Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity
2017; Elsevier BV; Volume: 16; Issue: 12 Linguagem: Inglês
10.1074/mcp.ra117.000296
ISSN1535-9484
AutoresXianghuo He, He‐wei Jiang, Hong Chen, Hainan Zhang, Yin Liu, Zhaowei Xu, Fanlin Wu, Shujuan Guo, Jingli Hou, Mingkun Yang, Wei Yan, Jiaoyu Deng, Lijun Bi, Xian‐En Zhang, Sheng‐ce Tao,
Tópico(s)vaccines and immunoinformatics approaches
ResumoMycobacterium tuberculosis (Mtb) has evolved multiple strategies to counter the human immune system. The effectors of Mtb play important roles in the interactions with the host. However, because of the lack of highly efficient strategies, there are only a handful of known Mtb effectors, thus hampering our understanding of Mtb pathogenesis. In this study, we probed Mtb proteome microarray with biotinylated whole-cell lysates of human macrophages, identifying 26 Mtb membrane proteins and secreted proteins that bind to macrophage proteins. Combining GST pull-down with mass spectroscopy then enabled the specific identification of all binders. We refer to this proteome microarray-based strategy as SOPHIE (Systematic unlOcking of Pathogen and Host Interacting Effectors). Detailed investigation of a novel effector identified here, the iron storage protein BfrB (Rv3841), revealed that BfrB inhibits NF-κB-dependent transcription through binding and reducing the nuclear abundance of the ribosomal protein S3 (RPS3), which is a functional subunit of NF- κB. The importance of this interaction was evidenced by the promotion of survival in macrophages of the mycobacteria, Mycobacterium smegmatis, by overexpression of BfrB. Thus, beyond demonstrating the power of SOPHIE in the discovery of novel effectors of human pathogens, we expect that the set of Mtb effectors identified in this work will greatly facilitate the understanding of the pathogenesis of Mtb, possibly leading to additional potential molecular targets in the battle against tuberculosis. Mycobacterium tuberculosis (Mtb) has evolved multiple strategies to counter the human immune system. The effectors of Mtb play important roles in the interactions with the host. However, because of the lack of highly efficient strategies, there are only a handful of known Mtb effectors, thus hampering our understanding of Mtb pathogenesis. In this study, we probed Mtb proteome microarray with biotinylated whole-cell lysates of human macrophages, identifying 26 Mtb membrane proteins and secreted proteins that bind to macrophage proteins. Combining GST pull-down with mass spectroscopy then enabled the specific identification of all binders. We refer to this proteome microarray-based strategy as SOPHIE (Systematic unlOcking of Pathogen and Host Interacting Effectors). Detailed investigation of a novel effector identified here, the iron storage protein BfrB (Rv3841), revealed that BfrB inhibits NF-κB-dependent transcription through binding and reducing the nuclear abundance of the ribosomal protein S3 (RPS3), which is a functional subunit of NF- κB. The importance of this interaction was evidenced by the promotion of survival in macrophages of the mycobacteria, Mycobacterium smegmatis, by overexpression of BfrB. Thus, beyond demonstrating the power of SOPHIE in the discovery of novel effectors of human pathogens, we expect that the set of Mtb effectors identified in this work will greatly facilitate the understanding of the pathogenesis of Mtb, possibly leading to additional potential molecular targets in the battle against tuberculosis. Mycobacterium tuberculosis (Mtb) 1The abbreviations used are: Mtb; Mycobacterium