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• W2023874344 abstract "Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG5, which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG5 interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG1 interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG5 and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG5 and EspG1 interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway. Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG5, which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG5 interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG1 interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG5 and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG5 and EspG1 interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway. Bacterial pathogens use dedicated protein secretion systems to export virulence factors that interfere with cellular processes of the host to ensure bacterial survival, multiplication, and spread (1Finlay B.B. Falkow S. Common themes in microbial pathogenicity revisited.Microbiol. Mol. Biol. Rev. 1997; 61: 136-169Crossref PubMed Scopus (1171) Google Scholar). Also, pathogenic mycobacteria, such as Mycobacterium tuberculosis, the causal agent of tuberculosis, secrete proteinaceous virulence factors. These bacteria are surrounded by a unique diderm cell envelope that requires a specialized secretion pathway, known as the ESX or type VII secretion pathway, to facilitate protein export (2Abdallah A.M. Gey van Pittius N.C. Champion P.A. Cox J. Luirink J. Vandenbroucke-Grauls C.M. Appelmelk B.J. Bitter W. Type VII secretion: mycobacteria show the way.Nat. Rev. Microbiol. 2007; 5: 883-891Crossref PubMed Scopus (520) Google Scholar). M. tuberculosis contains five ESX clusters, designated ESX-1 to ESX-5. The most well studied locus, ESX-1, is crucial for the virulence of M. tuberculosis and secretes two virulence factors of the WXG100 protein family, i.e. EsxA and EsxB. In addition, ESX-1 secretes a number of proteins referred to as ESX-1 secretion-associated proteins (Esp) 3The abbreviations used are: EspESX-1 secretion-associated protein(s)CBBCoomassie Brilliant BlueeccESX-conserved componentsMS/MStandem MSNi-NTAnickel-nitrilotriacetic acidRBSribosomal binding site. (3Fortune S.M. Jaeger A. Sarracino D.A. Chase M.R. Sassetti C.M. Sherman D.R. Bloom B.R. Rubin E.J. Mutually dependent secretion of proteins required for mycobacterial virulence.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10676-10681Crossref PubMed Scopus (304) Google Scholar, 4McLaughlin B. Chon J.S. MacGurn J.A. Carlsson F. Cheng T.L. Cox J.S. Brown E.J. A Mycobacterium ESX-1-secreted virulence factor with unique requirements for export.PLoS Pathog. 2007; 3: e105Crossref PubMed Scopus (146) Google Scholar, 5Carlsson F. Joshi S.A. Rangell L. Brown E.J. Polar localization of virulence-related Esx-1 secretion in mycobacteria.PLoS Pathog. 2009; 5: e1000285Crossref PubMed Scopus (86) Google Scholar, 6DiGiuseppe Champion P.A. Champion M.M. Manzanillo P. Cox J.S. ESX-1 secreted virulence factors are recognized by multiple cytosolic AAA ATPases in pathogenic mycobacteria.Mol. Microbiol. 2009; 73: 950-962Crossref PubMed Scopus (113) Google Scholar, 7Sani M. Houben E.N. Geurtsen J. Pierson J. de Punder K. van Zon M. Wever B. Piersma S.R. Jiménez C.R. Daffé M. Appelmelk B.J. Bitter W. van der Wel N. Peters P.J. Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.PLoS Pathog. 2010; 6: e1000794Crossref PubMed Scopus (222) Google Scholar, 8Bitter W. Houben E.N. Bottai D. Brodin P. Brown E.J. Cox J.S. Derbyshire K. Fortune S.M. Gao L.Y. Liu J. Gey van Pittius N.C. Pym A.S. Rubin E.J. Sherman D.R. Cole S.T. Brosch R. Systematic genetic nomenclature for type VII secretion systems.PLoS Pathog. 