scc1 (cp0432) and scc4 (cp0033) function as a type iii secretion

JOURNAL OF BACTERIOLOGY, July 2011, p. 3490–3496 Vol. 193, No. 140021-9193/11/$12.00 doi:10.1128/JB.00203-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Scc1 (CP0432) and Scc4 (CP0033) Function as a Type III SecretionChaperone for CopN of Chlamydia pneumoniae�†

Eugenia Silva-Herzog,2 Sabrina S. Joseph,3 Ann K. Avery,4,5 Jose A. Coba,1 Katerina Wolf,1Kenneth A. Fields,1 and Gregory V. Plano1*

Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, Florida1; Department ofMolecular Microbiology and Infectious Diseases, Florida International University, Miami, Florida2; Department of Microbiology andImmunology, Uniformed Services University, Bethesda, Maryland3; Department of Medicine, Case Western Reserve University,

Cleveland, Ohio4; and Division of Infectious Diseases, MetroHealth Medical Center, Cleveland, Ohio5

Received 11 February 2011/Accepted 4 May 2011

The Chlamydia pneumoniae CopN protein is a member of the YopN/TyeA/InvE/MxiC family of secretedproteins that function to regulate the secretion of type III secretion system (T3SS) translocator and effectorproteins. In this study, the Scc1 (CP0432) and Scc4 (CP0033) proteins of C. pneumoniae AR-39 were demon-strated to function together as a type III secretion chaperone that binds to an N-terminal region of CopN. TheScc1/Scc4 chaperone promoted the efficient secretion of CopN via a heterologous T3SS, whereas, the Scc3chaperone, which binds to a C-terminal region of CopN, reduced CopN secretion.

Numerous Gram-negative bacterial pathogens utilize typeIII secretion systems (T3SSs) to inject effector proteins intotheir host’s cells in order to manipulate signaling pathways thatnormally function to limit bacterial growth and/or dissemina-tion (19). Effector proteins are transported across the bacterialmembranes by a multicomponent type III secretion (T3S) ap-paratus or injectisome (4, 29). The injectisome consists of abase structure that spans both bacterial membranes and aneedle structure that extends 40 to 60 nm from the bacterialcell surface. The passage of effector proteins across the eu-karyotic membrane is enabled by a pore-forming transloconcomplex. Translocon assembly, as well as effector protein se-cretion and injection, is triggered by contact between a bacte-rium and a eukaryotic cell; therefore, the T3S process has beentermed “contact dependent” (33).

The regulation of the T3S process is complex and mani-fested at the levels of both substrate selection and T3S appa-ratus activation (13, 14). Following the assembly of a secretion-competent base structure, substrates required for the assemblyof the internal rod-like and external needle-like structures areselectively secreted (26). Upon completion of the needle/rodassembly, a substrate specificity switch is triggered, allowingthe secretion of translocon components and/or translocon andeffector proteins (1). In many T3SSs, the needle tip complex, incombination with other translocon components, plays a role inpreventing the spurious release of effector proteins prior tocontact with a eukaryotic cell (13). A separate protein com-plex, exemplified by the YopN/SycN/YscB/TyeA complex ofthe Yersinia sp. plasmid-encoded T3SS, also plays an essentialrole in preventing the premature release of effector proteins (7,11). Upon contact with a eukaryotic cell, translocon assembly

is completed and effector protein secretion and injection aretriggered.

In the Yersinia spp., the YopN/SycN/YscB/TyeA complex isrequired to prevent effector protein secretion prior to contactwith a eukaryotic cell in vivo and in the presence of extracel-lular calcium in vitro. The 293-residue secreted and injectedYopN protein interacts with the cytosolic SycN/YscB chaper-one at its N terminus and with the cytosolic TyeA protein at itsC terminus (12, 22). The SycN/YscB chaperone is required forstable expression of YopN and regulation of Yop secretion, aswell as for the efficient secretion and translocation of YopN.TyeA, on the other hand, functions with YopN to directlyregulate effector protein secretion (7, 11). In addition, TyeAfunctions to inhibit YopN translocation upon contact with aeukaryotic cell. Previous studies suggest that the cytosolicYopN/SycN/YscB/TyeA complex prevents effector protein se-cretion from a cytosolic location, possibly via direct interac-tions with the T3S apparatus (16, 30).

Proteins homologous to YopN and TyeA can be found inessentially all nonflagellar T3SSs; however, in the majority ofthese proteins, domains homologous to YopN and TyeA arepresent in a single protein, with sequences homologous toTyeA located in the C-terminal portion of the protein (31). Incontrast, proteins homologous to the Yersinia SycN and YscBproteins have been identified only in closely related T3SSs,such as those of Pseudomonas aeruginosa, Photorhabdus lumi-nescens, Vibrio parahaemolyticus, and Aeromonas spp. (38). Ingeneral, T3SSs that express separate proteins homologous toYopN and TyeA also encode chaperones homologous to SycNand YscB. On the contrary, most T3SSs that express a singleprotein with domains homologous to YopN and TyeA do notencode chaperones homologous to SycN and YscB. Indeed, noT3S chaperones have been identified for MxiC of Shigellaflexneri (5) or InvE of Salmonella enterica (28); however, re-cently, CesL of enteropathogenic Escherichia coli was shown tofunction as a T3S chaperone for SepL, a YopN/TyeA familyprotein with domains homologous to both YopN and TyeA(41). Another possible exception to this rule is the CopN pro-

* Corresponding author. Mailing address: P.O. Box 016960 (R-138),Miami, FL 33101. Phone: (305) 243-6310. Fax: (305) 243-4623. E-mail:[email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 13 May 2011.