tuberculosis; MOA, Mode of action; SOPHIE, Systematic unlOcking Pathogen and Host Interacting Effectors; Msm, Mycobacterium smegmatis; TB, tuberculosis; RPS3, ribosomal protein S3; BLI, Bio-Layer Interferometry; PPI, protein- protein interactions; NF-κB, Nuclear factor-kappa B. 1The abbreviations used are: Mtb; Mycobacterium tuberculosis; MOA, Mode of action; SOPHIE, Systematic unlOcking Pathogen and Host Interacting Effectors; Msm, Mycobacterium smegmatis; TB, tuberculosis; RPS3, ribosomal protein S3; BLI, Bio-Layer Interferometry; PPI, protein- protein interactions; NF-κB, Nuclear factor-kappa B. is the causative agent of tuberculosis (TB). There were an estimated 1.4 million TB deaths in 2015, and an additional 400, 000 deaths resulting from the co-infection of TB and HIV (1.World Health Organization (WHO) Global tuberculosis report 2016. WHO Press, 2016Google Scholar). Owing especially to the emergence of highly drug-resistant strains of Mtb, including multidrug-resistant (MDR) and extensively drug- resistant (XDR) strains, as well as co-infection with HIV, TB is still a serious infectious disease world-wide (2.Gandhi N.R. Shah N.S. Andrews J.R. Vella V. Moll A.P. Scott M. Weissman D. Marra C. Lalloo U.G. Friedland G.H. Tugela Ferry C. Research C. HIV coinfection in multidrug- and extensively drug-resistant tuberculosis results in high early mortality.Am. J. Respir. Crit. Care Med. 2010; 181: 80-86Crossref PubMed Scopus (249) Google Scholar, 3.Goldberg D.E. Siliciano R.F. Jacobs Jr., W.R. Outwitting evolution: fighting drug- resistant TB, malaria, and HIV.Cell. 2012; 148: 1271-1283Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Thus there is great urgency to identify novel proteins that can be targeted by drugs for effective treatment of TB, and especially MDR-TB and XDR-TB. Mtb is a highly successful human pathogen, surviving and multiplying within macrophages of infected people (4.Jean P. John G. Hijacking the host: survival of pathogenic mycobacteria inside macrophages.Trends Microbiol. 2002; 10: 142-146Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 5.Cambier C.J. Falkow S. Ramakrishnan L. Host evasion and exploitation schemes of Mycobacterium tuberculosis.Cell. 2014; 159: 1497-1509Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Host innate immune responses play crucial roles in the early protection against Mtb infection (6.MacMicking J.D. Cell-autonomous effector mechanisms against Mycobacterium tuberculosis.Cold Spring Harb. Perspect. Med. 2014; 4Crossref PubMed Scopus (25) Google Scholar). For one, the signaling pathway of the transcription factor NF-κB regulates innate immunity by controlling the expression of various immune-regulatory molecules and thus modulates the intracellular survival of pathogens (7.Behar S.M. Martin C.J. Booty M.G. Nishimura T. Zhao X. Gan H.X. Divangahi M. Remold H.G. Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis.Mucosal Immunol. 2011; 4: 279-287Crossref PubMed Scopus (281) Google Scholar, 8.Xu G. Wang J. Gao G.F. Liu C.H. Insights into battles between Mycobacterium tuberculosis and macrophages.Protein Cell. 2014; 5: 728-736Crossref PubMed Scopus (68) Google Scholar). On the pathogen side, Mtb membrane proteins and secreted proteins are important effectors that may play important roles in host-pathogen interactions (9.Poirier V. Av-Gay Y. Mycobacterium tuberculosis modulators of the macrophage's cellular events.Microbes Infect. 