2009; 5: e1000507Crossref PubMed Scopus (208) Google Scholar) and a few PE/PPE proteins (3Fortune S.M. Jaeger A. Sarracino D.A. Chase M.R. Sassetti C.M. Sherman D.R. Bloom B.R. Rubin E.J. Mutually dependent secretion of proteins required for mycobacterial virulence.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10676-10681Crossref PubMed Scopus (304) Google Scholar, 7Sani M. Houben E.N. Geurtsen J. Pierson J. de Punder K. van Zon M. Wever B. Piersma S.R. Jiménez C.R. Daffé M. Appelmelk B.J. Bitter W. van der Wel N. Peters P.J. Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.PLoS Pathog. 2010; 6: e1000794Crossref PubMed Scopus (222) Google Scholar, 9Daleke M.H. Ummels R. Bawono P. Heringa J. Vandenbroucke-Grauls C.M. Luirink J. Bitter W. General secretion signal for the mycobacterial type VII secretion pathway.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 11342-11347Crossref PubMed Scopus (140) Google Scholar). Pe/ppe genes, which are named after conserved Pro-Glu and Pro-Pro-Glu motifs near the N termini of their gene products, are present in high numbers in the genomes of several mycobacterial pathogens and are thought to contribute to mycobacterial virulence (10Sampson S.L. Mycobacterial PE/PPE proteins at the host-pathogen interface.Clin. Dev. Immunol. 2011; 2011: 497203Crossref PubMed Scopus (192) Google Scholar). Based on the presence of specific motifs in their C termini, the PE and PPE proteins are further divided into subfamilies, of which the PE_PGRS subfamily is the largest (11Gey van Pittius N.C. Sampson S.L. Lee H. Kim Y. van Helden P.D. Warren R.M. Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions.BMC Evol. Biol. 2006; 6: 95Crossref PubMed Scopus (317) Google Scholar). Although some PE/PPEs are secreted via ESX-1, ESX-5 is responsible for the secretion of most of these proteins, including many PE_PGRS proteins (12Abdallah A.M. Verboom T. Hannes F. Safi M. Strong M. Eisenberg D. Musters R.J. Vandenbroucke-Grauls C.M. Appelmelk B.J. Luirink J. Bitter W. A specific secretion system mediates PPE41 transport in pathogenic mycobacteria.Mol. Microbiol. 2006; 62: 667-679Crossref PubMed Scopus (186) Google Scholar, 13Abdallah A.M. Verboom T. Weerdenburg E.M. Gey van Pittius N.C. Mahasha P.W. Jiménez C. Parra M. Cadieux N. Brennan M.J. Appelmelk B.J. Bitter W. PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5.Mol. Microbiol. 2009; 73: 329-340Crossref PubMed Scopus (207) Google Scholar, 14Daleke M.H. Cascioferro A. de Punder K. Ummels R. Abdallah A.M. van der Wel N. Peters P.J. Luirink J. Manganelli R. Bitter W. Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway.J. Biol. Chem. 2011; 286: 19024-19034Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 15Bottai D. Di Luca M. Majlessi L. Frigui W. Simeone R. Sayes F. Bitter W. Brennan M.J. Leclerc C. Batoni G. Campa M. Brosch R. Esin S. Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation.Mol. Microbiol. 2012; 83: 1195-1209Crossref PubMed Scopus (143) Google Scholar). The ESX-5 locus is restricted to the genomes of slow growing mycobacteria. Importantly, all ESX substrates identified to date lack classical signal peptides, and the mechanism of substrate targeting is not yet fully understood (6DiGiuseppe Champion P.A. Champion M.M. Manzanillo P. Cox J.S. ESX-1 secreted virulence factors are recognized by multiple cytosolic AAA ATPases in pathogenic mycobacteria.Mol. Microbiol. 2009; 73: 950-962Crossref PubMed Scopus (113) Google Scholar, 16Champion P.A. Stanley S.A. Champion M.M. Brown E.J. Cox J.S. C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis.Science. 