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tein of Chlamydia spp. CopN is a secreted and translocatedprotein that is required for Chlamydia pneumoniae intracellu-lar growth (21). CopN, like MxiC, InvE, and SepL, containsdomains homologous to both YopN and TyeA (17). CopN haspreviously been shown to interact with a tetratricopeptide re-peat-containing class II chaperone termed Scc3 (specific chla-mydial chaperone 3); however, Scc3 binds to a C-terminalregion of CopN (35). Interestingly, the copN gene in Chla-mydia trachomatis is located adjacent to a gene that encodes aclass I T3S chaperone (Scc1) whose product was found tointeract with another predicted class I chaperone (CT663) in ayeast 2-hybrid screen (36). Although Scc1 and CT663 share nosignificant amino acid sequence homology with SycN andYscB, their gene locations (Fig. 1) and interaction are remi-niscent of the Yersinia SycN and YscB chaperones.

In this study, we demonstrate that C. pneumoniae AR-39encodes a T3S chaperone composed of Scc1 (CP0432) andScc4 (CP0033) that binds to an N-terminal region of CopN.Furthermore, we demonstrate a specific effect of the Scc1/Scc4chaperone complex, but not the individual chaperones, onCopN secretion using a heterologous Yersinia T3SS.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The Yersinia pestis KIM strains (Ta-ble 1) used in these studies are avirulent and are excluded from the NationalSelect Agent Registry due to deletion of the 102-kb pgm locus (39). In addition,the Y. pestis KIM strains used in CopN secretion assays carried a deletion of theyopE and sycE loci (3) and were cured of the plasminogen activator (Pla)protease-encoding pPCP1 plasmid (40). Experiments with attenuated Y. pestisstrains were reviewed and approved by the Institutional Biosafety Committee atthe University of Miami. Y. pestis strains were routinely grown in heart infusionbroth (HIB) or on tryptose blood agar (TBA) base plates (BD-Difco) at 27°C.For secretion assays, Y. pestis strains were grown in TMH medium in the pres-ence or absence of 2.5 mM CaCl2 as described previously (23). E. coli DH5� andBL21(DE3) were grown in HIB or Luria-Bertani (LB) medium. When appro-

priate, antibiotics were routinely used at the following concentrations: ampicillin,50 �g/ml; streptomycin, 50 �g/ml; kanamycin, 25 �g/ml; trimethoprim, 25 �g/ml;chloramphenicol, 20 �g/ml.

Construction of the Y. pestis yopN tyeA sycN yscB deletion strain. LambdaRed-mediated recombination was used to delete the coding sequences for yopN,tyeA, and sycN from a �yscB mutant Y. pestis strain (KIM8-3002.P1) (23) using apreviously described modification (24) of the procedure of Datsenko and Wan-ner (10). The oligonucleotide primers used to amplify the kanamycin cassettefrom plasmid pKD4 were YopN-SycN-P1 and YopN-SycN-P2 (see Table S1 inthe supplemental material). The resultant PCR product was gel purified andelectroporated into Y. pestis KIM8-3002.P1 carrying plasmid pKD46, and kana-mycin-resistant �yopN-tyeA-sycN-yscB deletion mutants were selected for byplating on TBA plates containing 25 �g/ml kanamycin and confirmed by PCRanalyses. Plasmid pCP20 (8), which encodes the FLP recombinase, was electro-porated into the �yopN-tyeA-sycN-yscB deletion mutant strain to remove theFLP recognition sequence-flanked kan cassette. Plasmids pCP20 and pKD46,which carry temperature-sensitive origins of replication, were cured by overnightgrowth at 39°C. The presence of the deletion and the absence of pCP20 andpKD46 were confirmed by PCR analyses and by agarose gel electrophoresis ofplasmids isolated by the method of Kado and Liu (27). The resultant Pgm�

