康涵，吳文娟

上海市(復(fù)旦大學(xué)附屬)公共衛(wèi)生臨床中心，上海 201508

絲氨酸/蘇氨酸蛋白激酶(serine/threonine protein kinase，STPK)是一類真核細胞樣的蛋白激酶，是分枝桿菌生長和代謝的重要調(diào)節(jié)因子。結(jié)核分枝桿菌共有11種STPK，分為跨膜蛋白和可溶性蛋白兩大類[1]。對多種分枝桿菌STPK基因序列進行分析，按系統(tǒng)發(fā)生可分為5群：ABL 群(pknA、pknB、pknL)，HED群(pknH、pknE、pknD)，F(xiàn)IJ群(pknF、pknI、pknJ)，pknG及pknK[2]。研究發(fā)現(xiàn)，STPK參與分枝桿菌多種細胞活動(如細胞和菌落形態(tài)、葡萄糖和谷氨酰胺轉(zhuǎn)運、吞噬體-溶酶體融合、轉(zhuǎn)錄因子活性等)的調(diào)節(jié)(表1)。STPK與其配體結(jié)合，二聚化后構(gòu)象改變引起自身磷酸化，并磷酸化下游蛋白，從而發(fā)揮信號轉(zhuǎn)導(dǎo)作用[3]。Cui等[4]對結(jié)核分枝桿菌蛋白間相互作用的預(yù)測顯示，STPK可與許多重要蛋白發(fā)生相互作用。Malhotra等[5]對結(jié)核分枝桿菌H37Rv和H37Ra的STPK基因表達水平比較分析發(fā)現(xiàn)，有毒株H37Rv的6個STPK基因pknD、pknG、pknH、pknJ、pknK和pknL有更高的表達。

表1 結(jié)核分枝桿菌的絲氨酸/蘇氨酸蛋白激酶
Fig.1 The serine/threonine protein kinases ofMycobacteriumtuberculosis

NameORFLengthClassificationRegulatory role PknARv0015c431 aaTransmembrane Cell division and morphologyPknBRv0014c626 aaTransmembraneCell division and morphologyPknLRv2176399 aaTransmembranePhosphorylation of Rv2175c, transcription?PknHRv1266c626 aaTransmembraneArabinan metabolism, growth in hostile environmentPknERv1743566 aaTransmembraneResponse to NO pressure, apoptosis of infected macrophagesPknDRv0931c664 aaTransmembraneTranscription, phosphate transport?PknFRv1746476 aaTransmembraneCell divisionPknIRv2914c585 aaTransmembraneGrowth in hostile environmentPknJRv2088589 aaTransmembranePhosphorylation of sorts of functional proteinsPknGRv0410c750 aaSolubleGlutamate/glutamine metabolism, intrinsic antibiotic resistance, in-hibition of phagosome-lysosome fusion, enhancing bacterial intra-cellular survivalPknKRv3080c1110 aaSolubleTranscription, cell wall ultrastructure, growth in vivo

1 跨膜STPK

1.1 ABL群

目前研究表明，PknA和PknB主要與細胞形態(tài)和分裂有關(guān)，PknL可磷酸化DNA結(jié)合蛋白Rv2175c。Schultz等[6]構(gòu)建了多種谷氨酸棒狀桿菌的STPK突變株，發(fā)現(xiàn)PknA、PknB、PknG和PknL對細菌的生存不是必需的，它們都能磷酸化2-酮戊二酸脫氫酶OdhI，其中又以PknG作用最強；此外，細胞分裂蛋白FtsZ也能被PknA、PknB和PknL磷酸化。但Fiuza等[7]認(rèn)為，PknA和 PknB對谷氨酸棒狀桿菌的生長是必需的，其過表達使桿菌呈小球狀生長。在結(jié)核分枝桿菌中，PknA和PknB也有相似作用：PknA能磷酸化細胞分裂蛋白FtsZ及分枝桿菌UDP-N-乙酰胞壁酰-L-丙氨酰-D-谷氨酸連接酶[8, 9]，PknB也有多種底物，包括GarA、N-乙酰葡糖胺-1-磷酸尿苷酰轉(zhuǎn)移酶、叉頭結(jié)構(gòu)域相關(guān)蛋白Rv0019c、PapA5等[10-12]；而PknA和PknB能被跨膜的Mstp蛋白去磷酸化[13]。這些底物多與分枝桿菌細胞壁合成、細胞分裂等有關(guān)，在大腸埃希菌中表達結(jié)核分枝桿菌pknA可引起細菌異常延伸[14]，表達結(jié)核分枝桿菌pknA和pknB的恥垢分枝桿菌生長變慢，敲除pknA和pknB的結(jié)核分枝桿菌菌體變得更狹窄[15]。PknL可磷酸化一種放線菌的保守DNA結(jié)合蛋白Rv2175c[16,17]，但其下游效應(yīng)還不清楚。

