Max von Pettenkofer-Institut, Pettenkoferstr. 9a, D-80336 Munich, Germany1
Author for correspondence: Jürgen Heesemann. Tel: +49 89 5160 5200. Fax: +49 89 5160 5202. e-mail: heesemann{at}m3401.mpk.med.uni-muenchen.de
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ABSTRACT |
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Keywords: YopQ-GFP accumulation, yopQ/yopK sequences, mRNA secondary structures
Abbreviations: BHI, brainheart infusion; Cm, chloramphenicol; GFP, green fluorescent protein; GST, glutathione S-transferase
The GenBank/EMBL accession numbers for the sequences reported in this paper are AJ421529 [yopQ gene fragment from Y. enterocolitica WA-314 (O:8)] and AJ421530 [yopQ gene fragment from Y. enterocolitica Y-108-P (O:3)].
a These authors contributed equally to this work.
b Present address: University of Ljubljana, Institute of Microbiology and Microbial Biotechnology, Groblje 3, SI-1230 Domale, Slovenia.
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INTRODUCTION |
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Anderson & Schneewind (1997) reported that several frameshift mutations, which completely altered the peptide sequence of the first 15 codons of the yopE and yopN genes, did not prevent secretion. This finding led to the assumption that the secretion signal is at the mRNA level. It was further suggested that an mRNA element signals transport of Yops by coupling translation and secretion (Anderson & Schneewind, 1999
). Recently, Lloyd et al. (2001b
) performed a systematic mutational analysis within the N-terminal sequence of YopE. Some of the mutations, changing the amino acid sequence, had significant effects on the secretion of YopE. Therefore, they concluded that secretion of YopE is directed by the N terminus and not by the 5' end of yopE mRNA.
YopQ of Yersinia enterocolitica (YopK of Yersinia pseudotuberculosis) is a secreted protein (ca 18 kDa) that is required for virulence (Holmström et al., 1995 ; Mulder at al., 1989
). YopK was shown to be located in association with bacteria infecting cultured cells and it has been suggested that YopK controls translocation of Yops by regulating the size of the translocation pore (Holmström et al., 1997
). No chaperone has been described for YopQ/K. Based on the results performed with Y. enterocolitica strain W22703, Anderson & Schneewind (1999)
predicted a co-translational mechanism of YopQ secretion. They proposed that an mRNA secretion signal at the 5' end is necessary and sufficient for secretion of YopQ. In accordance with their prediction they could not detect YopQ intracellularly. Moreover, they demonstrated lack of post-translational secretion of YopQ (Anderson & Schneewind, 1999
). In contrast, it has been shown that YopK of Y. pseudotuberculosis is accumulated intracellularly (Holmström et al., 1995
), indicating that the mRNA signal hypothesis cannot be applied to YopQ/K of all pYV-carrying Yersinia species.
In this study YopQ production and secretion was investigated in different serotypes of Y. enterocolitica. Cytosolic accumulation and secretion of YopQ was analysed by immunoblotting. yopQ translational fusions with gfp, encoding the green fluorescent protein (Jacobi et al., 1998 ), were constructed to study cytosolic accumulation of the YopQ-GFP hybrid protein in yersiniae under various culture conditions. The intensity of fluorescence was measured by flow cytometry. By infecting HeLa cells we tried to localize YopQ-GFP by confocal laser scanning microscopy.
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METHODS |
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Nucleic acid manipulation.
Plasmid DNA preparation, restriction enzyme digests, ligations and transformations were performed as described by Sambrook et al. (1989) . Plasmid DNA was sequenced using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and the ABI Prism 377XL DNA Sequencer (Applied Biosystems).
