreted. We have previously shown that secretion of LcrQ is tightly linked to regulation of Yop expression (32). LcrQ is located in the cytosol of the bacterium when it is grown in Ca2+-containing medium, while it is secreted in calcium-depleted medium. Thus, secretion lowers the intracellular concentration of the negative regulator LcrQ, and subsequently Yop expression is derepressed. The N263A and P264A mutants both secreted Yops regardless of the calcium concentration (Fig. (Fig.4A);4A); hence, we sought to analyze whether LcrQ is secreted under both growth conditions by the yscU mutants N263A, P264A, and T265A. Interestingly, the calcium-independent N263A and P264A mutants both secreted LcrQ under conditions that did not permit secretion, and in these mutants less LcrQ was retained in the pellet fractions under both growth conditions (Fig. (Fig.6).6). This was not the case for the wild type or the T265A mutant, since both of these strains were unable to secrete LcrQ in Ca2+-containing medium and there were elevated LcrQ levels in the pellet fractions compared to the levels under −Ca2+ conditions (Fig. (Fig.6).6). Therefore, the derepressed phenotype seen in the N263A and P264A mutants with respect to Yop secretion and expression is tightly connected to LcrQ secretion; accordingly, these results provide a plausible explanation for the phenotype of the mutants.

FIG. 6.
N263A and P264A mutants leak LcrQ when they are grown in calcium-containing medium. The wild type (wt) and the N263A, P264A, and T265A mutants were grown in TMH under +Ca2+and −Ca2+ conditions for 3 h, and culture supernatants ...

YscU is involved in T3SS needle regulation.

YscU has been connected to substrate specificity switching through its regulatory function with the T3SS needle protein, YscF. In line with this, mutants with single-site mutations in yscU which can suppress the yscP-null mutant phenotype have also been isolated (11). With the intention of determining the role of YscU autoproteolysis in secretion of the needle protein, we sheared YscF needles off bacterial surfaces of different yscU mutants and analyzed the preparations with anti-YscF antibodies (Fig. (Fig.7A).7A). The wild type displayed increased amounts of YscF on the bacterial surface under conditions that permitted secretion (−Ca2+ conditions) compared to the amounts under conditions that did not permit secretion (+Ca2+ conditions). This was also the case for the single-site mutant T265A (Fig. (Fig.7A).7A). In contrast, the yscU-null mutant, as well as the ΔNPTH mutant, lacked YscF on the exterior of the bacterium (data not shown). The N263A and P264A mutants, with altered YscU autoprocessing patterns, on the other hand, displayed YscF on the bacterial surface, but the levels were elevated (11- and 1.5-fold, respectively) compared to the wild-type levels when organisms were grown under +Ca2+ conditions (Fig. (Fig.7A).7A). The increased YscF export was not due to fragile needles that broke more easily, since there were still substantial amounts of YscF in the pellet fractions of these mutants after shearing (Fig. (Fig.7B).7B). As it is in the wild type, YscF secretion is calcium regulated in the mutants, with larger amounts of YscF exported under −Ca2+ conditions (Fig. (Fig.7A).7A). It should be noted, however, that although the N263A and P264A mutants are induced to secrete YscF under −Ca2+ conditions, the amount of YscF exported was smaller for the mutants than for the wild type (Fig. (Fig.7A).7A). Interestingly, the yscP mutant exhibits the same YscF secretion pattern as the N263A mutant, showing that autoproteolysis of YscU and YscP are linked to needle regulation (Fig. (Fig.7A7A).

FIG. 7.
YscU motif mutants display altered regulation of surface-localized YscF. The wild type (wt), the yscP mutant, and the N263A, P264A, and T265A mutants were grown in TMH under +Ca2+and −Ca2+ conditions for 3 h and sheared ...

Cytotoxic responses of N263A and P264A are delayed on HeLa cells.