1.2 HED群

此群STPK參與細菌的多種重要生理活動，并與致病性密切相關(guān)。PknH存在于致病的結(jié)核分枝桿菌和牛分枝桿菌，而不存在于恥垢分枝桿菌中。在壓力環(huán)境下，結(jié)核分枝桿菌pknH的表達水平改變[18]。重組表達pknH的恥垢分枝桿菌中，PknH能磷酸化EmbR，導(dǎo)致embCAB操縱子的轉(zhuǎn)錄水平增加，影響阿拉伯糖代謝，使脂化甘露聚醣與脂化阿拉伯甘露聚糖的比值升高，并對乙胺丁醇的抵抗性增加。結(jié)核分枝桿菌感染巨噬細胞后，pknH表達增高[19]。但另有研究發(fā)現(xiàn)，在結(jié)核分枝桿菌中敲除pknH并不影響embCAB操縱子的表達，僅減少乙胺丁醇誘導(dǎo)的embCAB操縱子的表達[20]。除EmbR外，Zheng等[21]在體外實驗中發(fā)現(xiàn)，PknH還能磷酸化Rv0681和DacB1。敲除pknH的結(jié)核分枝桿菌感染BALB/c小鼠后，在感染慢性期生存和復(fù)制能力增強[20]。對PknE的活性、定位、晶體結(jié)構(gòu)及結(jié)構(gòu)域的分析提示，它可能是跨膜的受體型蛋白激酶，能接收細胞外的信號并將其傳遞到細胞內(nèi)，引起細胞活動改變。該過程主要與其結(jié)合配體后二聚化變構(gòu)并自身磷酸化有關(guān)[22, 23]。體外實驗發(fā)現(xiàn)，PknE、PknD均能磷酸化ATP結(jié)合盒轉(zhuǎn)運蛋白(ATP-binding cassette transporter, ABC)[24]。PknE在結(jié)核分枝桿菌對一氧化氮(nitric oxide，NO)壓力反應(yīng)及人巨噬細胞感染后的凋亡有重要作用。與野生株相比，pknE突變株對NO供體的抵抗力增強，而對還原劑和某些金屬離子的抵抗力減弱，引起巨噬細胞凋亡增加，但巨噬細胞壞死和增殖減少[25]。結(jié)核分枝桿菌的PknD跨膜區(qū)C端有個β-propeller模體，而牛分枝桿菌中無此結(jié)構(gòu)。結(jié)核分枝桿菌毒力相關(guān)轉(zhuǎn)運蛋白MmpL7可能是PknD的底物之一[26]。PknD也許與磷酸鹽轉(zhuǎn)運有關(guān)，但尚未證實[27,28]。PknD具有與PknE相似的二聚化及變構(gòu)激活過程[29]。體外實驗發(fā)現(xiàn)，PknD過表達能改變多種基因轉(zhuǎn)錄，包括Rv0516c及一些受sigma因子F調(diào)節(jié)的基因等[30]。