2012; 14: 1211-1219Crossref PubMed Scopus (26) Google Scholar). Previous work has indeed shown that Mtb has evolved several highly effective strategies including the secretion of specific effectors into the host to subvert the signaling pathways of the innate immune system, thereby allowing Mtb to avoid elimination and survive within macrophages for long periods of time (10.Kim K.H. An D.R. Song J. Yoon J.Y. Kim H.S. Yoon H.J. Im H.N. Kim J. Kim D.J. Lee S.J. Kim K.H. Lee H.M. Kim H.J. Jo E.K. Lee J.Y. Suh S.W. Mycobacterium tuberculosis Eis protein initiates suppression of host immune responses by acetylation of DUSP16/MKP-7.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7729-7734Crossref PubMed Scopus (147) Google Scholar, 11.Wang J. Li B.X. Ge P.P. Li J. Wang Q. Gao G.F. Qiu X.B. Liu C.H. Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system.Nat. Immunol. 2015; 16: 237-245Crossref PubMed Scopus (110) Google Scholar). The presently best-characterized survival mechanisms of Mtb are the inhibition of phagosomal acidification and maturation, and the inhibition of apoptosis of infected macrophages (12.Liu M. Li W. Xiang X. Xie J. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.Apoptosis. 2015; 20: 883-891Crossref PubMed Scopus (25) Google Scholar, 13.Wong D. Bach H. Sun J. Hmama Z. Av-Gay Y. Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+-ATPase to inhibit phagosome acidification.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 19371-19376Crossref PubMed Scopus (268) Google Scholar). However, there are presently only a limited number of well- characterized Mtb effectors that have been described, and only a few whose corresponding host-interacting proteins have been identified (14.Wang J. Teng J.L. Zhao D. Ge P. Li B. Woo P.C. Liu C.H. The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection.Sci. Rep. 2016; 6: 34827Crossref PubMed Scopus (38) Google Scholar, 15.Bach H. Papavinasasundaram K.G. Wong D. Hmama Z. Av-Gay Y. Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B.Cell Host Microbe. 2008; 3: 316-322Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 16.Sun J. Wang X. Lau A. Liao T.Y. Bucci C. Hmama Z. Mycobacterial nucleoside diphosphate kinase blocks phagosome maturation in murine RAW 264.7 macrophages.PLoS ONE. 2010; 5: e8769Crossref PubMed Scopus (93) Google Scholar, 17.Vergne I. Chua J. Lee H.H. Lucas M. Belisle J. Deretic V. Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4033-4038Crossref PubMed Scopus (401) Google Scholar, 18.Sreejit G. Ahmed A. Parveen N. Jha V. Valluri V.L. Ghosh S. Mukhopadhyay S. The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (beta2M) affecting antigen presentation function of macrophage.PLoS Pathog. 2014; 10: e1004446Crossref PubMed Scopus (97) Google Scholar, 19.Mueller-Ortiz S.L. Wanger A.R. Norris S.J. Mycobacterial protein HbhA binds human complement component C3.Infect. Immun. 2001; 69: 7501-7511Crossref PubMed Scopus (41) Google Scholar, 20.Deghmane A.-E. Soualhine H. Bach H. Sendide K. Itoh S. Tam A. Noubir S. Talal A. Lo R. Toyoshima S. Av-Gay Y. Hmama Z. Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation.J. Cell Sci. 2007; 120: 3489Crossref Scopus (3) Google Scholar, 21.Mehra A. Zahra A. Thompson V. Sirisaengtaksin N. Wells A. Porto M. Koster S. Penberthy K. Kubota Y. Dricot A. Rogan D. Vidal M. Hill D.E. Bean A.J. Philips J.A. Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking.PLoS Pathog. 