A well-established assay for in vivo assessment of functional T3S in Y. pseudotuberculosis is the HeLa cell infection model. T3SS-mediated targeting of YopE into HeLa cells is scored by using altered (rounded) morphology of the infected cell as the actin cytoskeleton structure is disrupted (34-36). Strains with different yscU mutations were pregrown under T3SS-inducing conditions and later applied to HeLa cell monolayers. The cytotoxic response for both the N263A and P264A mutants was slightly delayed, and cell rounding was observed around 45 min later for cells infected with these mutants than for cells infected with the wild-type strain (Fig. (Fig.8).8). The ΔNPTH mutant failed to induce cell rounding, like the yscU-null mutant (Fig. (Fig.8).8). The T265A mutant, like the wild type, induced a rapid cytotoxic response (Fig. (Fig.8).8). These results show that yscU mutants whose autoproteolytic ability is affected have reduced expression or secretion of Yops in vitro and delayed translocation in vivo, while mutants with mutations in yscU that generate an yscU-null mutant phenotype can neither secrete nor translocate Yops. Importantly, this confirms that all translocators are indeed secreted at levels that promote functional effector translocation by the N263A, P264A, and T265A strains under in vivo-like conditions. Thus, autoproteolysis of YscU is not essential for a functional T3SS, while efficient autoproteolysis is required for proper regulation, secretion, and translocation of Yops.

FIG. 8.
NPTH motif mutants show altered cytotoxic responses on HeLa cells. The NPTH motif mutants, along with the wild type and the ΔyopD mutant, were pregrown in TMH under −Ca2+ conditions for 1 h at 26°C, followed by 1 h at 37°C. ...

DISCUSSION

It is known that proteins homologous to YscU in other species, such as EscU, SpaS, and Spa40 of E. coli, Salmonella, and Shigella, respectively, and FlhB in the Salmonella flagellar system undergo specific autoproteolysis in a conserved NPTH motif in their cytoplasmic C-terminal sequences (8, 13, 45). We now add YscU to this collection of proteins, as we showed that in Y. pseudotuberculosis YscU undergoes autoproteolysis between the asparagine at position 263 and the proline at position 264 of the NPTH motif. Interestingly, analogous to findings of Macnab and coworkers, we also found that YscUCN associated with YscUCC following autoproteolysis of the YscUC protein (Fig. (Fig.1B)1B) (27). This result suggests that there is a tight interaction between the two protein products, which likely is important for proper YscU function. Autoproteolysis of YscU, however, is not a prerequisite for Yop secretion in Yersinia, since mutants defective in processing are still able to express, secrete, and translocate Yops. However, when the relative ratio of secreted effector Yops to translocator Yops was determined for the wild-type strain and for the N263A processing mutant with impaired autoproteolysis, no difference in the ratio of secreted Yops and LcrV in the two strains was found, although the mutant expressed and secreted reduced amounts of Yops and LcrV compared to the wild type. Thus, we did not find any support for the idea that the secretion of translocator proteins is specifically diminished in a mutant defective in autoproteolysis, as has previously been suggested (33, 38). In agreement with previously reported data (33), we found that the N263A mutant was able to induce a cytotoxic response in HeLa cells, which clearly demonstrated that YopE, the major effector in this assay system (35), was translocated into the lumen of the HeLa cells. This result strongly supports the hypothesis that the translocator proteins are secreted by the N263A mutant, since the translocator proteins are absolutely required for translocation of effector proteins (17, 31, 36). Thus, we were unable to verify that YscU is an element that has a specific role in determination of translocator protein secretion, as was recently suggested (33, 38). A likely explanation for this apparent discrepancy is that only low levels of secreted translocators are required for functional delivery of Yop effectors. This is obvious from our work, where secretion of the translocator proteins by the N263A and P264A mutants was clearly decreased compared to the secretion by the wild type (Fig. 4A to C and Fig. Fig.5).5). Similar observations have been made in other studies (4, 10). Conversely, we show that YscU is an important protein both for Yop secretion and for Yop regulation. A plausible explanation for the differences between our results and those of other workers is likely that we used mutants constructed in cis exclusively, whereas other groups employed mostly or exclusively mutants generated in trans (33, 38). The use of in trans constructs may be risky, and we have previously reported that in trans expression of YscU affects the viability of the bacteria and, more importantly, affects regulation, which generates results that are difficult to interpret (21). In contrast to the wild type, the autoproteolytic N263A and P264A mutants were both calcium independent for growth and able to secrete Yops in calcium-containing medium, indicating that YscU is involved in regulation of expression and secretion of Yops. Surprisingly, both the N263A and P264A mutants show more surface-localized YscF than the wild type when the bacteria are grown in calcium-containing medium (Fig. (Fig.7).7). On the other hand, both the ΔNPTH mutant and the yscU-null mutant fail to express YscF on the bacterial surface. Thus, both Yop regulation and YscF export control are affected in the autoproteolytic mutants. Importantly, the regulation of YscF export by the wild type mimics the regulation of Yop protein secretion; i.e., export is increased under −Ca2+ conditions. Although the N263A mutant showed an 11-fold increase in YscF secretion under +Ca2+ conditions, YscF secretion was still induced in the mutant under −Ca2+ conditions, reaching a level that was about 50% of the wild-type level. Thus, autoproteolysis of YscU is not of major importance when the bacteria are grown under inducing conditions. Rather, these results indicate that autoproteolysis of YscU is important for negative regulation of YscF and Yop export under +Ca2+ conditions. Based on these results, we suggest that nonprocessed YscU is an inhibitor of Yop release and that autoproteolysis is required to relieve this negative block. Consequently, both YscF export and Yop export are induced under −Ca2+ conditions. This model is a minor modification of the switch model for the flagellum of Salmonella enterica serovar Typhimurium, since it has been shown that polymerization of the hook protein FlgE (which in this context is YscF) stops to the advantage of the FliC (Yops in Yersinia) subunit assembly to make up the flagellum (43). In contrast, we now suggest that YscF and Yops are secreted in parallel under inducing conditions. Our proposed model may at a first glance not appear to be compatible with the results that we obtained for the N263A and P264A mutants with respect to the observed secretion of Yops under +Ca2+ conditions and with respect to the fact that we did not observe any link between Ca2+-mediated regulation of YscU expression and autoproteolysis. It is possible, however, that the mutations influence the conformation of the YscU protein in such a way that Yop secretion, as well as LcrQ secretion, is only partially blocked. Indeed, a conformational change upon cleavage of YscU was described when crystal structure analyses of YscU in its cleaved and uncleaved forms were conducted (23). This conformational change may in turn allow the negative regulator LcrQ (32) to be secreted, and the block in Yop expression mediated by LcrQ is partially relieved, followed by elevated Yop expression and secretion, which is supported by our results (Fig. (Fig.66).