1.3 FIJ群

目前研究顯示，PknF與細菌分裂有關(guān)，PknI參與調(diào)節(jié)有害環(huán)境中的細菌生長，PknJ則可磷酸化多種功能蛋白，進而影響細菌的代謝活動。體外實驗發(fā)現(xiàn)，PknF也可磷酸化多種含有叉頭相關(guān)結(jié)構(gòu)域的蛋白，如ABC轉(zhuǎn)運子、髓磷脂堿性蛋白等[24,31,32]。PknF在結(jié)核分枝桿菌葡萄糖轉(zhuǎn)化、細胞生長及隔膜形成中可能起直接或間接作用[33]。PknF也表達于恥垢分枝桿菌，它能影響恥垢分枝桿菌的生物膜形成及運動性，這與結(jié)核分枝桿菌相似[34]。PknI雖然為跨膜STPK，但有報道發(fā)現(xiàn)其也存在于細胞質(zhì)中[35]?；蛐畔⒎治鲱A(yù)測，PknI可能參與細胞生長和延伸[36]。感染巨噬細胞后，pknI表達下調(diào)[33]。與野生株相比，PknI突變株在酸性條件及缺氧時生長更好，感染巨噬細胞后其生存力更強，在免疫缺陷小鼠中突變株的毒力也更高[37]。PknJ的底物較多，包括PknJ本身、轉(zhuǎn)錄調(diào)節(jié)因子EmbR、參與分枝菌酸合成的甲基轉(zhuǎn)移酶MmaA4/Hma、二肽酶PepE、丙酮酸激酶A等[38, 39]。

2 可溶性STPK

2.1 PknG

PknG與真核細胞的蛋白激酶Cα(protein kinase Cα, PKCα)很相似，可自我磷酸化，其磷酸化能被白屈菜赤堿阻斷。與其他大多數(shù)結(jié)核分枝桿菌的STPK不同，PknG中未發(fā)現(xiàn)已知的穿膜結(jié)構(gòu)域。恥垢分枝桿菌不表達PknG，結(jié)核分枝桿菌和牛分枝桿菌卡介苗(bacillus Calmette-Guerin，BCG)表達PknG[40]。亞細胞定位發(fā)現(xiàn)，PknG存在于分枝桿菌的胞質(zhì)溶膠和細胞膜中[41]，巨噬細胞攝入分枝桿菌后，在吞噬體和胞質(zhì)溶膠內(nèi)都能檢測到PknG存在[40]。結(jié)核分枝桿菌pknG缺陷株可造成谷氨酸鹽和谷氨酰胺堆積，使其細胞內(nèi)濃度達到野生株的3倍[41]。但Nguyen等發(fā)現(xiàn)，BCG中pknG缺陷并不影響谷氨酰胺的攝入及其細胞內(nèi)濃度，他們認(rèn)為BCG中的谷氨酰胺代謝不受PknG調(diào)節(jié)[42]。谷氨酸鹽和谷氨酰胺在分枝桿菌中的作用包括：①谷氨酸鹽和谷氨酰胺的合成及潛在的脫氫酶活性是分枝桿菌氨基酸氨基同化作用的唯一途徑；②聚合L-谷氨酸鹽/谷氨酰胺是結(jié)核分枝桿菌細胞膜結(jié)構(gòu)完整性的一部分[41]。PknG缺陷造成谷氨酸鹽/谷氨酰胺過度堆積可能是影響分枝桿菌生存和毒力的機制之一。比較結(jié)核分枝桿菌pknG缺陷株與野生株對多種抗生素的最低抑菌濃度發(fā)現(xiàn)，PknG在結(jié)核分枝桿菌的天然耐藥中起重要作用[43]。體外生長實驗發(fā)現(xiàn)，PknG對BCG體外生存不是必需的，突變株和野生株在細胞形態(tài)和生長上都沒有差別，其特異性抑制劑對分枝桿菌在培養(yǎng)物中的生長亦沒有影響[40,42]。而體內(nèi)或巨噬細胞受結(jié)核分枝桿菌感染后，情況則有所不同，重癥聯(lián)合免疫缺陷(severe combined immunodeficiency，SCID)小鼠感染突變株后死亡時間推遲，且突變株在SCID及BALB/c小鼠體內(nèi)的生存力均下降。這在分枝桿菌生長周期的靜止相或營養(yǎng)缺乏時尤為顯著[41]。導(dǎo)入pknG可延長恥垢分枝桿菌在巨噬細胞內(nèi)的存活。BCG感染巨噬細胞后，突變株主要存在于吞噬溶酶體內(nèi)，而野生株則主要存在于非溶酶體細胞器內(nèi)。PknG特異抑制劑可導(dǎo)致劑量依賴的、分枝桿菌感染的巨噬細胞內(nèi)吞噬溶酶體的形成，吞噬體迅速與溶酶體融合并殺死其中的分枝桿菌。進一步研究發(fā)現(xiàn)，這些與PknG的激酶活性有關(guān)[40]。此外，H37Rv、H37Ra及BCG感染巨噬細胞后，都發(fā)現(xiàn)PknG能下調(diào)巨噬細胞PKCα表達，從而影響巨噬細胞對分枝桿菌的吞噬，并增加分枝桿菌在細胞內(nèi)存活。重組了pknG的恥垢分枝桿菌在巨噬細胞內(nèi)的生存能力也顯著增強[44]。