2006; 313: 1632-1636Crossref PubMed Scopus (161) Google Scholar). Recently, we demonstrated that the C termini of PE proteins and several other ESX-1 substrates share a conserved YXXXD/E motif that is required for secretion. Intriguingly, this signal does not discriminate among the various ESX pathways (9Daleke M.H. Ummels R. Bawono P. Heringa J. Vandenbroucke-Grauls C.M. Luirink J. Bitter W. General secretion signal for the mycobacterial type VII secretion pathway.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 11342-11347Crossref PubMed Scopus (140) Google Scholar). ESX-1 secretion-associated protein(s) Coomassie Brilliant Blue ESX-conserved components tandem MS nickel-nitrilotriacetic acid ribosomal binding site. The ESX clusters contain a number of conserved genes, termed ESX conserved components (ecc), which are required for secretion (8Bitter W. Houben E.N. Bottai D. Brodin P. Brown E.J. Cox J.S. Derbyshire K. Fortune S.M. Gao L.Y. Liu J. Gey van Pittius N.C. Pym A.S. Rubin E.J. Sherman D.R. Cole S.T. Brosch R. Systematic genetic nomenclature for type VII secretion systems.PLoS Pathog. 2009; 5: e1000507Crossref PubMed Scopus (208) Google Scholar). In addition, there are components that are only present in one or a few ESX systems, one of which is EspG. EspG was originally thought to be specific for ESX-1, -2, and ESX-3, but a homologue with low similarity is also present in the ESX-5 locus. Disruption of this homologue in Mycobacterium marinum (mmar_2676) blocked secretion via ESX-5 (13Abdallah A.M. Verboom T. Weerdenburg E.M. Gey van Pittius N.C. Mahasha P.W. Jiménez C. Parra M. Cadieux N. Brennan M.J. Appelmelk B.J. Bitter W. PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5.Mol. Microbiol. 2009; 73: 329-340Crossref PubMed Scopus (207) Google Scholar) and affected intracellular levels of ESX-5 substrates (17van der Woude A.D. Sarkar D. Bhatt A. Sparrius M. Raadsen S.A. Boon L. Geurtsen J. van der Sar A.M. Luirink J. Houben E.N. Besra G.S. Bitter W. Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum.J. Biol. Chem. 2012; 287: 20417-20429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Here, we have investigated the role of MMAR_2676/EspG5 in ESX-5 secretion, and we show that this protein interacts selectively with PE/PPE proteins. M. marinum WT strain E11 (18Puttinaowarat S. Thompson K.D. Adams A. Fish Vet. J. 2000; 5: 6-21Google Scholar) and the ESX-5 mutants 7C1 (espG5::tn) (13Abdallah A.M. Verboom T. Weerdenburg E.M. Gey van Pittius N.C. Mahasha P.W. Jiménez C. Parra M. Cadieux N. Brennan M.J. Appelmelk B.J. Bitter W. PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5.Mol. Microbiol. 2009; 73: 329-340Crossref PubMed Scopus (207) Google Scholar) and Mx2 (eccA5::tn) (12Abdallah A.M. Verboom T. Hannes F. Safi M. Strong M. Eisenberg D. Musters R.J. Vandenbroucke-Grauls C.M. Appelmelk B.J. Luirink J. Bitter W. A specific secretion system mediates PPE41 transport in pathogenic mycobacteria.Mol. Microbiol. 2006; 62: 667-679Crossref PubMed Scopus (186) Google Scholar) were grown and electroporated as described (14Daleke M.H. Cascioferro A. de Punder K. Ummels R. Abdallah A.M. van der Wel N. Peters P.J. Luirink J. Manganelli R. Bitter W. Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway.J. Biol. Chem. 2011; 286: 19024-19034Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Escherichia coli BL21(DE3), BL21(DE3) pLysS, DH5α, TOP10F′, and XL10-Gold were grown at 37 °C in LB medium with shaking at 200 rpm, or on LB agar. When required, ampicillin was used at a concentration of 100 μg/ml, chloramphenicol at 30 μg/ml, kanamycin at 25 μg/ml, and hygromycin at 50 μg/ml for mycobacteria and 100 μg/ml for E. coli. PCRs were carried out with the Phusion High-Fidelity DNA polymerase (Finnzymes) using primers listed in supplemental Table S1. The mmar_2672-2676 operon was amplified by PCR from M. marinum E11 genomic DNA using 5′ primer AbMmMiddle-H5-F and 3′ primer 1794-His-SpeI-R, which contains a His6-encoding sequence and a SpeI restriction site. This fragment was cloned in the integrative pUC-Int-cat vector (13Abdallah A.M. Verboom T. Weerdenburg E.M. Gey van Pittius N.C. Mahasha P.W. Jiménez C. Parra M. Cadieux N. Brennan M.J. Appelmelk B.J. Bitter W. PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5.Mol. Microbiol. 