Plasmids pJTYopQO8-GFP3 and pJTYopQO9-GFP3 were constructed by cloning yopQ from Y. enterocolitica WA-314, serotype O:8 (Heesemann, 1987 ), and Y. enterocolitica MRS 40, serotype O:9 (Sory et al., 1995
), respectively. The yopQ PCR-amplified fragments were ligated into the HindIII/BamHI restriction sites of plasmid pCJ-G3 (Jacobi et al., 1998
). The yopQ gene, including 391 bp upstream of the translational start but without stop codon, was PCR-amplified using the forward primer 5'-CGGGATCCTCCCATAATACATTTTTGATC-3' and reverse primer 5'-ACGCAAGCTTTCTCTGCGTCAGAGTACATG-3', both preceded by appropriate restriction sites (underlined). yopQO8-gfp3 and yopQO9-gfp3 were cointegrated into virulence plasmids pYVO8 and pYVO9, respectively, by homologous recombination. Fragments yopQO8-gfp3 and yopQO9-gfp3 were also inserted into the SalI/XbaI restriction sites of suicide plasmid pKAS32 and transformed into E. coli S17-1
pir (Skorupski & Taylor, 1996
). After conjugation and single cross-over, suicide plasmids pKASYopQO8-GFP3 and pKASYopQO9-GFP3 were co-integrated into the virulence plasmids of Y. enterocolitica WA-314 and Y. enterocolitica MRS 40, respectively. The correct co-integration was confirmed by sequencing.
Flow cytometric measurement.
A Coulter Epics flow cytometer equipped with an argon 488 nm laser was used. Bacteria were diluted and detected by side scatter as described by Russo-Marie et al. (1993) and Jacobi et al. (1998)
. The scale was logarithmic, and fluorescence data and scatter data were collected for 10000 events.
Protein analyses.
Yersiniae were harvested by centrifugation (12000 g, 10 min) and the culture supernatant was collected. Released proteins were precipitated with trichloroacetic acid and prepared as described previously (Heesemann et al., 1986 ). To eliminate cell-surface-associated Yops, pelleted cells were resuspended in PBS containing 600 µg proteinase K ml-1. After incubation for 15 min at 30 °C, PMSF was added to 5 mM and cells were pelleted and dissolved in SDS sample buffer (see below) supplemented with 5 mM PMSF. Samples from supernatant and whole bacteria were dissolved in a modified SDS sample buffer consisting of 0·1 M MgCl2, 4% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0·001% bromophenol blue and 100 mM Tris/HCl, pH 9. MgCl2 was added to SDS sample buffer to precipitate DNA of whole-cell lysates (Chen & Christen, 1997
). Tris/HCl, pH 9, instead of pH 6·8, was used to eliminate hydrolysis of aspartyl-prolyl peptide bonds (Cannon-Carlson & Tang, 1997
). The volume of samples was adjusted in accordance to the OD600 values of bacterial cultures. Electrophoresis in 12% polyacrylamide gels in the presence of SDS was performed as described by Laemmli (1970)
. Proteins were transferred to a nitrocellulose membrane by electroblotting (Towbin et al., 1979
). The membrane sheets were blocked with 0·5% Tween 20 (Sigma-Aldrich) and 5% bovine serum albumin (Sigma-Aldrich) in PBS. Immunostaining of YopQ/K, YopE and GFP was performed with rabbit anti-glutathione S-transferase (GST)-YopQ polyclonal antibodies (dilution 1:10000), anti-YopE polyclonal antibodies (dilution 1:5000), or anti-GFP polyclonal antibodies (dilution 1:3000) and peroxidase-conjugated secondary anti-rabbit antibodies (dilution 1:5000). Detection was carried out using the ECL Western Blotting System (Amersham Pharmacia Biotech). For immunostaining of YadA the mAb 8D1 (Roggenkamp et al., 1995
) was used. Subsequently, secondary binding of alkaline-phosphatase-conjugated anti-mouse IgG was detected with BCIP/NBT substrate (Sigma).
Preparation of polyclonal antibodies.
Polyclonal antibodies against fusion protein GST-YopQ were generated by immunization of a rabbit. The yopQ gene was amplified by PCR and cloned in-frame downstream of gst in the vector pGEX-4T-3 (Amersham Pharmacia Biotech). The fusion protein could not be successfully expressed in E. coli; therefore wild-type Y. enterocolitica was used as host organism. Protein was purified by GST purification modules according to the manufacturers instructions (Amersham Pharmacia Biotech).