pPCP1� (Pla�) �yopN-tyeA-sycN-yscB deletion mutant, designated KIM8-3002.P108, was used for a second round of Lambda Red-mediated recombina-tion to remove the coding sequences for SycE and YopE. A ca.1-kb fragment wasamplified from plasmid pCD1-�4 of Y. pestis KIM8 �4 (3) using oligonucleotideprimers YopE-dhfr-F and YopE-dhfr-R. Plasmid pCD1-�4 carries a deletion ofpCD1 sequences from position 42,958 to position 47,891 (sycE and yopE codingsequences) and an insertion of dhfr. The resultant purified PCR product waselectroporated into KIM8-3002.P108 carrying plasmid pKD46, and tri-methoprim-resistant Pgm� pPCP1� (Pla�) �yopN-tyeA-sycN-yscB �sycE-yopEdeletion mutants were selected for on TBA plates containing 25 �g/ml tri-methoprim. Plasmid pKD46 was cured by overnight growth at 39°C, and thepresence of the �yopN-tyeA-sycN-yscB and �sycE-yopE deletions, as well as theabsence of plasmid pKD46, was confirmed by PCR analyses and by the methodof Kado and Liu (27). The resultant Pgm� pPCP1� (Pla�) �yopN-tyeA-sycN-yscB �sycE-yopE deletion strain, designated KIM8-3002.P109, and the parentKIM8 �4 strain were used for CopN, Scc1 (CP0432), Scc3 (CP0832), and Scc4(CP0033) expression and secretion studies.

Construction of plasmid pMAL-CopN/Scc1/Scc4. Plasmid pMAL-CopN/Scc1/Scc4 was constructed through multiple steps using the PCR-ligation-PCR tech-nique (2). A DNA fragment encoding 406 bp of malE and the TEV proteaserecognition site of plasmid pKm1255 (gift from David Waugh) was amplifiedusing oligonucleotide primers MBP-BlpI-F and TEV-R. A second DNA frag-ment encoding CopN (starting at codon 2) was amplified from C. pneumoniaeAR-39 chromosomal DNA using oligonucleotide primers CopN-F and CopN-BamHI-R. The two PCR products were ligated together and reamplified usingprimers MBP-BlpI-F and CopN-BamHI-R. The resultant PCR product wasdigested with BlpI and BamHI and inserted into BlpI- and BamHI-digestedpMAL-c2X (New England BioLabs). The resulting construct, encoding MBP-CopN, was termed pMAL-CopN.

The gene for Scc1 was amplified from C. pneumoniae AR-39 chromosomalDNA using primers Scc1-RBS-BamHI-F and Scc1-RBS-R. Likewise, the genefor Scc4 (CP0033) was amplified using primers Scc4-RBS-F and Scc4-HindIII-6xHIS-R. The Scc1-RBS-BamHI-F and Scc4-RBS-F primers were designed toinsert sequences encoding a strong ribosome-binding site (RBS) upstream of thescc1 and scc4 ATG start codons. The DNA fragments encoding Scc1 and Scc4were ligated together and reamplified using primers Scc1-RBS-BamHI-F andScc4-HindIII-6xHIS-R. The resulting DNA fragment was digested with BamHI

FIG. 1. Genetic map of C. pneumoniae chromosomal regions and Y. pestis plasmid pCD1 regions encoding CopN- and YopN-specificchaperones. The C-terminal region of CopN is homologous to TyeA of Y. pestis. The locations of the genes encoding Scc1 and Scc4 of C.pneumoniae are similar to those of sycN and yscB in Y. pestis. The C. pneumoniae YscC homolog CdsC is located downstream of the CdsEFG-encoding genes.

TABLE 1. Bacterial strains used in this study

Strain Description Source

E. coliDH5� F� �80dlacZ�M15 �(lacZYA argF)U169

endA1 recA1 hsdR17 deoR supE44 thi-1gyr96 relA1

6

BL21(DE3) F� ompT hsdSB(rB� mB

�) gal dcm (DE3) 37

Y. pestisKIM8-3002.P1 Smr pCD1 (�yscB)/pMT1 23KIM8-3002.P108 Smr pCD1 (�yopN-tyeA-sycN-yscB)/pMT1 This studyKIM8 �4 pCD1 (�sycE-yopE::dhfr)/pMT1 3KIM8-3002.P109 Smr pCD1 (�yopN-tyeA-sycN-yscB

�sycE-yopE::dhfr)/pMT1This study

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and HindIII and inserted into BamHI- and HindIII-digested pMAL-CopN, gen-erating plasmid pMAL-CopN/Scc1/Scc4. The pMAL-CopN/Scc1/Scc4 plasmidencodes a maltose-binding protein (MBP)-CopN hybrid protein with a TEVprotease site located between MBP and CopN. In addition, the plasmid encodesScc1 and Scc4 with a C-terminal 6-histidine tag.