2009; 73: 329-340Crossref PubMed Scopus (207) Google Scholar), resulting in pUC-Int-cat::Mm2672–76His, which was used to express C-terminally His-tagged EspG5 under the ag85 promoter in M. marinum. Mmar_1460 (ppe33) was amplified from E11 DNA using primers PPE1460-XbaI-F and PPE1460-HindIII-HA-R, which contained a 3′ HA epitope. After digestion with XbaI, this fragment was ligated into the E. coli-mycobacterial shuttle vector pSMT3::LipYtub (14Daleke M.H. Cascioferro A. de Punder K. Ummels R. Abdallah A.M. van der Wel N. Peters P.J. Luirink J. Manganelli R. Bitter W. Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway.J. Biol. Chem. 2011; 286: 19024-19034Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), which had been digested with NheI and EcoRV to remove the lipYtub gene, resulting in vector pSMT3::Mm1460-HA. For expression in E. coli, espG5 and espG1 were cloned both with and without C-terminal His tag under control of the tet promoter in the pASK-IBA3c vector (IBA Gmbh). The genes were amplified using a 5′ primer containing an E. coli ribosomal binding site (RBS) and XbaI digestion site (1794-RBS-XbaI-F or EspG1-RBS-XbaI-F) and a 3′ primer with HindIII digestion site (1794-R, 1794His-HindIII-R, or EspG1His-HindIII-R). After digestion, the fragments were ligated into XbaI and HindIII-digested pASK-IBA3c, resulting in pIBA::EspG5, pIBA::EspG5-His, pIBA::EspG1-His. For expression of PE25 and C-terminally His6-tagged PPE41 in E. coli a previously described construct was used in which the rv2431c and rv2430c genes were each placed behind a RBS, i.e. pET29b(+)::Rv2430-31c-His (19Strong M. Sawaya M.R. Wang S. Phillips M. Cascio D. Eisenberg D. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8060-8065Crossref PubMed Scopus (575) Google Scholar). To create an unlabeled version of PPE41, the rv2430c gene was amplified with primers pET41_F and pET41noHis_R containing NcoI and HindIII sites from pSMT3::PE25-HA-PPE41 (9Daleke M.H. Ummels R. Bawono P. Heringa J. Vandenbroucke-Grauls C.M. Luirink J. Bitter W. General secretion signal for the mycobacterial type VII secretion pathway.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 11342-11347Crossref PubMed Scopus (140) Google Scholar). This fragment was subsequently used to replace the rv2430c-His sequence in pET29b(+)::Rv2430–31c-His, thereby generating pET29b(+)::Rv2430–31c. Similarly, a fragment coding for C-terminally His-tagged PPE41 without its last 20 amino acids was amplified from pSMT3::PE25-HA-PPE41 with primers pET41_F and pET41d20C_R. Subsequently, replacement of the rv2430c-His fragment in pET29b(+)::Rv2430–31c-His as described above resulted in pET29b(+)::Rv2430–31cΔ20C-His. Plasmids for E. coli expression of PE35 and PPE68_1, with and without a C-terminal His tag, were constructed by first amplifying mmar_0185 from pSMT3::MmPE35-HA-MmPPE68_1 (9Daleke M.H. Ummels R. Bawono P. Heringa J. Vandenbroucke-Grauls C.M. Luirink J. Bitter W. General secretion signal for the mycobacterial type VII secretion pathway.