In addition, when the expression and autoproteolysis of YscU were studied, we found that smaller amounts of YscU were expressed in Ca2+-containing medium than in a medium deprived of Ca2+. In this respect the pattern of expression mimicked that of the Yops. Moreover, the relative level of autoproteolysis of YscU was the same irrespective of the concentration of Ca2+ in the growth medium, suggesting that processing is not regulated by calcium. In order to adapt these data to the model presented above, we suggest that autoproteolysis is not enough to separate the two products of YscU (YscUCN and YscUCC) from each other. This is indicated in Fig. Fig.1B,1B, which shows that YscUCN and YscUCC were still bound to each other after autoproteolysis occurred. Thus, an additional factor that allows separation of YscUCC and YscUCN may be involved. We suggest that YscP, through an interaction with YscU, acts to regulate the formation of the needle on the bacterial surface. Longer needles, made up of YscF monomers, are formed in a yscP-null mutant, while no Yop secretion is detectable (11). With respect to YscF secretion the N263A mutant behaves like a yscP-null mutant, further supporting the idea that YscP and YscU interact. In addition, specific point mutations in the YscUCC sequence generate extragenic yscP mutant phenotype suppressor mutants, allowing Yop secretion, albeit at a reduced level (11). Altogether, these findings strongly support the hypothesis that YscUCC interacts with YscP. We suggest, therefore, that the YscP function is required to separate YscUCC from the remaining part of YscU and that this activity is a response to a lower concentration of calcium in the growth medium or to target cell contact. The YscUCC suppressor mutants may, as suggested above, exhibit a changed conformation of YscU, resulting in a destabilized interaction and an incomplete block of Yop secretion. We are now trying to answer these questions in our laboratory.

Acknowledgments

This work was supported by grants from the Swedish Research Council to Hans Wolf-Watz and Åke Forsberg. Ann-Catrin Björnfot thanks the J. C. Kempes Memorial Fund for financial support.

Footnotes

[down-pointing small open triangle]Published ahead of print on 24 April 2009.

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