Construction of plasmids pBAD18-Scc1, pBAD18-Scc1/Scc4, pBAD33-Scc3,and pFLAG-Scc4. The scc1 gene was amplified from plasmid pMAL-CopN/Scc1/Scc4 DNA using oligonucleotide primers Scc1-XbaI-F and Scc1-HindIII-R. TheDNA fragment encoding both Scc1 and Scc4 was also amplified from pMAL-CopN/Scc1/Scc4 DNA using oligonucleotide primers Scc1-XbaI-F and Scc4-HindIII-R. The resultant DNA fragments were digested with XbaI and HindIIIand inserted into XbaI- and HindIII-digested pBAD18 (20), generating plasmidspBAD18-Scc1 and pBAD18-Scc1/Scc4. Plasmid pFLAG-CTC was used for con-struction of pFLAG-Scc4. The scc4 gene was amplified from C. pneumoniaeAR-39 chromosomal DNA using primers Scc4-HindIII-F and Scc4-BamHI-R.The resultant DNA fragment was digested with HindIII and KpnI and insertedinto HindIII- and KpnI-digested pFLAG-CTC (Sigma Aldrich), generating plas-mid pFLAG-Scc4. Plasmid pBAD33-Scc3 expresses Scc3 under the control ofthe PBAD promoter of plasmid pBAD33 (20). The DNA fragment encoding Scc3was amplified from C. pneumoniae AR-39 chromosomal DNA with primersScc3-KpnI-F and Scc3-XhoI-R. The resultant PCR product was digested withKpnI and XhoI and inserted into KpnI- and SalI-digested pBAD33, generatingplasmid pBAD33-Scc3.

Construction of plasmids pCopN, pCopN/Scc1, pCopN/Scc4, pCopN/Scc1/Scc4, and pScc3. Plasmids pCopN, pCopN/Scc1, and pCopN/Scc1/Scc4 carry thecopN gene alone, with the scc1 gene, or with both the scc1 and scc4 genes underthe control of the PBAD promoter of plasmid pBAD24 (20). DNA fragmentsencoding CopN, CopN, and Scc1 or CopN, Scc1, and Scc4 were amplified frompMAL-CopN/Scc1/Scc4 using primer CopN-MfeI-F paired with primer CopN-HindIII-R (copN), Scc1-KpnI-R (copN and scc1), or Scc4-HindIII-R2 (copN,scc1, and scc4). The resultant DNA fragments were digested with MfeI andHindIII (or KpnI) and inserted into EcoRI- and HindIII (or KpnI)-digestedpBAD24, generating plasmids pCopN, pCopN/Scc1, and pCopN/Scc1/Scc4. Plas-mid pCopN/Scc4 was generated by the PCR-ligation-PCR technique using prim-ers CopN-MfeI-F and CopN-R to amplify copN and primers Scc4-RBS-F andScc4-HindIII-R2 to amplify scc4. The resultant PCR products were ligated to-gether and reamplified with primers CopN-MfeI-F and Scc4-HindIII-R2. Theresultant DNA fragment was digested with MfeI and HindIII and inserted intoEcoRI- and HindIII-digested pBAD24. The scc3 gene was amplified from the C.pneumoniae AR-39 genomic DNA using primers Scc3-F and Scc3-R. The frag-ment was cloned into a Gateway compatible pBAD33-based vector using stan-dard Gateway cloning techniques (Invitrogen Life Technologies) to yield pScc3.

Construction of plasmids pGST-CopN, pGST-CopN1-320, pGST-CopN1-220,pGST-CopN1-100, pGST-CopN101-399, pGST-CopN201-399, and pGST-CopN301-399.Plasmid pET42b (Novagen) was used for the construction of glutathioneS-transferase (GST)-CopN expression vectors. DNA fragments encodingCopN1-399, CopN1-320, CopN1-220, or CopN1-100 were amplified from C. pneu-moniae AR-39 chromosomal DNA using primer CopN-PshA-F paired withprimer CopN-399-HindIII-R (CopN1-399), CopN-320-HindIII-R (CopN1-320),CopN-220-HindIII-R (CopN1-220), or CopN-100-HindIII-R (CopN1-100). Theresultant DNA fragments were digested with PshAI and HindIII and insertedinto PshAI- and HindIII-digested pET42b, generating plasmids pGST-CopN,pGST-CopN1-320, pGST-CopN1-220, and pGST-CopN1-100. DNA fragmentsencoding CopN101-399, CopN201-399, or CopN101-399 were amplified from plas-mid pMAL-CopN/Scc1/Scc4 using primer CopN-399-HindIII-R paired withprimer CopN-101-EcoRV-F (CopN101-399), CopN-201-EcoRV-F (CopN201-399),or CopN-301-EcoRV-F (CopN301-399). The resultant DNA fragments were digestedwith EcoRV and HindIII and inserted into PshAI- and HindIII-digested pET42b, gen-erating plasmids pGST-CopN101-399, pGST-CopN201-399, and pGST-CopN301-399.

Construction of pGAD-CopN, pGAD-Scc1, pGAD-Scc3, pGAD-Scc4, pGBT-CopN, pGBT-Scc1, pGBT-Scc3, and pGBT-Scc4. DNA fragments encodingCopN, Scc1, Scc3, and Scc4 were amplified from C. pneumoniae AR-39 chro-mosomal DNA using primers CopN-MfeI-F, CopN-NsiI-R, Scc1-MfeI-F, Scc1-PstI-R, Scc3-EcoRI-F, Scc3-NsiI-R, Scc4-EcoRI-F, and Scc4-PstI-R. The resul-tant DNA fragments were digested with MfeI or EcoRI and PstI or NsiI andinserted into EcoRI- and PstI-digested pGAD424 and pGBT9, generating plas-mids pGAD-CopN, pGAD-Scc1, pGAD-Scc3, pGAD-Scc4, pGBT-CopN,pGBT-Scc1, pGBT-Scc3, and pGBT-Scc4.