2013; 9: e1003734Crossref PubMed Scopus (114) Google Scholar, 22.Dziadek B. Brzostek A. Grzybowski M. Fol M. Krupa A. Kryczka J. Plocinski P. Kurdowska A. Dziadek J. Mycobacterium tuberculosis AtsG (Rv0296c), GlmU (Rv1018c) and SahH (Rv3248c) proteins function as the human IL-8-binding effectors and contribute to pathogen entry into human neutrophils.PLoS ONE. 2016; 11: e0148030Crossref PubMed Scopus (8) Google Scholar, 23.Danelishvili L. Yamazaki Y. Selker J. Bermudez L.E. Secreted Mycobacterium tuberculosis Rv3654c and Rv3655c proteins participate in the suppression of macrophage apoptosis.PLoS ONE. 2010; 5: e10474Crossref PubMed Scopus (64) Google Scholar) (supplemental Table S1). These interactions were mainly discovered by yeast two-hybrid (Y2H) (21.Mehra A. Zahra A. Thompson V. Sirisaengtaksin N. Wells A. Porto M. Koster S. Penberthy K. Kubota Y. Dricot A. Rogan D. Vidal M. Hill D.E. Bean A.J. Philips J.A. Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking.PLoS Pathog. 2013; 9: e1003734Crossref PubMed Scopus (114) Google Scholar, 24.Rajagopala S.V. Sikorski P. Kumar A. Mosca R. Vlasblom J. Arnold R. Franca-Koh J. Pakala S.B. Phanse S. Ceol A. Hauser R. Siszler G. Wuchty S. Emili A. Babu M. Aloy P. Pieper R. Uetz P. The binary protein-protein interaction landscape of Escherichia coli.Nat. Biotech. 2014; 32: 285-290Crossref PubMed Scopus (148) Google Scholar) and affinity purification followed by mass spectrometry analysis (AP/MS) (22.Dziadek B. Brzostek A. Grzybowski M. Fol M. Krupa A. Kryczka J. Plocinski P. Kurdowska A. Dziadek J. Mycobacterium tuberculosis AtsG (Rv0296c), GlmU (Rv1018c) and SahH (Rv3248c) proteins function as the human IL-8-binding effectors and contribute to pathogen entry into human neutrophils.PLoS ONE. 2016; 11: e0148030Crossref PubMed Scopus (8) Google Scholar, 25.Jäger S. Cimermancic P. Gulbahce N. Johnson J.R. McGovern K.E. Clarke S.C. Shales M. Mercenne G. Pache L. Li K. Hernandez H. Jang G.M. Roth S.L. Akiva E. Marlett J. Stephens M. D'Orso I. Fernandes J. Fahey M. Mahon C. O'Donoghue A.J. Todorovic A. Morris J.H. Maltby D.A. Alber T. Cagney G. Bushman F.D. Young J.A. Chanda S.K. Sundquist W.I. Kortemme T. Hernandez R.D. Craik C.S. Burlingame A. Sali A. Frankel A.D. Krogan N.J. Global landscape of HIV- human protein complexes.Nature. 2011; 481: 365-370Crossref PubMed Scopus (511) Google Scholar). For example, Mehra et al. applied a high- throughput Y2H platform to identify Mtb secreted virulence factors and Mtb-host interacting proteins (21.Mehra A. Zahra A. Thompson V. Sirisaengtaksin N. Wells A. Porto M. Koster S. Penberthy K. Kubota Y. Dricot A. Rogan D. Vidal M. Hill D.E. Bean A.J. Philips J.A. Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking.PLoS Pathog. 2013; 9: e1003734Crossref PubMed Scopus (114) Google Scholar). However, one of the disadvantages of Y2H is that it detects only about 20% of all interactions because of its high false-negative rate (21.Mehra A. Zahra A. Thompson V. Sirisaengtaksin N. Wells A. Porto M. Koster S. Penberthy K. Kubota Y. Dricot A. Rogan D. Vidal M. Hill D.E. Bean A.J. Philips J.A. Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking.PLoS Pathog. 2013; 9: e1003734Crossref PubMed Scopus (114) Google Scholar, 26.Mendez-Rios J. Uetz P. Global approaches to study protein-protein interactions among viruses and hosts.Future Microbiol. 2010; 5: 289-301Crossref PubMed Scopus (25) Google Scholar, 27.Yu H.Y. Braun P. Yıldırım M.A. Lemmens I. Venkatesan K. Sahalie J. Hirozane-Kishikawa T. Gebreab F. Li N. Simonis N. Hao T. Rual J.F. Dricot A. Vazquez A. Ryan Murray R. Christophe S. Tardivo L. Tam S. Svrzikapa N. Fan C. de Smet A. Motyl. A. Hudson M. Park J. Xin X. Cusick M. Moore T. Boone C. Snyder M. Roth F. Barabási A. Tavernier J. Hill D. Vida M. High- quality binary protein interaction map of the yeast interactome network.Science. 