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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Human pathogenic Yersinia resist host defences, in part through the expression and delivery of a set of plasmid-encoded virulence proteins termed Yops. A number of these Yops are exported from the bacteria directly into the cytoplasm of their eukaryotic host's cells upon contact with these cells. The secreted YopN protein (also known as LcrE) is required to block Yop secretion in the presence of calcium in vitro or before contact with a eukaryotic cell in vivo. In this study, we characterize the role of the tyeA, sycN and yscB gene products in the regulation of Yop secretion in Yersinia pestis. Mutants specifically defective in the expression of TyeA, SycN or YscB were no longer able to block Yop secretion in the presence of calcium. In addition, the secretion of YopN was specifically reduced in both the sycN and the yscB deletion mutants. Protein cross-linking and immunoprecipitation studies in conjunction with yeast two-hybrid analyses showed that SycN and YscB interact with one another to form a SycN/YscB complex. Yeast three-hybrid analyses demonstrated that the SycN/YscB complex, but not SycN or YscB alone, specifically associates with YopN. SycN and YscB share amino acid sequence similarity and structural similarities with the specific Yop chaperones SycE and SycH. Together, these results indicate that a complex composed of SycN and YscB functions as a specific chaperone for YopN in Y. pestis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The three human pathogenic species of Yersinia (Y. pestis, Y. enterocolitica and Y. pseudotuberculosis) avoid phagocytic destruction, in part through the expression and delivery of virulence factors called Yops (Forsberg et al., 1994; Fallman et al., 1995). The Yops and proteins required to regulate the expression and delivery of Yops are encoded on closely related approximately 70 kb virulence plasmids (Ben-Gurion and Shafferman, 1981; Ferber and Brubaker, 1981). Secretion of Yops across the bacterial membranes occurs via a type III or ‘contact-dependent’ secretion mechanism (Michiels et al., 1990; Straley et al., 1993a; Cornelis and Wolf-Watz, 1997). After cell contact, several of the Yops are translocated directly from the bacterium into the cytoplasm of the eukaryotic cell (Rosqvist et al., 1994; Persson et al., 1995; Cornelis and Wolf-Watz, 1997). A number of the Yops function directly as anti-phagocytic agents by interfering with actin microfilament rearrangement (YopE: Rosqvist et al., 1991), by disrupting signal transduction (YopH: Bliska et al., 1991, Fallman et al., 1997, Persson et al., 1997; YpkA: Hakansson et al., 1996) or by inducing programmed cell death (YopJ: Mills et al., 1997, Monack et al., 1997). These capabilities enable the yersiniae to avoid the primary immune responses of the host and ensure survival of the bacteria within host tissues.

Yersinia growing in calcium-depleted media secrete large amounts of Yops into the surrounding media. In the presence of calcium or before cell contact in vivo, virulence plasmid-encoded operons are transcriptionally downregulated (Goguen et al., 1984; Straley and Bowmer, 1986; Straley et al., 1993b), and Yop secretion is blocked (Michiels et al., 1990; Cornelis and Wolf-Watz, 1997). The secreted YopN (also known as LcrE; Viitanen et al., 1990; Forsberg et al., 1991), LcrG (Skrzypek and Straley, 1993) and the surface-bound TyeA (Iriarte et al., 1998) proteins are required to block Yop secretion before contact with a eukaryotic cell. The co-ordinate regulation of Yop expression and Yop secretion is maintained via a negative feedback mechanism that involves the secreted LcrQ [YscM] (Rimpiläinen et al., 1992; Pettersson et al., 1996; Stainier et al., 1997) and YopD (Williams and Straley, 1998) proteins and the cytoplasmic LcrH [SycD] protein (Price and Straley, 1989; Wattiau et al., 1994). The YopN-, LcrG- and TyeA-dependent block in Yop secretion prevents the release of LcrQ and YopD, which in turn prevents full transcriptional induction of virulence plasmid operons by the transcriptional activator LcrF (Lambert de Rouvroit et al., 1992; Pettersson et al., 1996). In the absence of calcium or upon contact with a eukaryotic cell, the block in secretion is removed, LcrQ and YopD are released and the transcription of virulence plasmid operons is induced. The secreted V antigen interacts directly with LcrG within the cell and is required to counteract LcrG's role in blocking Yop secretion (Nilles et al., 1997). Together, these gene products control the expression and delivery of Yops in response to specific triggering signals associated with either growth at 37°C in the absence of calcium in vitro or contact with a eukaryotic cell in vivo.

The sequence elements that target Yops for secretion have been localized to the amino-terminal region of these proteins. These signals are distinct from those recognized by the general secretion system (type II systems) or by members of the ATP binding cassette transport family (type I systems) (Michiels et al., 1990). Sequences encoding the amino-terminal 15 and 17 amino acid residues of YopE (Sory et al., 1995; Schesser et al., 1996) and YopH (Sory et al., 1995; Woestyn et al., 1996), respectively, have been shown to be sufficient for the secretion of chimeric reporter proteins into the extracellular medium; however, deletion of these amino-terminal regions does not fully abrogate Yop secretion (Cheng et al., 1997). Recent evidence suggests that the secretion signal targeting YopE chimeric proteins containing the coding region for only the amino-terminal 15 residues of YopE is encoded in the messenger RNA sequence rather than the peptide sequence, thus implicating a co-translational mechanism for Yop secretion (Anderson and Schneewind, 1997). Cheng et al. (1997) demonstrated recently that YopE contains a second independent secretion signal located between amino acid residues 15 and 100. The function of the second secretion signal was dependent upon a functional SycE protein (Wattiau and Cornelis, 1993), the specific Yop chaperone for YopE. SycE and its binding site on YopE have also been shown to be necessary for translocation of YopE into the eukaryotic cell (Rosqvist et al., 1994; Sory et al., 1995; Schesser et al., 1996; Woestyn et al., 1996; Lee et al., 1998).