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 11342-11347Crossref PubMed Scopus (140) Google Scholar) with primers pET35_F and pET35_R containing an NdeI and a KpnI site, respectively. This fragment was ligated into pET29b(+)::Rv2430–31c-His after NdeI/KpnI digestion, thereby replacing the rv2431c gene. Subsequently, the rv2430c-His fragment of this vector was substituted for either WT or His-labeled PPE68_1-encoding fragments, amplified with the 5′ NcoI site-containing primer pET68_F and either pET68noHis_R or pET68_R, both with HindIII sites, from pSMT3::PE35-HA-PPE68_1. This resulted in plasmids pET29b(+)::PE35-PPE68_1 and pET29b(+)::PE35-PPE68_1-His. To generate the construct for expression of PE25 lacking the last 15 amino acids, together with C-terminally His-tagged PPE41, the pET29b(+)::Rv2430–31c-His vector was used as template in a nested PCR approach. A fragment containing the first RBS and the first 255 bp of rv2431c was amplified using primers pET-31cF, which contained a PstI restriction site, and pET-d15cR. This fragment was fused to a PCR amplicon containing the second E. coli-optimized RBS, the entire rv2430c, and the sequence coding for a His6 epitope, using primers pET-d15cF and pET-30cHisR, which had an EcoRV site. The resulting fragment was digested with PstI and EcoRV and ligated into the E. coli-mycobacterial shuttle vector pSMT3 (20Hayward C.M. O'Gaora P. Young D.B. Griffin G.E. Thole J. Hirst T.R. Castello-Branco L.R. Lewis D.J. Construction and murine immunogenicity of recombinant Bacille Calmette Guérin vaccines expressing the B subunit of Escherichia coli heat-labile enterotoxin.Vaccine. 1999; 17: 1272-1281Crossref PubMed Scopus (39) Google Scholar), cut with the same enzymes, resulting in pSMT3::PE25Δ15C-PPE41-His. To express EsxM and EsxN in E. coli, the encoding genomic region was amplified by PCR and introduced into cloning vector pJet1 (Fermentas). After digestion by XbaI and XhoI, the resulting fragment was introduced in pET15b(+), which was cut with the same enzymes, resulting in pET15b(+)::esxMN. Preparation of whole cell lysates, extraction of surface proteins by treatment with Genapol X-080, and precipitation of proteins secreted into the medium of M. marinum were carried out as described (14Daleke M.H. Cascioferro A. de Punder K. Ummels R. Abdallah A.M. van der Wel N. Peters P.J. Luirink J. Manganelli R. Bitter W. Conserved Pro-Glu (PE) and Pro-Pro-Glu (PPE) protein domains target LipY lipases of pathogenic mycobacteria to the cell surface via the ESX-5 pathway.J. Biol. Chem. 2011; 286: 19024-19034Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Mycobacterial cultures grown to an A600 of ∼1.0 were incubated for 1 h with 100 μg/ml ciprofloxacin (Sigma), washed twice with PBS, and subsequently resuspended in PBS supplemented with Complete protease inhibitor mixture (Roche Applied Science), 1 mm EDTA, and optionally 10 mm imidazole. Cells were broken by two-times passage through a One Shot cell disrupter (Constant Systems) at 0.83 kbar, adding 1 mm DTT after the first press. Unbroken cells were spun down by repeated centrifugation at 3000 × g, and subsequently the cell envelope and soluble fractions were separated by ultracentrifugation at 100,000 × g. Overnight cultures of E. coli TOP10F′ harboring pASK-IBA3 plasmids were diluted to an OD660 of 0.05 and incubated until an OD660 of 0.3, at which expression of recombinant proteins was induced by the addition of 200 μg/ml anhydrotetracycline for 2 h. Proteins were expressed from pET29b(+) vectors in E. coli BL21(DE3)pLysS essentially as described in Ref. 19Strong M. Sawaya M.R. Wang S. Phillips M. Cascio D. Eisenberg D. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8060-8065Crossref PubMed Scopus (575) Google Scholar. Briefly, overnight cultures were diluted to an OD660 of 0.05 in medium containing 0.2% dextrose to repress premature expression of recombinant proteins. At an OD660 of 0.6, expression of the recombinant proteins was induced for 3 h in the presence of 0.4 mm isopropyl β-d-thiogalactopyranoside. EsxM/EsxN were expressed from pET15b(+) using the same strain and conditions, but for induction 0.1 mm isopropyl β-d-thiogalactopyranoside was used. PE25Δ15C and C-terminally His-tagged PPE41 were expressed constitutively in E. coli XL10-Gold from the Plac promoter in the pSMT3::PE25Δ15C-PPE41-His vector. Cells were harvested by centrifugation, resuspended to a concentration of 50 OD units/ml in a buffer containing 50 mm NaH2PO4 and 300 mm NaCl (pH 8.0), and lysed in a One Shot cell disrupter at 1.72 kbar. Unbroken cells were removed by low speed centrifugation, and soluble proteins were obtained by ultracentrifugation. Subsequently, the E. coli lysates were diluted in 50 mm NaH2PO4 and 300 mm NaCl (pH 8.0) in such a way that the concentration of soluble recombinant protein was similar in all samples. Ni2+ purifications of His-labeled proteins expressed in M. marinum were performed by loading filtered soluble lysates on washed HisTrap HP columns (GE Healthcare) at 0.5 ml/min. Optionally, the filtered M. marinum lysates were precleared on HisTrap-Sepharose columns that had been stripped from Ni2+, prior to loading on a Ni2+-loaded column. After extensive washing of the column with phosphate buffer (20 mm phosphate, 150 mm NaCl, pH 8.0) containing 50 mm imidazole, bound proteins were eluted with 5 ml phosphate buffer containing 150 mm imidazole, and collected in 500-μl fractions. Wash and elution fractions were precipitated with 10% TCA. Alternatively, lysates of M. marinum-expressing proteins of interest were incubated with Ni-NTA-agarose beads (Qiagen) for 1 h at room temperature with head-over-head rotation. After washing the beads five times with phosphate buffer containing 20 mm imidazole, bound proteins were eluted by incubation with 100, 200, and 500 mm imidazole, respectively. Immunoprecipitation of HA-tagged proteins was performed using the HA Tag IP/Co-IP kit (Pierce). For the in vitro pulldowns, soluble lysates of E. coli containing similar amounts of recombinant proteins were mixed with Ni-NTA-agarose beads (Qiagen) for 1 h at room temperature. Washing was performed as described above using a buffer containing 50 mm NaH2PO4, 300 mm NaCl, and 20 mm imidazole (pH 8.0), and proteins were eluted using this buffer supplemented with 100 or 250 mm imidazole. espG5 was cloned with an N-terminal His tag under control of the T7 promoter in the pETM-11 vector (EMBL). The gene was amplified using a 5′ primer containing a NcoI digestion site (AP-303) and a 3′ primer with XhoI digestion site (AP-304). After digestion, the fragments were ligated into NcoI and XhoI-digested pETM-11, resulting in pAP200. For expression of PE25 and C-terminally His-tagged PPE41 in E. coli a previously described construct was used in which the rv2431c and rv2430c genes were each placed behind a RBS (19Strong M. Sawaya M.R. Wang S. Phillips M. Cascio D. Eisenberg D. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8060-8065Crossref PubMed Scopus (575) Google Scholar). All proteins were produced in E. coli BL21(DE3) after induction with 0.2 mm isopropyl β-d-thiogalactopyranoside and overnight incubation at 20 °C. Cells were lysed by sonication in 25 mm HEPES (pH 7.5), 150 mm NaCl supplemented with protease inhibitor mix (Serva). Lysates were cleared by centrifugation and subjected to standard immobilized metal ion affinity chromatography (IMAC) using HisTrap HP columns. After washing with 20 mm imidazole, recombinant proteins were eluted with a linear gradient of 20–250 mm imidazole in 25 mm HEPES (pH 7.5), 150 mm NaCl. Relevant protein fractions were pooled and concentrated and subjected to protease treatment for tag removal. His tags were cleaved from EspG5 