2008; 322: 104-110Crossref PubMed Scopus (1109) Google Scholar). AP/MS can detect proteins in complexes but is unable to distinguish between direct from indirect interactions when several proteins are purified together (24.Rajagopala S.V. Sikorski P. Kumar A. Mosca R. Vlasblom J. Arnold R. Franca-Koh J. Pakala S.B. Phanse S. Ceol A. Hauser R. Siszler G. Wuchty S. Emili A. Babu M. Aloy P. Pieper R. Uetz P. The binary protein-protein interaction landscape of Escherichia coli.Nat. Biotech. 2014; 32: 285-290Crossref PubMed Scopus (148) Google Scholar, 26.Mendez-Rios J. Uetz P. Global approaches to study protein-protein interactions among viruses and hosts.Future Microbiol. 2010; 5: 289-301Crossref PubMed Scopus (25) Google Scholar). Thus, because of the intrinsic limitations of Y2H and AP/MS, these methods are not well-suited for the global screening of protein-protein interactions (PPI) between Mtb and the host. Systematic identification of Mtb and macrophage interactions holds the promise for revealing the complex mechanism underlying pathogenesis of Mtb (28.Boshoff H.I. Lun D.S. Systems biology approaches to understanding mycobacterial survival mechanisms.Drug Discov. Today Dis. Mech. 2010; 7: e75-e82Crossref PubMed Scopus (15) Google Scholar, 29.Barczak A.K. Avraham R. Singh S. Luo S.S. Zhang W.R. Bray M.-A. Hinman A.E. Thompson M. Nietupski R.M. Golas A. Montgomery P. Fitzgerald M. Smith R.S. White D.W. Tischler A.D. Carpenter A.E. Hung D.T. Systematic, multiparametric analysis of Mycobacterium tuberculosis intracellular infection offers insight into coordinated virulence.PLoS Pathog. 2017; 13: e1006363Crossref PubMed Scopus (58) Google Scholar). Because of the lack of suitable proteome-scale techniques, we do not yet have a global picture of the Mtb-host interactions at the protein level. Proteome microarrays, usually composed of thousands of proteins from one species that are affinity purified and functionally active, are high- throughput platforms for the global profiling of thousands of molecular interactions in a single experiment (30.Zhu H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Mitchell T. Miller P. Dean R.A. Gerstein M. Snyder M. Global Analysis of Protein Activities Using Proteome Chips.Science. 2001; 293: 2101-2105Crossref PubMed Scopus (1935) Google Scholar). They provide a versatile platform for investigating many aspects at the systems-level, such as discovering serum biomarkers for various diseases (31.Gnjatic S. Ritter E. Buchler M.W. Giese N.A. Brors B. Frei C. Murray A. Halama N. Zornig I. Chen Y.T. Andrews C. Ritter G. Old L.J. Odunsi K. Jager D. Seromic profiling of ovarian and pancreatic cancer.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 5088-5093Crossref PubMed Scopus (146) Google Scholar) and globally investigating interactions with proteins (32.Deng R.P. He X. Guo S.J. Liu W.F. Tao Y. Tao S.C. Global identification of O- GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome.Proteomics. 2014; 14: 1020-1030Crossref PubMed Scopus (24) Google Scholar), DNA (33.Lin Y.Y. Lu J.Y. Zhang J. Walter W. Dang W. Wan J. Tao S.C. Qian J. Zhao Y. Boeke J.D. Berger S.L. Zhu H. Protein acetylation microarray reveals that NuA4 controls key metabolic target regulating gluconeogenesis.Cell. 2009; 136: 1073-1084Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), RNA (34.Zhu J. Gopinath K. Murali A. Yi G. Hayward S.D. Zhu H. Kao C. RNA-binding proteins that inhibit RNA virus infection.