In addition to the SycE protein, specific Yop chaperones for YopH (SycH: Wattiau et al., 1994), YopN (YscB: Jackson et al., 1998) and Yops B and D (SycD: Wattiau et al., 1994) have been identified and characterized. Syc-like proteins have also been identified in other bacteria equipped with type III secretion pathways (Frank, 1997; Gaudriault et al., 1997). Efficient secretion of the protein recognized by the Syc or Syc-like protein is dependent upon the chaperone and the binding site for the chaperone on the secreted protein.

Structural genes for Syc proteins are usually found adjacent or close to the structural gene of their target protein. Syc and Syc-like proteins are generally small (15–20 kDa) acidic proteins that exhibit only limited amino acid similarity (Wattiau et al., 1994; 1996). Structurally, they are all predicted to contain an amphipathic alpha-helical sequence near their carboxyl-terminus; however, this region does not structurally resemble a leucine zipper motif, nor is this region predicted to form a coiled-coil conformation.

The present study investigates the function of TyeA, SycN and YscB in the regulation of Yop secretion and, specifically, in the secretion of YopN. YopN, SycN, YscB and TyeA were all necessary to block Yop secretion in the presence of calcium. We demonstrate that SycN and YscB form a heteromultimeric complex that binds to YopN and functions as a specific chaperone for YopN in Y. pestis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Construction of non-polar tyeA and sycN deletion mutants

DNA sequence analysis of the 2.316 kb ClaI–EcoRV fragment upstream of lcrD in plasmid pGP2 (Plano et al., 1991) revealed the Y. pestis yopN and tyeA loci and three open reading frames (ORFs) that coincided with previously identified ORFs from the corresponding region of plasmid pYV of Y. enterocolitica (Viitanen et al., 1990) and Y. pseudotuberculosis (Forsberg et al., 1991) [Fig. 1]. The nucleotide sequence of this region was identical to the corresponding Y. pestis KIM plasmid pCD1 sequence compiled recently and annotated by Hu et al. (unpublished, GenBank accession number AF053946). The function of the three ORFs encoded downstream of tyeA have not been investigated previously. In this report, we characterize the role of TyeA (previously designated ORF1) and SycN (previously designated ORF2) of Y. pestis in the expression and delivery of pCD1-encoded gene products. Deletions eliminating the regions encoding amino acid residues 10–55 of TyeA and 34–65 of SycN were generated using the polymerase chain reaction (PCR)–ligation–PCR technique (Ali and Steinkasserer, 1995) and moved into plasmid pCD1 of Y. pestis KIM5-3001 by allelic exchange generating strains KIM5-3001.P2 (ΔtyeA) and KIM5-3001.P3 (ΔsycN ). Plasmids pTYEA1 and pSYCN1 were used in complementation studies.

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Figure 1. . Genetic organization of the yopN region of pCD1 in Y. pestis. DNA sequence analysis of a 2316 bp ClaI–EcoRV fragment of plasmid pGP2 revealed the coding sequence for yopN, the recently characterized tyeA locus (formally known as orf1), a gene we designated sycN (formally known as orf2) and two uncharacterized genes encoding ORFs designated ORF3 and ORF4. The location of in frame deletions within tyeA and sycN are shown (see Experimental procedures). Plasmids pTYEA1 and pSYCN1 were used in complementation experiments.

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Growth phenotype of the tyeA and sycN deletion mutants

The growth of the parent Y. pestis KIM5-3001, the tyeA deletion mutant KIM5-3001.P2 with and without pTYEA1 and the sycN deletion mutant KIM5-3001.P3 with and without pSYCN1 was determined after a temperature shift from 26°C to 37°C in the presence and absence of calcium (growth data not shown). As expected, the parent showed calcium-dependent growth typical of TMH growth media (Goguen et al., 1984). In contrast, both the tyeA and sycN deletion mutants underwent growth restriction at 37°C in the presence and absence of calcium (calcium-blind or temperature-sensitive growth phenotype; Viitanen et al., 1990; Forsberg et al., 1991). These results demonstrate that TyeA and SycN are essential for the normal calcium-dependent growth phenotype. The addition of plasmids pTYEA1 and pSYCN1 to the tyeA and sycN deletion mutants, respectively, completely restored the calcium-dependent growth phenotype, indicating that the calcium-blind growth phenotype of these mutants was caused by the inactivation of TyeA and SycN and not by polar effects on downstream genes.

Secretion of YopN, V antigen and YopM by the tyeA and sycN deletion mutants

The parent strain KIM5-3001 secreted YopM, YopN and V antigen into the culture supernatant when grown at 37°C in the absence of calcium (Fig. 2). However, as expected, no secretion of Yops and V antigen was detected in the presence of calcium. The yopN, tyeA, sycN and yscB deletion mutants grown at 37°C in the presence or absence of calcium expressed levels of YopM, V antigen and YopN similar to those expressed by the parent strain grown in the absence of calcium. In addition, these mutants secreted YopM and V antigen in the presence and absence of calcium at levels similar to that of the parent strain grown at 37°C in the absence of calcium (more than 80% of the total YopM secreted). These results demonstrate that the secretion of YopM and V antigen by these deletion mutants was no longer blocked in the presence of calcium (calcium-blind secretion phenotype; Forsberg et al., 1991). Interestingly, the secretion of YopN by the sycN deletion mutant grown at 37°C in the presence or absence of calcium was specifically reduced (23% [+Ca2+] to 29% [−Ca2+] of the total YopN secreted) compared with the parent strain grown in the absence of calcium (more than 85% of the total YopN secreted). As expected, the amount of YopN found associated with the cell pellet was increased in the sycN deletion mutant, indicating that the reduction in YopN found in the culture supernatant was most probably caused by a reduction in YopN export and not by a defect in YopN expression. We observed a similar defect in YopN secretion in a Y. pestis yscB deletion mutant (Fig. 2; Jackson et al., 1998).