Yeast two-hybrid assays. Yeast two-hybrid assays were performed using meth-ods recommended by the commercial supplier (Matchmaker Yeast Two-HybridSystem; Clontech). Plasmids were transformed into Saccharomyces cerevisiaeSFY596 according to the manufacturer’s instructions and plated on appropriatesynthetic defined medium (SD) plates. Colony lift assays for detection of �-ga-

lactosidase activity were performed essentially as described elsewhere (Clon-tech).

Secretion assays. Y. pestis KIM8 �4 or KIM8-P109 carrying plasmids pCopN,pCopN/Scc1, pCopN/Scc4, pCopN/Scc1/Scc4, and pCopN with Scc3 or pCopN/Scc1/Scc4 with pScc3 was grown overnight at 27°C in TMH medium with theappropriate antibiotics. The next day, overnight cultures were used to inoculatefresh TMH cultures, with or without 2.5 mM CaCl2, to an optical density at 620nm (OD620) of 0.2. Cultures were grown for 1 h at 27°C and then for 5 h at 37°C.Bacterial cell pellets and culture supernatants were separated by centrifugationat 12,200 � g for 10 min at room temperature (RT). Culture supernatantproteins were precipitated on ice overnight with 10% (vol/vol) trichloroaceticacid and collected by centrifugation at 21,920 � g for 15 min at 4°C. Volumes ofcellular fractions corresponding to equal numbers of bacteria were mixed 1:1with 2� electrophoresis sample buffer and analyzed by SDS-PAGE and immu-noblotting with antibodies specific for C. pneumoniae CopN, Scc1, Scc3, or Scc4and Y. pestis YopM or H-NS.

GST pulldown assays. E. coli BL21(DE3) was transformed with protein ex-pression vector pET42b, pGST-CopN, pGST-CopN1-320, pGST-CopN1-220,pGST-CopN101-399, pGST-CopN201-399, pGST-CopN301-399, pBAD18-Scc1,pBAD18-Scc1/Scc4, pBAD33-Scc3, or pFLAG-Scc4. Overnight cultures wereinoculated into 35 ml HIB to an OD620 of 0.2 and incubated with shaking at 37°Cto an OD620 of 0.4 to 0.6. The production of recombinant proteins was initiatedwith the addition of 0.1 mM isopropyl-�-D-thiogalactopyranoside (IPTG) or0.2% L-arabinose for 2 h at 37°C. Cultures were harvested by centrifugation at6,000 � g for 20 min at 4°C, and the resulting pellets were washed with 200 mlof cold phosphate-buffered saline (140 mM NaCl, 16 mM Na2HPO4, 4 mMNaH2PO4). Washed pellets were resuspended in 10 ml ice-cold GST bindingbuffer (5 mM Na2PO4, 1.5 mM KH2PO4, 140 mM NaCl, 3 mM KCl, pH 7.4) andlysed by passage through a chilled French pressure cell at 20,000 lb/in2. Deter-gent (0.1% [final concentration] NP-40) was added, and the lysates were centri-fuged at 6,000 � g for 20 min at 4°C to remove unlysed cells. Appropriatecombinations of the lysates (2 ml of each lysate) were mixed and incubated for5 min at RT prior to loading onto a 1-ml bed volume column of glutathioneagarose beads (GST-Bind Resin; Novagen). The column was washed with 10 mlof GST binding buffer containing 0.1% NP-40 and eluted with 1 ml GST bindingbuffer containing 20 mM reduced glutathione. Initial lysates and eluted sampleswere analyzed by SDS-PAGE and immunoblotting.

SDS-PAGE and immunoblotting. Volumes of cellular fractions correspondingto equal numbers of bacteria or equal amounts of protein were mixed 1:1(vol/vol) with 2� electrophoresis sample buffer and analyzed by SDS-PAGE andimmunoblotting essentially as previously described (23). CopN, Scc1, Scc3, andScc4 were detected with affinity-purified antipeptide antibodies raised in rabbitsagainst peptides corresponding to residues 385 to 399 of CopN, 31 to 45 of Scc1,158 to 172 of Scc3, and 32 to 46 of Scc4 (ProteinTech Group). Y. pestis proteinswere visualized as previously described, using polyclonal antiserum specific forYopM and H-NS. Band intensities were quantitated using an Alpha Innotech5500 imaging system (Cell Biosciences).