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 3129-3134Crossref PubMed Scopus (88) Google Scholar), lipids (35.Lu K.Y. Tao S.C. Tzu-Ching Y. Yu-Hsuan H. Chia-Hsien L. Chen-Ching L. Hsueh-Fen J. Hsuan Cheng -H. Chin-Yu Y. Ming-Shuo C. Yu-Yi L. Jin-Ying L. Heng Z. Chien-Sheng C. Profiling lipid–protein interactions using nonquenched fluorescent liposomal nanovesicles and proteome microarrays.Mol. Cell. Proteomics. 2012; 11: 1177-1190Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and small molecules (36.Zhang H.N. Yang L. Jian-ya Czajkowsky L.D.M. Wang J.F. Zhang X.W. Zhou Y.M. Ge FYang M-k Xiong Q. Guo S.J. Lea H.Y. Wu S.F. Yan W. Liu B. Zhu H. Chen Z. Tao S.C. Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 15084-15089Crossref PubMed Scopus (95) Google Scholar, 37.Huang J. Zhu H. Haggarty S.J. Spring D.R. Hwang H. Jin F. Snyder M. Schreiber S.L. Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16594-16599Crossref PubMed Scopus (210) Google Scholar). Recently, we constructed a functional Mtb proteome microarray (with >95% coverage of the Mtb proteome) (38.Deng J.Y. Bi L.J. Zhou L. Guo S.J. Fleming J. Jiang H.W. Zhou Y. Gu J. Zhong Q. Wang Z.X. Liu Z. Deng R.P. Gao J. Chen T. Li W. Wang J.F. Wang X. Li H. Ge F. Zhu G. Zhang H.N. Gu J. Wu F.L. Zhang Z. Wang D. Hang H. Li Y. Cheng L. He X. Tao S.C. Zhang X.E. Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity.Cell Rep. 2014; 9: 2317-2329Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The power of this Mtb proteome microarray has already been demonstrated in investigations of PPI (39.Wu Fan-Lin Yin Liu He-Wei Jiang Yi-Zhao Luan Hai-Nan Zhang Xiang He Zhao-Wei Xu Jing-Li Hou Ji L.-Y. Zhi Xie Daniel Czajkowsky M. Wei Yan Jiao-Yu Deng Li-Jun Bi Xian-En Zhang Tao S.-C. The Ser/Thr protein kinase protein-protein interaction map of M.tuberculosis.Mol. Cell. Proteomics. 2017; 16: 1491-1506Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and small molecule/drug-protein binding (40.Zhang H.-N. Zhao-wei Jiang X.H.W. Fan-Lin He WXYin Guo L.S.J. Yang Bi L.L. Deng J. Xian-en Z. Tao S.C. Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide.Sci Rep. 2017; 7: 5860Crossref PubMed Scopus (26) Google Scholar). Herein, we took advantage of this Mtb proteome microarray and established a strategy for the global identification of the Mtb proteins involved in Mtb-macrophage interactions. With this, we identified a total of 26 Mtb membrane proteins and secreted proteins that may play important roles in Mtb-host interactions. We focused on BfrB (Rv3841), a ferritin-like protein that was known to play a key role in the maintenance of Mtb iron homeostasis (41.Pandey R. Rodriguez G.M. A ferritin mutant of Mycobacterium tuberculosis is highly susceptible to killing by antibiotics and is unable to establish a chronic infection in mice.Infect. Immun. 2012; 80: 3650-3659Crossref PubMed Scopus (78) Google Scholar). Interestingly, BfrB was recently identified as a secreted protein that is enriched from infected macrophages and is also present in the exosomes of patients, suggesting that it may play additional roles in the interactions with the host (42.Mattow J. Schaible U.E. Schmidt F. Hagens K. Siejak F. Brestrich G. Haeselbarth G. Muller E.C. Jungblut P.R. Kaufmann S.H. Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis BCG Copenhagen.Electrophoresis. 