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Figure 2. . Immunoblot analysis of culture supernatant and cell pellet fractions of Y. pestis strains grown at 37°C in the presence (+) or absence (−) of calcium. Antisera specific for YopM, V antigen or YopN were used to detect the presence of these proteins in the supernatant (S) and cell pellet (P) fractions (as indicated by the arrow to the right of each blot). Lanes: 1, KIM5-3001 (parent); 2, KIM5-3001.6 (ΔyopN ); 3, KIM5-3001.P2 (ΔtyeA); 4. KIM5-3001.P2 + pTYEA1; 5, KIM5-3001.P3 (ΔsycN ); 6, KIM5-3001.P3 + pSYCN1; 7, KIM5-3001.P1 (ΔyscB); 8, KIM5-3001.P1 + pYSCB1.

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Providing complementing plasmids pTYEA1, pSYCN1 and pYSCB1 in trans to the tyeA, sycN and yscB deletion mutants, respectively, completely restored calcium-regulated expression and secretion of YopM, YopN and V antigen. These data indicate that the defects in the regulation of V antigen and Yop secretion in the tyeA, sycN and yscB deletion mutants, as well as the specific reduction in YopN secretion in the sycN and yscB deletion mutants, resulted solely from the disruption of tyeA, sycN and yscB and not from polar effects on downstream genes or from spontaneous mutations in other ysc or lcr loci. Together, these data support a role for YopN, TyeA, SycN and YscB in the regulation of V antigen and Yop export in Y. pestis and point specifically to a joint role for SycN and YscB in the secretion of YopN.

Identification and localization of the sycN gene product

Polyclonal antiserum raised against a polyhistidine-tagged sycN gene product was used to identify SycN in immunoblots from whole-cell and fractionated Y. pestis cultures (Fig. 3). An approximately 12 kDa protein was identified as SycN in immunoblots from the parent Y. pestis KIM5-3001 and from the sycN deletion mutant KIM5-3001.P3 complemented with plasmid pSYCN1. As expected, the approximately 12 kDa SycN protein was missing in immunoblots from the sycN deletion mutant.

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Figure 3. . Identification and localization of SycN. A. Polyclonal antiserum specific for SycN was used to identify the SycN protein by immunoblot analysis of SDS–PAGE-separated whole bacterial cell proteins. Lanes: 1, KIM5-3001 (parent); 2, KIM5-3001.P3 (ΔsycN ); 3, KIM5-3001.P3 + pSYCN1. Antiserum specific for YscB was used to confirm the identity of the yscB gene product. Lanes: 4, KIM5-3001 (parent); 2, KIM5-3001.P1 (ΔyscB); 3, KIM5-3001.P1 + pYSCB1. B. Soluble, membrane and culture supernatant fractions of Y. pestis KIM10 grown at 37°C in the absence (−) of calcium and of Y. pestis KIM8 grown at 37°C in the presence (+) and absence (−) of calcium were subjected to SDS–PAGE and immunoblot analysis with antisera specific for SycN or YscB.

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Soluble (cytoplasmic and periplasmic proteins), total membrane and culture supernatant fractions from Y. pestis KIM10 (pCD1−, pPCP1−) grown at 37°C in the absence of calcium and from Y. pestis KIM8 (pCD1+, pPCP1−) grown at 37°C in the presence and absence of calcium were analysed for the presence of SycN and YscB. SycN was found primarily in the soluble fraction of KIM8. However, a small amount of SycN was also found associated with the membrane fraction. No SycN was found in the culture supernatant fractions. These results, in conjunction with the lack of a classical sec-dependent secretion signal and no predicted transmembrane domains, strongly suggest that SycN is a cytoplasmic or peripheral membrane protein that is not exported from the cell. The localization of SycN closely parallels that of the previously characterized YscB protein (Fig. 3; Jackson et al., 1998), providing further evidence that these proteins might function together in Y. pestis.

SycN shows similarities with Pcr2 of Pseudomonas aeruginosa and SycE, SycH, and YscB of Y. enterocolitica, Y. pseudotuberculosis and Y. pestis

The effect of the sycN deletion on the regulation of Yop secretion and, specifically, on the secretion of YopN was similar to the phenotype observed previously for a Y. pestis yscB deletion mutant (Jackson et al., 1998). YscB exhibited significant amino acid similarity to SycE and SycH and was shown to function as a specific chaperone for YopN in Y. pestis. A comparison of the SycN amino acid sequence with other reported Syc and Syc-like proteins revealed significant sequence and structural similarities between SycN and Pcr2 of P. aeruginosa (Yahr et al., 1997) and the Yersinia SycE (Wattiau and Cornelis, 1993), SycH (Wattiau et al., 1994) and YscB (Haddix and Straley, 1992) proteins. SycN showed the greatest overall similarity to Pcr2 of P. aeruginosa (47% identity) and, to a lesser extent, with SycE (18% identity), SycH (18% identity) and YscB (15% identity).

SycN also shares many of the common structural and functional features of the Syc and Syc-like family of proteins. Similar to the other Syc and Syc-like proteins, SycN is a small (13.5 kDa), acidic (pI = 5.0), cytoplasmic (or peripheral) protein with a predicted carboxyl-terminal amphipathic alpha-helix (amino acid residues 106–122 of SycN; Wattiau et al., 1994). These data, in conjunction with the effect of the sycN deletion on YopN export, indicate that SycN, like YscB (Jackson et al., 1998), functions as a specific chaperone for YopN in Y. pestis.