2.2 PknK

與PknG不同，PknK主要存在于細胞壁和細胞膜(后者雜交信號很弱)，在感染巨噬細胞中僅溶酶體內(nèi)有信號檢出[5, 45]。已知PknK底物包括VirS和mymA操縱子編碼的4個蛋白。PknK能磷酸化轉(zhuǎn)錄因子VirS，增強其與DNA的結(jié)合能力，進而引起mymA的轉(zhuǎn)錄及表達[45]。mymA操縱子編碼多個酶，參與分枝桿菌脂類代謝。virS和(或)mymA敲除的結(jié)核分枝桿菌細胞壁超微結(jié)構(gòu)發(fā)生改變，分枝菌酸的合成受影響；對去污劑、酸性環(huán)境及抗生素的敏感性增加；在活化巨噬細胞中的生存能力及對豚鼠的毒力也發(fā)生改變[46]。掃描電鏡觀察發(fā)現(xiàn)，pknK突變株比野生株菌體小，細胞壁薄。僅在結(jié)核分枝桿菌H37Rv感染巨噬細胞后18 h，pknK有顯著表達。體外生長實驗發(fā)現(xiàn)，PknK能抑制結(jié)核分枝桿菌在穩(wěn)定期的生長。感染C57BL/6小鼠后，突變株在感染早期生長受限而后期增強；但在靜息巨噬細胞內(nèi)，突變株與野生株的生長情況相似，說明其在小鼠體內(nèi)生長不同并不是由于其內(nèi)在的感染性不同。分析不同時間點細胞因子的mRNA 表達發(fā)現(xiàn)，在突變株感染早期，白細胞介素2(interleukin 2，IL-12)、腫瘤壞死因子α(tumor necrosis factor α，TNF-α)、γ干擾素(interferon γ，IFN-γ)、誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase，iNOS)的表達均低于野生株，后期則差異不顯著。表明PknK可能在調(diào)節(jié)體內(nèi)環(huán)境壓力下的生長中起重要作用[5]。

3 結(jié)語

面對結(jié)核病對人類健康的重大挑戰(zhàn)，結(jié)核分枝桿菌致病機制的明確將給抗結(jié)核病帶來重大突破，如新藥物靶點的發(fā)現(xiàn)、新型疫苗的開發(fā)等。雖然已有大量研究發(fā)現(xiàn)了結(jié)核病相關(guān)致病物質(zhì)[47-49]，但具體機制尚不明確。STPK在結(jié)核分枝桿菌的許多細胞活動中起重要作用，能感知細菌所處的環(huán)境并作出反應(yīng)，且可能與結(jié)核分枝桿菌-宿主免疫平衡的維持有關(guān)。對于多數(shù)STPK，其作用機制還很不清楚，需進一步研究。

[1] Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence [J]. Nature, 1998, 393(6685): 537-544.

[2] Narayan A, Sachdeva P, Sharma K, Saini AK, Tyagi AK, Singh Y. Serine threonine protein kinases of mycobacterial genus: phylogeny to function [J]. Physiol Genomics, 2007, 29(1): 66-75.

[3] Alber T. Signaling mechanisms of the Mycobacterium tuberculosis receptor Ser/Thr protein kinases [J]. Curr Opin Struct Biol, 2009, 19(6): 650-657.