RESULTS

To confirm the previously identified interaction of Scc1(CP0432) with Scc4 (CP0033; CT663 in C. trachomatis) (36), aswell as that of CopN (CP0433) with Scc3 (CP0832) (35), thecopN, scc1, scc4, and scc3 genes from C. pneumoniae AR-39were amplified (see Table S1 in the supplemental material)and inserted into the pGAD424 and pGBT9 yeast two-hybridvectors. Yeast two-hybrid studies confirmed that CopN(CP0433) interacts with Scc3 (CP0832) and that Scc1 (CP0432)interacts with Scc4 (CP0033) as previously reported (35, 36);however, no interaction of Scc1 or Scc4 with CopN was de-tected (data not shown). These findings indicate that C. pneu-moniae Scc1 and Scc4 interact directly and could function as aheterodimeric CopN-specific chaperone similar to the Yersiniasp. YopN-specific SycN/YscB chaperone.

The C. pneumoniae Scc1/Scc4 chaperone and Scc3 chaper-one bind to CopN. To determine if the Scc1/Scc4 complexinteracts with CopN, GST pulldown studies were conductedusing GST and GST-CopN. Whole-cell lysates from E. coli

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BL21(DE3) expressing GST or GST-CopN were mixed withlysates expressing Scc1, FLAG-tagged Scc4, Scc3, Scc1, andScc4 or Scc1, Scc4, and Scc3. Scc4 without a FLAG tag wasunstable and poorly expressed in the absence of Scc1. Lysateswere combined, incubated for 5 min at RT to allow complexformation, and applied to glutathione-Sepharose columns. Thecolumns were washed and eluted with 20 mM reduced gluta-thione, and initial lysates along with elutions were analyzed bySDS-PAGE and immunoblotting (Fig. 2). No interaction ofthe individual Scc1 or Scc4 protein with GST-CopN was de-tected; however, Scc3 efficiently bound to and coeluted withGST-CopN as previously reported (35). Interestingly, Scc1 andScc4 coeluted with GST-CopN when lysates containing bothScc1 and Scc4 or all three chaperone-like proteins (Scc1, Scc4,and Scc3) were used in pulldown experiments. No interactionof Scc1, Scc4, or Scc3 with GST alone was detected (Fig. 2A).These results demonstrate that a complex composed of Scc1and Scc4, but not the individual proteins, directly interacts withCopN, suggesting that Scc1 and Scc4 form a heterodimericchaperone similar to the Yersinia sp. SycN/YscB chaperone.

Localization of the Scc1/Scc4 and Scc3 binding sites onCopN. The majority of class 1 T3S chaperones interact with anN-terminal region of their cognate substrate termed the chap-erone-binding domain (CBD) (15). The CBD of YopN consistsof YopN residues 32 to 77 (34). To begin to locate the bindingsite on CopN for the Scc1/Scc4 complex, deletions in pGST-CopN were constructed that removed the coding sequence forresidues 321 to 399 (pGST-CopN1-320), residues 221 to 399(pGST-CopN1-220), residues 101 to 399 (pGST-CopN1-100),residues 1 to 100 (pGST-CopN101-399), residues 1 to 200(pGST-CopN201-399), and residues 1 to 300 (pGST-CopN301-399).The resulting constructs were moved into E. coli BL21(DE3)for pulldown experiments. The pGST-CopN1-100 constructfailed to express a stable CopN product and was not used insubsequent studies. BL21(DE3) lysates containing GST, GST-CopN, GST-CopN1-320, GST-CopN1-220, GST-CopN101-399,GST-CopN201-399, or GST-CopN301-399 were combined withlysates containing Scc1, Scc4, and Scc3 and analyzed as de-scribed for Fig. 2. As expected, Scc1, Scc4, and Scc3 all coe-luted with full-length GST-CopN but not with GST alone (Fig.

3). Removal of the CopN C-terminal 79 (GST-CopN1-320) or179 (GST-CopN1-220) residues abolished the binding of Scc3 toCopN but had no effect on the binding of Scc1 and Scc4. Incontrast, deletion of the CopN N-terminal 100, 200, or 300residues eliminated the binding of Scc1 and Scc4 to CopN buthad little or no effect on the interaction of Scc3 with CopN.These results indicate that the Scc1/Scc4 complex binds to anN-terminal region of CopN, whereas Scc3 binds to a C-termi-nal region of CopN as previously reported (35).

The Scc1/Scc4 chaperone promotes efficient CopN secretionvia the Yersinia sp. T3SS. T3S chaperones have been shown tohave a variety of activities that promote the secretion of theircognate substrate or substrates. These include preventing the

FIG. 2. The Scc1/Scc4 chaperone and the Scc3 chaperone directly interact with CopN. E. coli BL21(DE3) lysates expressing GST (A) orGST-CopN (B) were combined with lysates expressing Scc1, FLAG-Scc4, Scc1/Scc4, Scc3, or Scc1/Scc4/Scc3. GST, GST-CopN, and interactingproteins were purified using glutathione agarose beads. Initial lysates and elutions were analyzed by SDS-PAGE and immunoblotting with antiseraspecific for GST, Scc1, Scc3, and Scc4.