Interaction of SycN and YscB with YopN

Recent studies using protein cross-linking reagents and immunoprecipitation techniques identified a specific interaction between YscB and YopN in Y. pestis (Jackson et al., 1998). To understand the roles of SycN, YscB, YopN and TyeA in the secretion of YopN and in the regulation of Yop secretion, we used similar techniques to identify and characterize the interactions among these proteins. Soluble proteins (cytoplasmic and periplasmic) from the Pla− parent strain Y. pestis KIM8-3002, the yopN deletion mutant KIM8-3002.P7 (Pla−), the tyeA deletion mutant KIM8-3002.P8 (Pla−), the sycN deletion mutant KIM8-3002.P9 (Pla−) and the yscB deletion mutant KIM8-3002.P10 (Pla−) grown at 37°C in the absence of calcium were cross-linked by the addition of the thio-cleavable, amine-reactive cross-linker dithiobis(succinimidyl propionate) (DSP) to a final concentration of 1 mM for 30 min at room temperature. Polyclonal antisera specific for YopN, SycN and YscB were used to immunoprecipitate the corresponding protein and proteins cross-linked with the immunoprecipitated protein (co-precipitating proteins). Cross-linked immunoprecipitates were collected using protein A–Sepharose CL-4B, washed, cleaved by the addition of solubilization buffer containing 5% β-mercaptoethanol and analysed by SDS–PAGE and immunoblot analysis (Fig. 4).

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Figure 4. . Co-immunoprecipitation of SycN, YscB and YopN. Soluble extracts of plasmid pPCP1 cured (Pla−) Y. pestis strains grown at 37°C in the absence of calcium were cross-linked with DSP and immunoprecipitated with antisera specific for YopN (A), SycN (B) or YscB (C). Washed immunoprecipitates were boiled for 3 min in the presence of 5% β-mercaptoethanol to break chemical cross-links and analysed by SDS–PAGE and immunoblot analysis. Antisera specific for YopN, SycN and YscB were used to detect the immunoprecipitated and co-precipitated proteins. Lanes: 1, KIM8-3002 (parent); 2, KIM8-3002.P7 (ΔyopN ); 3, KIM8-3002.P8 (ΔtyeA); 4, KIM8-3002.P9 (ΔsycN ); 5, KIM8-3002.P10 (ΔyscB).

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Antiserum specific for YopN immunoprecipitated YopN from the parent strain, the tyeA deletion mutant, the sycN deletion mutant and the yscB deletion mutant (Fig. 4A). As expected, no yopN gene product was immunoprecipitated from the yopN deletion strain. SycN and YscB co-precipitated with YopN in both the parent strain and the tyeA deletion strain; however, these proteins failed to co-precipitate with YopN in the sycN and yscB deletion mutants. Antiserum specific for SycN immunoprecipitated SycN from the parent strain, the yopN deletion mutant, the tyeA deletion mutant and the yscB deletion mutant (Fig. 4B). Likewise, antiserum specific for YscB immunoprecipitated YscB from the parent strain, the yopN deletion mutant, the tyeA deletion mutant and the sycN deletion mutant (Fig. 4C). YopN co-precipitated with both immunoprecipitated SycN (Fig. 4B) and YscB (Fig. 4C) in the parent strain and in the tyeA deletion strain. No significant co-precipitation of YopN with either SycN or YscB was observed in the yopN deletion mutant, the sycN deletion mutant or the yscB deletion mutant. A small amount of YopN was detected in samples from the sycN and yscB deletion strains immunoprecipitated with antisera specific for YscB or SycN respectively (Fig. 4B and C). However, a similar background level of YopN was also found in control immunoprecipitations with preimmune antiserum (data not shown; Jackson et al., 1998). These data indicate that cross-linking with DSP stabilized a complex of YopN and SycN and/or YscB. The complex or complexes could be immunoprecipitated efficiently with antisera specific for either YopN, SycN or YscB, suggesting that these proteins are closely associated or directly interact with one another in Y. pestis. The fact that SycN did not co-precipitate with YopN in a yscB deletion mutant and, likewise, that YscB did not co-precipitate with YopN in a sycN deletion mutant suggests that both SycN and YscB are required to form a stable interaction with YopN. The absence of functional TyeA (Fig. 4, lane 2) did not effect the interactions between SycN, YscB and YopN, suggesting that the interaction of SycN and YscB with YopN is independent of the interaction between TyeA and YopN (Iriarte et al., 1998).

Interaction of SycN with YscB

The fact that SycN and YscB did not co-precipitate with YopN in either a sycN or a yscB deletion mutant suggested that these proteins interact with one another before interaction with YopN. Although SycE is thought to function as a homodimer, no interaction between different individual Syc and Syc-like proteins has been reported. We used protein cross-linking and immunoprecipitation techniques to test for interactions between SycN and YscB (Fig. 4). SycN co-precipitated with immunoprecipitated YscB in the parent strain, in the yopN deletion mutant and in the tyeA deletion mutant (Fig. 4C). Likewise, YscB co-precipitated with immunoprecipitated SycN in these same three strains (Fig. 4B). The fact that SycN and YscB co-precipitated with each other in both the yopN and tyeA deletion mutants suggests that SycN and YscB interact with one another independently of YopN and TyeA. Interestingly, the amount of SycN immunoprecipitated from the yscB deletion mutant with the anti-SycN antiserum was significantly less than that immunoprecipitated from the parent strain, the yopN deletion mutant and the tyeA deletion mutant, suggesting that SycN is either expressed at a lower level or is less stable in the absence of YscB. Similarly, less YscB was immunoprecipitated from the sycN deletion mutant than from the other YscB-expressing strains. Often, the stability of proteins that function together as a complex are compromised in the absence of their interacting partners; thus, the decrease in SycN and YscB in the yscB and sycN deletion mutants, respectively, also suggests that SycN and YscB function together as a heteromultimer.