[4] Cui T, Zhang L, Wang X, He ZG. Uncovering new signaling proteins and potential drug targets through the interactome analysis of Mycobacterium tuberculosis [J]. BMC Genomics, 2009, 10: 118.

[5] Malhotra V, Arteaga-Cortés LT, Clay G, Clark-Curtiss JE. Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation [J]. Microbiology, 2010, 156(Pt 9): 2829-2841.

[6] Schultz C, Niebisch A, Schwaiger A, Viets U, Metzger S, Bramkamp M, Bott M. Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases [J]. Mol Microbiol, 2009, 74(3): 724-741.

[7] Fiuza M, Canova MJ, Zanella-Cléon I, Becchi M, Cozzone AJ, Mateos LM, Kremer L, Gil JA, Molle V. From the characterization of the four serine/threonine protein kinases (PknA/B/G/L) of Corynebacterium glutamicum toward the role of PknA and PknB in cell division [J]. J Biol Chem, 2008, 283(26): 18099-18112.

[8] Thakur M，Chakraborti PK. GTPase activity of mycobacterial FtsZ is impaired due to its transphosphorylation by the eukaryotic-type Ser/Thr kinase, PknA [J]. J Biol Chem, 2006, 281(52): 40107-40113.

[9] Thakur M，Chakraborti PK. Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis [J]. Biochem J, 2008, 415(1): 27-33.

[10] Gupta M, Sajid A, Arora G, Tandon V, Singh Y. Forkhead-associated domain-containing protein Rv0019c and polyketide-associated protein PapA5, from substrates of serine/threonine protein kinase PknB to interacting proteins of Mycobacterium tuberculosis [J]. J Biol Chem, 2009, 284(50): 34723-34734.

[11] Parikh A, Verma SK, Khan S, Prakash B, Nandicoori VK. PknB-mediated phosphorylation of a novel substrate, N-acetylglucosamine-1-phosphate uridyltransferase, modulates its acetyltransferase activity [J]. J Mol Biol, 2009, 386(2): 451-464.

[12] Villarino A, Duran R, Wehenkel A, Fernandez P, England P, Brodin P, Cole S T,Zimny-Arndt U, Jungblut PR, Cervenansky C, Alzari PM. Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions [J]. J Mol Biol, 2005, 350(5): 953-963.

[13] Chopra P, Singh B, Singh R, Vohra R, Koul A, Meena LS, Koduri H, Ghildiyal M, Deol P, Das TK, Tyagi AK, Singh Y. Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB [J]. Biochem Biophys Res Commun, 2003, 311(1): 112-120.

[14] Chaba R, Raje M, Chakraborti PK. Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division [J]. Eur J Biochem, 2002, 269(4): 1078-1085.

[15] Kang CM, Abbott DW, Park ST, Dascher CC, Cantley LC, Husson RN. The Mycobacterium tuberculosis serine/threonine kinases PknA and PknB: substrate identification and regulation of cell shape[J]. Genes Dev, 2005, 19(14): 1692-1704.

[16] Canova MJ, Veyron-Churlet R, Zanella-Cleon I, Cohen-Gonsaud M, Cozzone AJ, Becchi M, Kremer L, Molle V. The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c [J]. Proteomics, 2008, 8(3): 521-533.

[17] Cohen-Gonsaud M, Barthe P, Canova MJ, Stagier-Simon C, Kremer L, Roumestand C, Molle V. The Mycobacterium tuberculosis Ser/Thr kinase substrate Rv2175c is a DNA-binding protein regulated by phosphorylation [J]. J Biol Chem, 2009, 284(29): 19290-19300

[18] Sharma K, Chandra H, Gupta PK, Pathak M, Narayan A, Meena LS, D’Souza RC, Chopra P, Ramachandran S, Singh Y. PknH, a transmembrane Hank’s type serine/threonine kinase from Mycobacterium tuberculosis is differentially expressed under stress conditions [J]. FEMS Microbiol Lett, 2004, 233(1): 107-113.