FIG. 3. Localization of the binding sites for the Scc1/Scc4 chaper-one and the Scc3 chaperone on CopN. E. coli BL21(DE3) lysatesexpressing GST, GST-CopN, GST-CopN1-320, GST-CopN1-220, GST-CopN101-399, GST-CopN201-399, or GST-CopN301-399 were combinedwith lysates expressing Scc1, Scc3, and Scc4. GST-CopN hybrid pro-teins and interacting proteins were purified using glutathione agarosebeads. Elutions were analyzed by SDS-PAGE and immunoblottingwith antisera specific for GST, Scc1, Scc3, and Scc4.



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degradation and/or aggregation of their substrates, maintain-ing substrates in a partially unfolded secretion-competentstate, and/or directly targeting substrates to the T3S apparatus(15). Previous studies have demonstrated that CopN can besecreted via the Yersinia sp. plasmid-encoded T3SS (17); there-fore, the effects of Scc1, Scc4, and Scc3 on CopN expressionand secretion was determined by expressing various combina-tions of these proteins in avirulent Y. pestis KIM8 �4, whichexpresses a functional calcium-regulated T3SS.

As demonstrated previously (17), CopN expressed in theabsence of all other chlamydial proteins is secreted by theYersinia T3SS at low levels in the absence of calcium (21% oftotal CopN secreted) but not in the presence of calcium, con-firming that CopN is a T3S substrate (Fig. 4A). Coexpressionof the individual Scc1 or Scc4 protein with CopN had no effecton CopN secretion; in contrast, coexpression of both Scc1 andScc4 dramatically increased CopN secretion (91% of totalCopN secreted), indicating that Scc1 and Scc4 function to-gether as a T3S chaperone that specifically promotes efficientsecretion of CopN. Expression of Scc3 in the presence of Scc1and Scc4 resulted in a slight decrease in the expression of Scc1and Scc4 and in a modest reduction in CopN secretion (63% oftotal CopN secreted), suggesting that Scc3 may have a direct orindirect inhibitory effect on CopN export.

To determine if the inhibitory effect of Scc3 on CopN se-cretion was dependent upon the Scc1/Scc4 chaperone, secre-tion of CopN was measured in both the presence and theabsence of the Scc1/Scc4 and Scc3 chaperones (Fig. 5). Asexpected, only low levels of CopN were secreted in the absenceof Scc1 and Scc4; however, even less CopN was secreted in the

presence of the Scc3 chaperone, suggesting that Scc3 functionsto limit CopN secretion independently of the Scc1/Scc4 chap-erone.

Expression of CopN with or without its cognate chaperoneshas no effect on the calcium-dependent regulation of the Yer-sinia sp. T3SS. The YopN/SycN/YscB/TyeA complex of Yer-sinia spp. is required to prevent Yop effector protein secretion

FIG. 4. Expression and secretion of CopN in the presence or absence of Scc1, Scc4, and Scc3. Y. pestis KIM8 �4 (parent) (A) and KIM8-P109�yopN-sycN-yscB-tyeA) (B) alone or carrying plasmids pCopN, pCopN/Scc1, CopN/Scc4, and pCopN/Scc1/Scc4 or plasmids pCopN/Scc1/Scc4 andpScc3 were cultured in TMH medium with (�) and without (�) 2.5 mM calcium for 5 h at 37°C. Secreted (S) and/or cell pellet (P) proteins wereanalyzed by SDS-PAGE and immunoblotting with antisera specific for CopN, Scc1, Scc4, Scc3, YopM, or H-NS.

FIG. 5. Expression and secretion of CopN in the presence or ab-sence of Scc3. Y. pestis KIM8 �4 carrying plasmid pCopN or pCopN/Scc1/Scc4 with and without plasmid pScc3 was cultured in TMH me-dium with (�) and without (�) 2.5 mM calcium for 5 h at 37°C.Secreted (S) and/or cell pellet (P) proteins were analyzed by SDS-PAGE and immunoblotting with antisera specific for CopN, Scc1,Scc4, and Scc3.



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in the presence of calcium; therefore, it is possible that expres-sion of CopN, Scc1, Scc4, and/or Scc3 could augment or inter-fere with the regulatory function of these proteins. The expres-sion and secretion of the Yersinia YopM protein wereexamined to assess the effects of the chlamydial proteins on thefunction of the Yersinia T3SS. The only significant effect ob-served was a modest decrease in YopM expression and secre-tion in strains expressing CopN and Scc1 (Fig. 4A). The cyto-plasmic H-NS protein was used as a lysis control protein. NoH-NS was observed in the culture supernatant fractions, con-firming that the CopN present in the culture supernatants wasdue to the T3S process and not cell lysis.