Use of the yeast two- and three-hybrid systems to confirm interactions between SycN, YscB and YopN

The protein cross-linking and immunoprecipitation studies described above suggest that SycN and YscB form a complex or are part of a larger complex that interacts directly or indirectly with YopN within the cell. In order to confirm these interactions and to demonstrate that no additional Yersinia proteins are required for these interactions, we used yeast two-and three-hybrid systems to examine the interactions of SycN, YscB and YopN. Plasmids pGAD424 and pGBT9 (Clontech) were used to create gene fusions to the yeast GAL4 activation and DNA binding domains respectively. Plasmid pDela (Zhang and Lautar, 1996), which allows the construction of gene fusions to the SV40 large T antigen nuclear localization sequence, was used to target a third fusion protein to the yeast nucleus for three-hybrid experiments. Gene fusions of sycE and yopE to the sequences encoding GAL4 activation and DNA binding domains were also constructed and used to analyse the interaction of SycE with itself and YopE. Expression of the lacZ gene product in Saccharomyces cerevisiae SFY596 and BY3161 was measured by both colony lift and quantitative liquid β-galactosidase assays (Miller, 1992).

S. cerevisiae SFY596 cells containing both the pGAD424 and pGBT9 cloning vectors were used as negative controls. Cultures of these cells produced no β-galactosidase activity (< 1.0 U) (Table 1), indicating that the isolated GAL4 activation and DNA binding domains do not interact with each other. SFY596 cells containing pGAD-SycE and pGBT-YopE produced 111.9 ± 3.9 U of β-galactosidase, confirming that these proteins interact directly with each other, as shown previously using protein affinity blots (Wattiau and Cornelis, 1993). The specificity of the SycE–YopE interaction was apparently maintained in the yeast system, because pairing pGAD-SycE with pGBT-YopN or pGBT-YopE with pGAD-YopN, pGAD-SycN or pGAD-YscB produced no significant β-galactosidase activity. SFY596 cells containing pGAD-SycE and pGBT-SycE produced 91.7 ± 3.4 U of β-galactosidase, verifying that SycE has the capacity to interact (dimerize) with itself, as suggested by Wattiau and Cornelis (1993). These results indicate that the two-hybrid system is a practical tool for demonstrating specific interactions between Syc proteins and their cognate Yop.

Table 1. . Yeast two-hybrid analysis of YopN, SycN, YscB, YopE and SycE interactions. a. The SFY596 reporter strain was transformed with derivatives of the indicated plasmids.b.+ and − signs indicate the relative intensity of the blue colour developed after exposure of lysed cells to Xgal for 1 h.c. Expression of the reporter lacZ gene was measured according to the method of Miller (1992). The values given represent averages (± standard deviations) assayed in triplicate.Thumbnail image of

The interaction of hybrid proteins encoded for by fusions between the entire sycN, yscB, yopN and yopE (negative control) genes to sequences of pGAD424 and pGBT9 encoding the GAL4 activation and DNA binding domains, respectively, were measured by both colony lifts and quantitative liquid β-galactosidase assays (Table 1). In all except one case, plasmids encoding single hybrid proteins were unable to activate lacZ transcription above background in the absence of a second fusion protein (data not shown). The exception was when YopN was fused to the DNA binding domain encoded for on pGBT9, in which case low-level β-galactosidase activity (≤ 4 U) was detected. The combinations of pGAD-SycN and pGBT-YscB, together with the reciprocal combination of pGAD-YscB and pGBT-SycN, produced readily detectable β-galactosidase activity (Table 1), confirming that SycN and YscB associate readily with each other. Providing pGAD-SycN and pGBT-SycN, pGAD-YscB and pGBT-YscB or pGAD-YopN and pGBT-YopN to S. cerevisiae SFY596 failed to activate lacZ transcription (Table 1), suggesting that SycN, YscB and YopN, unlike SycE, do not dimerize or multimerize on their own. Finally, combinations of pGAD-YopN with either pGBT-SycN or pGBT-YscB also failed to activate lacZ transcription in S. cerevisiae SFY596, confirming that SycN or YscB alone do not interact with YopN. These data are consistent with a model requiring the formation of a SycN–YscB heteromultimer for binding to YopN.

To test this model, we used the three-hybrid system described by Zhang and Lautar (1996) to detect the formation of ternary protein complexes. In this system, the third hybrid protein is provided by plasmid pDela as a fusion to the SV40 T-antigen nuclear localization sequence. This system allows for the addition of a third hybrid protein that is essential to bring together two other hybrid proteins expressed as fusions to the GAL4 activation and DNA binding domains of pGAD424 and pGBT9 respectively. Positive control transformants of S. cerevisiae BY3161 carrying plasmids pGAD-SycN, pGBT-YscB and pDela or pGAD-YscB, pGBT-SycN and pDela produced significant levels of β-galactosidase (26.7 ± 0.9 and 47.4 ± 0.3 U respectively) confirming the utility of BY3161 as a reporter strain (Table 2). BY3161 carrying plasmids pGAD-YopN, pGBT-YscB and the pDela vector failed to produce significant β-galactosidase activity (< 1.0 U). However, substituting pDela-SycN for the pDela vector resulted in the production of significant β-galactosidase (31.9 ± 0.6 U) (Table 2). In a similar manner, BY3161 carrying plasmids pGAD-YopN and pGBT-SycN produced only background levels of β-galactosidase unless also provided with pDela-YscB. These results confirm that a complex composed of both SycN and YscB is required to interact with YopN.