Microscopic examination.
In the HeLa cell infection model the recombinant yersiniae with GFP constructs were visualized by using an Aristoplan epifluorescence and confocal laser scanning microscope (Leica TCS 4D) (CLSM) as described by Jacobi et al. (1998) .
Computer data analysis.
The sequences were aligned by using the CLUSTAL X 1.8 program (Thompson et al., 1994 ) and folded by using the RNAdraw program (Matzura & Wennborg, 1996
); in both cases using the default settings.
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RESULTS AND DISCUSSION |
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As a second approach, we studied the production of YopQ-GFP hybrid proteins by Y. enterocolitica WA-314 and Y. enterocolitica MRS 40. Reporter constructs were under the control of their autochthonous YopQ promoter and were inserted into the low-copy-number plasmid pACYC184 (Chang & Cohen, 1978 ). Both bacteria induced for secretion of Yops, as well as those blocked for secretion, fluoresced. The flow cytometry analysis showed that Y. enterocolitica WA-314 carrying plasmid pJTYopQO8-GFP3 fluoresced more strongly than Y. enterocolitica MRS 40 harbouring pJTYopQO9-GFP3 (Fig. 2b
). These results are supported by immunoblot analysis (Fig. 2a
). However, bacteria induced for secretion fluoresced more strongly than those in which secretion was blocked, although secretion of the hybrids was detected. This was surprising, since in the case of an open secretion pore, one would expect the majority of the fusion protein to be released from the bacteria. This might be due to impaired secretion of the hybrid protein compared to native YopQ (Fig. 3a
). In accordance with this, the secreted YopQ-GFP did not differ significantly among the strains compared (Fig. 2a
).
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Thus, all these results contradict the co-translational model of YopQ secretion which predicts no YopQ accumulation in the cytosol of yersiniae (Anderson & Schneewind, 1999 ). One reason for the discrepancy might be that Anderson & Schneewind established the model for YopQ secretion based on the characteristics of one single strain of Y. enterocolitica and its mutants, that is Y. enterocolitica W22703, serotype O:9 (Cornelis & Colson, 1975
). Our comparative analysis of Y. enterocolitica strains of different serotypes shows that YopQ expression appears to be strain-dependent and that significant amounts of YopQ are not co-translationally secreted. The contradictory results may also be caused by the instability of YopQ during sample preparation for SDS-PAGE.
The specificity and efficiency of plasmid-encoded type III secretion was proven for strain Y. enterocolitica WA-314(pJTYopQO8-GFP3) and was further used to monitor translocation of YopQ into HeLa cells (Fig. 3). A comparison with the secretion-deficient lcrD mutant WA-C(pYV-515) harbouring the same plasmid served as a control for specificity of protein release. Fig. 3(a)
additionally reveals that YopQ-GFP detected in whole-cell lysates is not associated with the cell surface. Approximately 35% of the intracellular amount of YopQ-GFP was detected in the supernatant (Fig. 3a
). However, we could not demonstrate translocation of YopQ-GFP fusion protein into eukaryotic cells (Fig. 3b
). This is in line with the results of Holmström et al. (1997)
who could not find translocated YopK.
Comparison of the yopQ sequences among the Y. enterocolitica strains
To explain the differences in the level of yopQ expression among the Y. enterocolitica strains, we sequenced the 5' end as well as the upstream region of the yopQ genes from Y. enterocolitica WA-314, serotype O:8, and Y. enterocolitica Y-108-P, serotype O:3. We compared the sequences to each other and to the homologous sequences of Y. enterocolitica serotype O:9 (accession no. NC 002120), Y. pseudotuberculosis (U18804) and Y. pestis (NC_001882). Downstream of the transcriptional start site of yopQ, which was determined by Anderson & Schneewind (1999) , an obvious difference exists between the sequences. The sequence of Y. enterocolitica O:8, 5 nt after the transcriptional start, shows a deletion of 8 nt compared to the homologous sequences of Y. enterocolitica serotype O:9, Y. enterocolitica serotype O:3 and Y. pseudotuberculosis. We speculate that this difference might be responsible for a modulation of transcription efficiency. To test this hypothesis, we isolated total RNA from different Yersinia serotypes and performed semiquantitative RT-PCR. However, we could not detect significant differences in the intensity of the RT-PCR signals among the analysed serotypes of yersiniae (data not shown).