2003; 24: 3405-3420Crossref PubMed Scopus (139) Google Scholar, 43.Chande A.G. Siddiqui Z. Midha M.K. Sirohi V. Ravichandran S. Rao K.V.S. Selective enrichment of mycobacterial proteins from infected host macrophages.Sci Rep. 2015; 5: 13430Crossref PubMed Scopus (18) Google Scholar, 44.Kruh-Garcia N.A. Wolfe L.M. Chaisson L.H. Worodria W.O. Nahid P. Schorey J.S. Davis J.L. Dobos K.M. Detection of Mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M.tuberculosis infection using MRM-MS.PLoS ONE. 2014; 9: e103811Crossref PubMed Scopus (104) Google Scholar). However, the mechanism by which BfrB functions in the modulation of the host immune response is poorly known. Therefore, we sought to identify crucial host target proteins to which BfrB binds. We found that BfrB subverts NF-κB function by binding and reducing the nuclear abundance of its subunit RPS3 (45.Wan F. Anderson D.E. Barnitz R.A. Snow A. Bidere N. Zheng L. Hegde V. Lam L.T. Staudt L.M. Levens D. Deutsch W.A. Lenardo M.J. Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.Cell. 2007; 131: 927-939Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) to inhibit host immune defense. Thus, our findings reveal a previously unknown mechanism by which BfrB, as a newly identified effector, subverts innate immunity. THP-1 and U937 cell were differentiated into macrophage-like cells using 100 ng/ml PMA (Sigma-Aldrich, Saint Louis, MO) for 24 h. Then the medium was replaced and cells cultured in RPMI 1640 medium supplemented with 10% FBS for 24 h. The cells were washed twice, and the cell lysates were prepared using cell lysis buffer as described (46.Schroder C. Alhamdani M.S. Fellenberg K. Bauer A. Jacob A. Hoheisel J.D. Robust protein profiling with complex antibody microarrays in a dual-colour mode.Protein Microarrays. 2011; 785: 203-221Crossref Scopus (15) Google Scholar, 47.Vizcaino J.A. Csordas A. del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q.W. Wang R. Hermjakob H. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2775) Google Scholar). The whole cell lysates were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin protein biotinylation kit (Thermo Scientific, Bremen, Germany) according to the manual. Mtb proteome microarrays were blocked for 1 h at room temperature with shaking in blocking buffer as described (46.Schroder C. Alhamdani M.S. Fellenberg K. Bauer A. Jacob A. Hoheisel J.D. Robust protein profiling with complex antibody microarrays in a dual-colour mode.Protein Microarrays. 2011; 785: 203-221Crossref Scopus (15) Google Scholar). Microarrays were incubated in the biotinylated whole cell lysates for 12 h at 4 °C with shaking, then washed three times in 1× PBS (pH 7.4) with 0.1% Tween 20 (PBST). Arrays were probed with Cy3-streptavidin and incubated in a humidified chamber for 1 h at room temperature. After washing three times in PBST and once ddH2O, microarrays were dried in a SlideWasher (CapitalBio, Beijing, China) and then scanned with a GenePix 4200A microarray scanner (Molecular Devices, CA). Data were analyzed with GenePix Pro 6.1 (Molecular Devices, CA). To identity the host targets of Mtb effectors, GST-tagged BfrB were purified as previously described (38.Deng J.Y. Bi L.J. Zhou L. Guo S.J. Fleming J. Jiang H.W. Zhou Y. Gu J. Zhong Q. Wang Z.X. Liu Z. Deng R.P. Gao J. Chen T. Li W. Wang J.F. Wang X. Li H. Ge F. Zhu G. Zhang H.N. Gu J. Wu F.L. Zhang Z. Wang D. Hang H. Li Y. Cheng L. He X. Tao S.C. Zhang X.E. Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity.Cell Rep. 2014; 9: 2317-2329Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). PMA-differentiated THP-1 cells lysed in Nonidet P-40 lysis buffer (Beyotime, Shanghai, China) supplemented with 0.5 mm PMSF (Sigma-Aldrich). The GST pull- down was performed as described (11.Wang J. Li B.X. Ge P.P. Li J. Wang Q. Gao G.F. Qiu X.B. Liu C.H. Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system.Nat. Immunol. 