Table 2. . Yeast three-hybrid analysis of YopN, SycN and YscB interactions. a. The BY3161 reporter strain was transformed with derivatives of the indicated plasmids.b.+ and − signs indicate the relative intensity of the blue colour developed after exposure of lysed cells to Xgal for 1 h.c. Expression of the reporter lacZ gene was measured according to the method of Miller (1992). The values given represent averages (± standard deviations) assayed in triplicate.Thumbnail image of

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The secretion and delivery of Y. pestis pCD1-encoded virulence proteins is blocked in the presence of calcium before contact with a eukaryotic cell (Michiels et al., 1990). Previous studies have established a role for the yopN (Viitenan et al., 1990; Forsberg et al., 1991), lcrG (Skrzypek and Straley, 1993), yscB (Jackson et al., 1998) and tyeA (Iriarte et al., 1998) gene products in blocking Yop secretion. In this study, we have established a role for the sycN gene product in the regulation of Yop secretion and, specifically, in the secretion and/or presentation of YopN in Y. pestis. Through protein cross-linking and immunoprecipitation analyses in conjunction with yeast two- and three-hybrid studies, we have begun to understand how these proteins interact with one another to regulate the expression and delivery of virulence proteins.

The direct interaction between SycN and YscB represents the first example of two separate Syc or Syc-like proteins that function together as a chaperone for a secreted virulence protein (YopN). The inability of the individual SycN or YscB proteins to interact with YopN suggests that only a SycN/YscB heteromultimer is capable of interacting with YopN. The stoichiometry of the SycN/YscB complex has yet to be determined. However, limited evidence suggests that SycN and YscB function as a heterodimer. The fact that SycE has previously been hypothesized to function as a homodimer (Wattiau and Cornelis, 1993) suggests that the structurally similar SycN and YscB proteins interact in a similar or related manner. Furthermore, two-hybrid studies indicated that SycN and YscB, unlike SycE, do not self-associate, but interact only with each other, arguing against the formation of a larger complex composed of SycN and YscB homodimers. The functional significance of Syc heteromultimerization (SycN/YscB) and homomultimerization (SycE/SycE) is unknown.

Previous studies with YscB determined that a region of YopN between amino acid residues 51 and 85 was necessary for co-immunoprecipitation of a YscB/YopN-containing complex (Jackson et al., 1998). We now know that SycN was also part of this complex and that both SycN and YscB are required to interact with YopN. The region eliminated by the 51–85 yopN deletion mutant includes a portion of a predicted coiled-coil domain (Pallen et al., 1997) that extends from amino acid residue 70 to 100. As coiled-coil domains are implicated in protein–protein interactions, this region might be involved in the interaction between the SycN/YscB complex and YopN. The MULTICOIL program (Pallen et al., 1997; Wolf et al., 1997) predicted the coiled-coil domain in YopN to be trimer forming; however, our two-hybrid experiments with YopN indicate that YopN does not multimerize. Therefore, we suggest that it is the interaction of YscB, SycN and YopN that occurs in this region.

The exact function of any individual Syc or Syc-like protein has yet to be determined. Mutational inactivation of individual syc gene products results in a marked reduction in the export of each individual Syc's cognate Yop. Cheng et al. (1997) demonstrated that a region encompassing the SycE-binding region of YopE could target YopE for secretion in a SycE-dependent manner, thus suggesting a direct role for SycE in YopE secretion. In addition, the specific Syc and Syc-binding region of YopE and YopH has been shown to be required for translocation of YopE and YopH hybrid proteins into eukaryotic target cells (Rosqvist et al., 1994; Sory et al., 1995; Schesser et al., 1996; Woestyn et al., 1996; Lee et al., 1998). Individual Syc proteins have also been suggested as playing a role in preventing proteolytic degradation of their specific Yop before secretion (Frithz-Lindsten et al., 1995).

Evidence provided by Boland et al. (1996) indicates that YopN of Y. enterocolitica is not translocated into eukaryotic cells. If this data is confirmed in Y. pestis, a function of the SycN/YscB complex in YopN translocation can be ruled out. Interestingly, Syc and Syc-like proteins have primarily been found associated with proteins that are translocated into eukaryotic cells, suggesting that the issue of YopN translocation should be re-examined. Indeed, recent evidence from Lee et al. (1998) indicates that Y. enterocolitica translocates a small, but significant, amount of YopN into HeLa cells.

The absence of SycN or YscB resulted in an increase in YopN breakdown products in the cell pellet fraction (Fig. 2). However, the majority of YopN appeared to remain proteolytically stable, indicating that the SycN/YscB complex does not play a major role in protecting YopN from proteolytic degradation within the cell. Thus, the primary consequence of eliminating SycN and/or YscB appears to be on the secretion and/or surface presentation of functional YopN. The amount of YopN secreted into the culture supernatant is reduced significantly in both the sycN and yscB deletion mutants. However, secreted YopN can still be detected easily in both mutant strains (see Fig. 2). The remaining YopN secretion in the sycN and yscB deletion mutants is most probably dependent upon the previously identified mRNA secretion signal (Anderson and Schneewind, 1997). Although the loss of SycN and/or YscB did not completely prevent YopN secretion, the ability to block secretion of the remaining Yops was lost completely. These results suggest that YopN must be efficiently targeted to the Yop secretion apparatus in order to block Yop secretion, that SycN and YscB are required to present YopN in a conformation capable of blocking secretion or that SycN and YscB participate directly in blocking Yop secretion.