Anderson & Schneewind (1997) had predicted a stemloop in the secretion signal of YopE and YopN, with the start codon and a possible ShineDalgarno ribosome-binding site being base-paired. They suggested that such an RNA structure inhibits a successful translation of yopQ by preventing ribosome loading (Anderson & Schneewind, 1997
). By folding yopQ mRNA sequences, we searched for such structures in the putative YopQ secretion signal. Three different mRNA secondary structures were predicted (Fig. 4
). Since the nucleotide sequences of YopQ from the Y. enterocolitica serotype O:9 and Y. enterocolitica serotype O:3 are the same, they could form the same predicted mRNA secondary structure. In that structure (Fig. 4a
) the first two nucleotides of the start codon as well as the upstream sequence with the putative ribosome-binding site are base-paired in a stem. The situation is different in the predicted mRNA secondary structure of yopQ from serotype O:8, where the start codon as well as the putative ribosome-binding site are not base-paired (Fig. 4b
). The predicted yopQ mRNA secondary structures of Y. pseudotuberculosis and Y. pestis are the same (Fig. 4c
). In that structure the first two nucleotides of the start codon are not base-paired, whereas in contrast to the O:8 sequence, the third nucleotide is part of a duplex structure. The ribosome-binding site is not base-paired in Y. pseudotuberculosis and Y. enterocolitica O:8.
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Post-translational secretion of YopQ
Based on the detection of cytosolic YopQ we addressed the question of post-translational secretion of this cytosolic pool. We inhibited protein synthesis by adding Cm after induction of secretion. To ensure a complete inhibition of protein synthesis, antibiotic treatment lasted for 20 min. Supernatant was subsequently removed and the cells were washed. Then they were further incubated in fresh medium under inducing conditions in the presence of antibiotics. Finally, supernatants were collected to analyse post-translationally secreted proteins. As shown in Fig. 5, YopQ of Y. enterocolitica serotypes O:8 and O:9 was still secreted after blocking translation, indicating possible post-translational secretion. Intracellularly, YopQ was not detectable any more after inhibition of protein synthesis, proving the efficiency of inhibition of YopQ biosynthesis and indicating that the cells were bled out by the procedure. This cannot be explained by the half-life of YopQ alone, since the secretion-deficient control strain WA-C(pYV-515) still harbours YopQ after the procedure (Fig. 5
). This is additional evidence that the intracellular YopQ pool is secretion-competent. For YopE a post-translational secretion has been demonstrated (Lloyd et al., 2001b
). Here, analysis of supernatants for YopE secretion shows comparable results to those found for YopQ (Fig. 5
).
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These findings are in contradiction to the work presented by Anderson & Schneewind (1999) , who could not demonstrate post-translational secretion. However, their experimental approach is not comparable as they used a tac promoter for induction of yopQ. This overproduction has been shown by the same authors to influence the regulation of YopQ secretion. It might also be that YopQ overproduction leads to overtitration of an unknown chaperone for YopQ. The role of chaperones in creating a hierarchy of Yop secretion has recently been discussed by Lloyd et al. (2001a
). However, we have no indication that YopQ secretion is delayed compared to YopE. As discussed by Anderson & Schneewind (1999)
, the predicted regulatory role of YopQ/K would suggest an early requirement for secretion. A chaperone for YopQ could also explain such apparent contradictions.
In conclusion our comparative analysis of different serotypes of Y. enterocolitica demonstrates (i) strain-dependent YopQ production and accumulation in the cytosol that might be due to differences in folding structure of the 5' end of the corresponding mRNA, and (ii) that a significant portion of YopQ is post-translationally secreted.
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ACKNOWLEDGEMENTS |
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Received 20 August 2001;
revised 3 January 2002;
accepted 15 January 2002.