Although it is inefficient, recent studies have demonstratedthat calcium chelation stimulates the release of chlamydial T3Ssubstrates from purified elementary bodies (25), indicatingthat the activity of the chlamydial T3SS, like that of the Yersiniasp. T3SS, may be regulated, in part, by extracellular calcium.To determine if the CopN/Scc1/Scc4 or CopN/Scc1/Scc4/Scc3complex can substitute for the YopN/SycN/YscB/TyeA com-plex and restore calcium-regulated Yop secretion, the variousCopN-expressing constructs were moved into an avirulent Y.pestis �yopN-sycN-yscB-tyeA deletion strain. The �yopN-sycN-yscB-tyeA deletion strain expressing CopN, the CopN/Scc1/Scc4 complex, or the CopN/Scc1/Scc4/Scc3 complex secretedYops in the presence or absence of calcium, indicating that thechlamydial CopN/Scc1/Scc4 and CopN/Scc1/Scc4/Scc3 com-plexes are unable to complement the regulatory function of theYopN/SycN/YscB/TyeA complex.(Fig. 4B). Importantly, theeffects of the Scc1/Scc4 complex and Scc3 on CopN secretionwere maintained in the �yopN-sycN-yscB-tyeA background.Overall, these studies demonstrate that Scc1 and Scc4 form achaperone complex that binds to an N-terminal region ofCopN and facilitates CopN secretion. In contrast, Scc3 binds toa C-terminal region of CopN and appears to have a smallinhibitory affect on CopN secretion.

DISCUSSION

Members of the YopN/TyeA/InvE/MxiC family of proteinsare found in essentially all nonflagellar T3SSs and function toregulate the secretion of translocator and effector proteins (13,31). The majority of the members of this family of proteins areexpressed as single proteins, with domains homologous toYopN and TyeA, and interact with no identified T3S chaper-ones. In contrast, the Yersinia YopN and TyeA proteins func-tion as part of a larger cytosolic YopN/TyeA/SycN/YscB com-plex (34). The SycN and YscB proteins form a heterodimericT3S chaperone that binds to an N-terminal CBD on YopN andfunctions to prevent YopN degradation and promote the effi-cient secretion of YopN (12). CopN of Chlamydia spp. is asecreted protein with domains homologous to YopN and TyeAthat is encoded directly upstream of a gene encoding a pre-dicted T3S chaperone (Scc1) (17), suggesting that CopN mayrepresent an example of a single protein YopN/TyeA/InvE/MxiC family member that utilizes a T3S chaperone. Interest-ingly, previous yeast two-hybrid studies have indicated that theScc1 chaperone does not interact with CopN but does interactwith another potential T3S chaperone (CT663) (36). Notably,the locations of the genes encoding the two interacting chap-erones closely matched the locations of the genes encoding

SycN and YscB in the yersiniae (Fig. 1). In this study, weconfirm that the C. pneumoniae Scc1 and Scc4 (CT663 ho-molog) proteins function together as a T3S chaperone forCopN, suggesting that CopN, like YopN, utilizes a chaperonecomplex for its efficient secretion.

GST and GST-CopN pulldown experiments indicated thatthe Scc1/Scc4 complex interacts with an N-terminal region ofCopN. Furthermore, expression of CopN with both Scc1 andScc4 but not the individual proteins facilitated efficient secre-tion of CopN via the Yersinia sp. T3SS. These studies representthe first direct demonstration of a T3S chaperone function fora chlamydial class I chaperone and establish a function forboth Scc1 and Scc4. Interestingly, the Scc3 chaperone has beenpreviously shown to bind to a C-terminal region of CopN (35).We confirmed this finding and demonstrated that coexpressionof Scc3 with CopN alone or with CopN, Scc1, and Scc4 had nostimulatory effect on the secretion of CopN but instead ap-peared to reduce CopN secretion. Importantly, the C-terminalregion of CopN recognized by Scc3 corresponds to the regionwith homology to the Yersinia TyeA protein. TyeA has beenshown to specifically reduce the translocation or injection ofYopN by Yersinia spp. (7, 11). The C-terminal region of CopNthat corresponds to TyeA might have a similar function, and itis possible that the binding of Scc3 to this region may enhancethis function. In this regard, the CopN-Scc3 interaction may beprimarily regulatory in nature. This assumption is supported bythe fact that expression of Scc3 had no positive effect on CopNexpression, stability, or secretion. Furthermore, tetratricopep-tide repeat-containing chaperones, like Scc3, have previouslybeen shown to be involved in a variety of regulatory proteininteractions aside from their interaction with their cognatesubstrate (9, 18, 32).

Finally, expression of CopN, Scc1, and Scc4 with and with-out Scc3 in a Yersinia �yopN-sycN-yscB-tyeA deletion strainfailed to restore any calcium-dependent regulation of Yopsecretion. This is not surprising, as these proteins share onlylimited amino acid sequence homology; however, homologs ofCopN, including YopN and TyeA, have a role in controllingthe secretion of translocators and effector proteins in all T3SSswhere their function has been investigated. Therefore, CopNand its interacting Scc1, Scc4, and Scc3 chaperones likely func-tion in a similar manner to coordinate the secretion of chla-mydial translocator and effector proteins.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant AI050552from the National Institutes of Health and a Grant-in-Aid(AHA0051373B) from the American Heart Association.

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scc1 (cp0432) and scc4 (cp0033) function as a type iii secretion

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