Interestingly, proteins similar to SycN, YscB and YopN, termed Pcr2, PscB and PopN, respectively, have been identified in the opportunistic pathogen Pseudomonas aeruginosa (Yahr et al., 1997). The amino acid sequence similarity of these proteins and the similarity in triggering signals between the Yersinia and P. aeruginosa type III secretion systems suggests that these proteins perform similar functions in both of these organisms. Proteins with significant amino acid similarity to YopN have also been identified in Chlamydia psittaci (Hsia et al., 1997), Salmonella typhimurium (Ginocchio et al., 1992) and in Erwinia amylovora (Bogdanove et al., 1996), suggesting that these organisms use similar mechanisms to control virulence protein secretion.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains and culture conditions

All Y. pestis strains used in this study are Pgm− (Une and Brubaker, 1984). Y. pestis KIM5-3001 (Lindler et al., 1990) and KIM8-3002 (Pla−) (Nilles et al., 1997) were used as parent strains. These strains and derivatives of these strains were grown routinely in heart infusion broth (HIB) liquid media or on tryptose blood agar (TBA) plates (Difco) at a temperature of 28°C. Y. pestis KIM8 (pCD1, pPCP1-[Pla−], pMT1) and KIM10 (pCD1-[Lcr−], pPCP1-[Pla−], pMT1) were used in fractionation and localization studies. Plasmid pPCP1 encodes the outer membrane plasminogen activator protease (Pla) that has been shown to degrade exported Yops (Protsenko et al., 1983). For growth experiments, Yersinia were grown in the presence or absence of 2.5 mM calcium chloride in the defined medium TMH as described previously (Goguen et al., 1984; Plano and Straley, 1995). Escherichia coli SY327 λpir (Miller and Mekalanos, 1988) was used to propagate derivatives of the suicide vector pUK4134. E. coli XL1 blue (Stratagene) was used for routine cloning experiments, and E. coli BL21(DE3) (Novagen) was used for overexpression of polyhistidine-tagged TyeA and SycN.

Construction of tyeA and sycN deletion mutants

In frame deletions in tyeA and sycN eliminating the coding regions for amino acids 10–55 and 34–65, respectively, were constructed by the PCR–ligation–PCR technique (Ali and Steinkasserer, 1995) and cloned into pT7BLUE (Novagen), generating plasmids pΔTYEA1 and pΔSYCN1. Oligonucleotides used in the construction of pΔTYEA1 (5′-AAT-

TAACCCTCACTAAAGGG-3′, 5′-CCCATAAACTCAGAAAGGTC-3′, 5′-CCCTCTGGGGGTATTTAGCG-3′ and 5′-ACTGTTCGAAAGCTTGATGT-3′) and pΔSYCN (5′-CCCTCT-

GGGGGTATTTAGCG-3′, 5′-TTGAGCCATCTCTAATTGAA-3′, 5′-GCGCTAACGCTCACGGCGGC-3′ and 5′-GTGCCTCAGCGGCCGCTAATCC-3′) were obtained from The Great American Gene Company. A 0.9 kb MscI–Bsi WI fragment of pΔTYEA1 carrying the deletion in tyeA was inserted into MscI–Bsi WI-digested pGP2K generating plasmid pGP2K-ΔTYEA. A 0.6 kb Bsi WI–BstBI fragment of pΔSYCN1 containing the sycN deletion was inserted into Bsi WI–BstBI-digested pGP2K generating plasmid pGP2k-ΔSYCN. Plasmid pGP2K carries a 3580 bp BamHI–KpnI fragment of pGP2 encoding YopN, TyeA, SycN, ORF3 and ORF4. An approximately 2.7 kb EcoRV fragment of both pGP2K-ΔTYEA and pGP2k-ΔSYCN was inserted into the EcoRV site of the suicide vector pUK4134 (Skrzypek et al., 1993), generating plasmids pUK4134.P2 and pUK4134.P3 respectively. These plasmids were introduced into Y. pestis KIM5-3001 and KIM8-3002 by electroporation, and recipient bacteria that had integrated the clones into pCD1 by homologous recombination were selected for by their resistance to ampicillin as described previously (Skrzypek et al., 1993). After passage under non-selective conditions, clones that had resolved the co-integrate by excision of the vector sequences were selected for by their resistance to streptomycin. Streptomycin-resistant resolvants were screened for the correct deletion by PCR and restriction endonuclease digestion. Y. pestis KIM5-3001.P2 and KIM8-3002.P8 contained the correct in frame deletion in tyeA. Y. pestis KIM5-3001.P3 and KIM8-3002.P9 contained the correct in frame deletion in sycN.

The TyeA-expressing plasmid pTYEA1 was constructed by inserting a 0.67 kb BglII–PvuII fragment of pGP2 (Plano et al., 1991) carrying the entire tyeA gene into BamHI–EcoRV-digested pBluescript KS−, such that the gene is transcribed from the vector lac promoter. Plasmid pSYCN1 contains a sycN-encoding, 0.7 kb DraI–SacI fragment of pGP2 inserted into EcoRV–SacI-digested pBluescript KS− downstream of the vector lac promoter. Plasmids pTYEA1, pSYCN1 and pYSCB1 (Jackson et al., 1998) were used in complementation experiments.

Generation of antisera specific for TyeA and SycN

Overexpression and purification of TyeA and SycN was facilitated by the construction of expression plasmids pET-TYEA1 and pET-SYCN1. The entire tyeA and sycN genes were amplified by PCR using the oligonucleotide primers TyeA-H1 (5′-TCATATGGCGTACGACCTTTCTGA-3′), TyeA-H2 (5′-AGGATCCTCAATCCTCAATCCAACTCACTCAATT-3′), SycN-H1 (5′-TCATATGGTGAGTTGGATTGAACCCA-3′) and SycN-H2 (5′-AGGATCCTCACGGCGCAAGCACCTCT-3′). The oligonucleotides were tailed with NdeI and BamHI restriction endonuclease sites (underlined) to facilitate insertion of the amplified fragment into NdeI–BamHI-digested pET15b (Novagen). Plasmids pET-TYEA1 and pET-SYCN1 were electroporated into E. coli BL21 (DE3) for overexpression of polyhistidine-tagged TyeA and SycN. Overexpression of polyhistidine-tagged TyeA and SycN was induced by the addition of IPTG to a final concentration of 1 mM. Purification of the polyhistidine-tagged proteins was performed according to the manufacturer's protocol using the His-Bind resin and buffer kit (Novagen). Polyclonal antisera specific for TyeA and SycN were raised in female New Zealand White rabbits using the purified TyeA and SycN proteins (Animal Pharm Services).