by overnight incubation with tobacco etch virus protease at 4 °C. Protease and uncleaved protein were removed by a second IMAC step. In case of PE25/PPE41-His, biotinylated thrombin (Novagen) was used which was subsequently removed by streptavidin beads (Invitrogen) according to the manufacturer's recommendations. All proteins were further purified to homogeneity by preparative size exclusion chromatography (HiLoad 16/600 Superdex 200 pg; GE Healthcare) in 20 mm HEPES (pH 7.5), 150 mm NaCl. EspG5 as well as PE25/PPE41 were concentrated using Vivaspin centrifugal concentrators (Corning) to a final concentration of 8.6 and 5.3 mg/ml, respectively. Protein concentrations were estimated by measurements of absorbance at 280 nm using theoretical molar absorption coefficients calculated by ProtParam. Proteins were analyzed with a Viscotek 305 tridetector (Malvern Instruments, Malvern, UK) which monitors light scattering, refractive index, and UV absorbance. This setup was connected to an analytical size exclusion column (Superdex 200 10/300 GL; GE Healthcare) equilibrated at 20 °C in 20 mm HEPES (pH 7.5), 150 mm NaCl as running buffer. Flow rate was set to 0.5 ml/min, and the sample volume was 100 μl. Proteins were diluted to 1 mg/ml in running buffer prior to analysis. To detect heterotrimeric complex formation, EspG5 was mixed with PE25/PPE41 at a 3-fold molar excess and incubated 30 min at 20 °C before injection. The provided OmniSEC software was used to acquire and evaluate all data. Molecular masses were estimated using refractive index combined with light-scattering data using BSA as an internal control using a refractive index increment with protein concentration (dn/dc) of 0.185 ml/g. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue G-250 (CBB; Bio-Rad), or transferred to nitrocellulose membranes by Western blotting. The membranes were incubated with mouse monoclonal antibodies directed against the conserved repeat of PE_PGRS proteins (mAb 7C4.1F7) (13Abdallah A.M. Verboom T. Weerdenburg E.M. Gey van Pittius N.C. Mahasha P.W. Jiménez C. Parra M. Cadieux N. Brennan M.J. Appelmelk B.J. Bitter W. PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5.Mol. Microbiol. 2009; 73: 329-340Crossref PubMed Scopus (207) Google Scholar), the influenza hemagglutinin epitope (HA.11; Covance), the C-terminal His6 epitope (11922416001; Roche Applied Science), EsxA (Hyb76-8; Statens Serum Institut, Copenhagen, Denmark), or GroEL2 (CS44; John Belisle, National Institutes of Health, Bethesda, MD, Contract AI-75320); or with rabbit polyclonal serum reactive against PPE41 (12Abdallah A.M. Verboom T. Hannes F. Safi M. Strong M. Eisenberg D. Musters R.J. Vandenbroucke-Grauls C.M. Appelmelk B.J. Luirink J. Bitter W. A specific secretion system mediates PPE41 transport in pathogenic mycobacteria.Mol. Microbiol. 2006; 62: 667-679Crossref PubMed Scopus (186) Google Scholar), PPE68 (21Pym A.S. Brodin P. Brosch R. Huerre M. Cole S.T. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti.Mol. Microbiol. 2002; 46: 709-717Crossref PubMed Scopus (551) Google Scholar), EspG5, 4E. N. G. Houben and W. Bitter, manuscript in preparation. FtsH/Mmar_0752 (22van Bloois E. Dekker H.L. Fröderberg L. Houben E.N. Urbanus M.L. de Koster C.G. de Gier J.W. Luirink J. Detection of cross-links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins.FEBS Lett. 2008; 582: 1419-1424Crossref PubMed Scopus (57) Google Scholar), or EsxN (Mtb9.9A) (23Alderson M.R. Bement T. Day C.H. Zhu L. Molesh D. Skeiky Y.A. Coler R. Lewinsohn D.M. Reed S.G. Dillon D.C. Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4+ T cells.J. Exp. Med. 2000; 191: 551-560Crossref PubMed Scopus (105) Goo" @default.
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