2015; 16: 237-245Crossref PubMed Scopus (110) Google Scholar). The pull-down proteins were eluted by 2× SDS sampling buffer and denatured at 95 °C for 10 min and analyzed by standard silver staining. To determine the proteins interacted with BfrB, the human proteins enriched by BfrB were subjected for electrophoresis and silver staining, the major bands in the gel were excised, destained and reduced by incubation in a solution of 10 mm DTT at 60 °C for 20 min, followed by alkylating in a solution of 25 mm IAM at room temperature for 15 min. The pieces were digested in-gel with trypsin (Promega, Madison, WI) overnight at 37 °C. The tryptic peptide digests of the proteins were analyzed using a LC system (Nano Pump, Ultimate 3000, Dionex, Thermofisher) coupled with an ESI- Q-TOF mass spectrometer (maXis Impact, Bruker Daltonik, Bremen Area, Germany). Tandem mass spectra were extracted, charge state deconvoluted and deisotoped by Compass Data Analysis version 4.1 (Bruker Daltonics). The peak list was directly generated from raw data using centroid algorithm with peak width set as 0.1 m/z and intensity above 100. No peak smooth or filter process was applied. After the charge states were calculated, the de-isotoped. Peak list was exported as mgf file for further mascot search. Mascot (version 2.4, Matrix Science, Boston, MA) was set up to search the SwissProt 2013_12 Homo sapiens database (20,274 entries). The following parameters were considered for the searches: peptide mass tolerance was set to 20 ppm, fragment mass tolerance was set to 0.05 Da, and a maximum of two missed cleavage of trypsin was chosen. Carbamidomethyl (C) was set as fixed modification, and oxidation (M) was set as variable modifications. The threshold score was greater than or equal to 30, expectation value was 0.01 and the number of peptides was more than 2 for accepting individual spectra. The binding kinetics of BfrB and RPS3 were measured using ForteBio Octet system (Pall, Menlo Park, USA). Affinity purified BfrB was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin protein biotinylation kit as described above. Biotinylated BfrB was tethered on the tip surface of a streptavidin-coated sensor. The binding partner, RPS3, in S.D. buffer (1× PBS [pH 7.4] with 0.02% Tween-20 and 0.1% BSA) was then exposed to tethered biotinylated BfrB, and binding was measured by coincident change in the interference pattern. Transfection was performed using Attractene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer's instruction. HEK293T cells were collected 24 h after transfection and lysed in Nonidet P-40 lysis buffer (Beyotime, Shanghai, China) supplemented with 0.5 mm PMSF (Sigma-Aldrich). The immunoprecipitation assay was performed as (11.Wang J. Li B.X. Ge P.P. Li J. Wang Q. Gao G.F. Qiu X.B. Liu C.H. Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system.Nat. Immunol. 2015; 16: 237-245Crossref PubMed Scopus (110) Google Scholar). The immunoblot analysis was followed as previously described (32.Deng R.P. He X. Guo S.J. Liu W.F. Tao Y. Tao S.C. Global identification of O- GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome.Proteomics. 2014; 14: 1020-1030Crossref PubMed Scopus (24) Google Scholar). The results were then recorded by the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Cytosolic and nuclear protein extracts were prepared from HEK293T cells transfected with FLAG-BfrB/FLAG plasmids as previously described (48.Gao X. Wan F. Mateo K. Callegari E. Wang D. Deng W. Puente J. Li F. Chaussee M.S. Finlay B.B. Lenardo M.J. Hardwidge P.R. Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function.PLoS Pathog. 2009; 5: e1000708Crossre
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