SDS–PAGE and immunoblotting

Volumes of cellular fractions corresponding to equal numbers of bacteria were mixed 1:1 (v/v) with 2× electrophoresis sample buffer and analysed by SDS–PAGE and immunoblot analysis essentially as described previously (Plano and Straley, 1995). The samples to be analysed with polyclonal antisera specific for SycN or YscB were electrophoresed on 14% (w/v) acrylamide gels. Y. pestis LCR proteins were visualized as described previously using polyclonal antisera or purified antibodies specific for YopM, V antigen and YopN (Plano and Straley, 1995).

Cross-linking and immunoprecipitation of SycN, YscB and YopN

Soluble proteins from Yersinia cultures grown at 37°C in the absence of calcium were cross-linked with the thio-cleavable, amine-reactive cross-linker DSP (Pierce Chemical) at a final concentration of 1 mM for 30 min at room temperature. Samples were immunoprecipitated with 10 μl of rabbit polyclonal antisera specific for YopN, SycN or YscB. Antigen–antibody complexes were collected by the addition of 100 μl of 10% (w/v) protein A–Sepharose CL-4B (Pharmacia) in immunoprecipitation buffer (20 mM HEPES, 250 mM NaCl, 0.5% Triton X-100, 0.1% SDS, pH 7.4) for 2 h at 4°C. Protein A–Sepharose antigen–antibody complexes were pelleted by centrifugation in a microfuge for 15 s at room temperature, washed three times with 1 ml of immunoprecipitation buffer, eluted with 100 μl of electrophoresis sample buffer containing 5% BME (cleaves DSP cross-links), boiled for 3 min and subjected to SDS–PAGE and immunoblot analysis.

Yeast two- and three-hybrid systems

The yeast two-hybrid assay was performed using methods recommended by the commercial supplier (Clontech). Vectors pGAD424 and pGBT9 encoding the activation domain and DNA binding domain, respectively, of the yeast GAL4 transcriptional activator were obtained from Clontech as part of the Matchmaker two-hybrid system kit. The YopN (5′-CCGGAATTCATGACGACGCTTCATAACCTATC-3′, 5′-TCCGATGCATTCAGAAAGGTCGTACGCCATTAG-3′), SycN (5′-CCGGAATTCAGTTGGATTGAACCCATCAT-3′, 5′-TCCGCTGCAGTCACGGCGCAAGCACCTCTTGCT-3′), YscB (5′-CCGGAATTCATGCAAAATTTACTAAAAAACTT-3′, 5′-TCCGCTGCAGTTAATTCCACCCCACGCGAGACG-3′), YopE (5′-CCGGAATTCATGAAAATATCATCATTTATTTC-3′, 5′-TCCGATGCATTCACATCAATGACAGTAATTTCT-3′) and SycE (5′-CCGCAATTGATGTATTCATTTGAACAAGCT-3′, 5′-TCCGCTGCAGTCAACTAAATGACCGTGGTGG-3′) coding sequences were obtained by high-fidelity PCR, digested with EcoRI (or MfeI) and PstI (or NdeI) and inserted into EcoRI–PstI-digested pGAD424 and pGBT9, generating plasmids pGAD-YopN, pGAD-SycN, pGAD-YscB, pGAD-YopE, pGAD-SycE, pGBT-YopN, pGBT-SycN, pGBT-YscB, pGBT-YopE and pGBT-SycE. Plasmids were transformed into S. cerevisiae SFY596 according to the manufacturer's instruction (Clontech) and plated on appropriate SD plates. Colony lift assays and liquid assays for detecting β-galactosidase activity (Miller, 1992) were performed essentially as described by Clontech.

Interactions between three Yersinia proteins were detected using a yeast three-hybrid system (Zhang and Lautar, 1996), consisting of the yeast strain BY3161 (MATα, leu2-3, trp1-901, his3-200, ura3-52, ade2-101, gal4-542, gal80-538, GAL1-lacZ, GAL1-His3) and plasmids pGBT9, pGAD424 and pDela. BY3161 was a kind gift from Dr Jef Boeke (Johns Hopkins University School of Medicine), and plasmid pDela was provided by Dr Jie Zhang of Guilford Pharmaceuticals. Plasmid pDela allows the construction of gene fusions to the sequence encoding the SV40 T-antigen nuclear localization sequence. The Ura3+ gene of pDela allows for the selection of triple transformants of pGAD424, pGBT9 and pDela in the ura− trp− leu− yeast strain BY3161. The entire sycN and yscB genes were obtained by high-fidelity PCR using oligonucleotide primers pDela-N1 (5′-AAAGGATCCGTGAGTTGGATTGAACCCATC-3′), pDela-N2 (5′-AAAGAATTCTCACGGCG-

CAAGCACCTCTTG-3′), pDela-B1 (5′-AAAGGATCCATGCAAAATTTACTAAAAAAC-3′) and pDela-B2 (5′-AAAGAATTCTTATTCCACCCCACGCGAGAC-3′). Primers were designed such that the amplified gene products contained flanking EcoRI and BamHI restriction endonuclease sites (underlined). Amplified sycN and yscB products were digested with EcoRI and BamHI and inserted into EcoRI–BamHI-digested pDela, creating plasmids pDela-SycN and pDela-YscB respectively. These plasmids were used in conjunction with the pGBT9 and pGAD424 vectors described above to detect the formation of ternary protein complexes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Dr Jef Boeke for the BY3161 yeast strain, and Dr Jie Zhang and Guilford Pharmaceuticals for plasmid pDela. We also thank Dr Diana Tomchick for critical reading of the manuscript and valuable comments. This work was supported by Public Health Service grant AI39575.

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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