[19] Sharma K, Gupta M, Pathak M, Gupta N, Koul A, Sarangi S, Baweja R, Singh Y. Transcriptional control of the mycobacterial embCAB operon by PknH through a regulatory protein, EmbR, in vivo [J]. J Bacteriol, 2006, 188(8): 2936-2944.

[20] Papavinasasundaram KG, Chan B, Chung JH, Colston MJ, Davis EO, Av-Gay Y. Deletion of the Mycobacterium tuberculosis pknH gene confers a higher bacillary load during the chronic phase of infection in BALB/c mice [J]. J Bacteriol, 2005, 187(16): 5751-5760.

[21] Zheng X, Papavinasasundaram KG, Av-Gay Y. Novel substrates of Mycobacterium tuberculosis PknH Ser/Thr kinase [J]. Biochem Biophys Res Commun, 2007, 355(1): 162-168.

[22] Molle V, Girard-Blanc C, Kremer L, Doublet P, Cozzone AJ, Prost JF. Protein PknE, a novel transmembrane eukaryotic-like serine/threonine kinase from Mycobacterium tuberculosis [J]. Biochem Biophys Res Commun, 2003, 308(4): 820-825.

[23] Gay LM, Ng HL, Alber T. A conserved dimer and global conformational changes in the structure of apo-PknE Ser/Thr protein kinase from Mycobacterium tuberculosis [J]. J Mol Biol, 2006, 360(2): 409-420.

[24] Grundner C, Gay LM, Alber T. Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains [J]. Protein Sci, 2005, 14(7): 1918-1921.

[25] Jayakumar D, Jacobs WR Jr, Narayanan S. Protein kinase E of Mycobacterium tuberculosis has a role in the nitric oxide stress response and apoptosis in a human macrophage model of infection [J]. Cell Microbiol, 2008, 10(2): 365-374.

[26] Pérez J, Garcia R, Bach H, de Waard JH, Jacobs WR Jr, Av-Gay Y, Bubis J, Takiff HE. Mycobacterium tuberculosis transporter MmpL7 is a potential substrate for kinase PknD [J]. Biochem Biophys Res Commun, 2006, 348(1): 6-12.

[27] Av-Gay Y, Everett M. The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis [J]. Trends Microbiol, 2000, 8(5): 238-244.

[28] Lefèvre P, Braibant M, de Wit L, Kalai M, R?eper D, Gr?tzinger J, Delville JP, Peirs P, Ooms J, Huygen K, Content J. Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG [J]. J Bacteriol, 1997, 179(9): 2900-2906.

[29] Greenstein AE, Echols N, Lombana TN, King DS, Alber T. Allosteric activation by dimerization of the PknD receptor Ser/Thr protein kinase from Mycobacterium tuberculosis [J]. J Biol Chem, 2007, 282(15): 11427-11435.

[30] Greenstein AE, MacGurn JA, Baer CE, Falick AM, Cox JS, Alber T. M. tuberculosis Ser/Thr protein kinase D phosphorylates an anti-anti-sigma factor homolog [J]. PLoS Pathog, 2007, 3(4): e49.

[31] Koul A, Choidas A, Tyagi AK, Drlica K, Singh Y, Ullrich A. Serine/threonine protein kinases PknF and PknG of Mycobacterium tuberculosis: characterization and localization [J]. Microbiology, 2001, 147(Pt 8): 2307-2314.

[32] Molle V, Soulat D, Jault JM, Grangeasse C, Cozzone AJ, Prost JF. Two FHA domains on an ABC transporter, Rv1747, mediate its phosphorylation by PknF, a Ser/Thr protein kinase from Mycobacterium tuberculosis [J]. FEMS Microbiol Lett, 2004, 234(2): 215-223.

[33] Deol P, Vohra R, Saini AK, Singh A, Chandra H, Chopra P, Das TK, Tyagi AK, Singh Y. Role of Mycobacterium tuberculosis Ser/Thr kinase PknF: implications in glucose transport and cell division [J]. J Bacteriol, 2005, 187(10): 3415-3420.

[34] Gopalaswamy R, Narayanan S, Jacobs WR Jr, Av-Gay Y. Mycobacterium smegmatis biofilm formation and sliding motility are affected by the serine/threonine protein kinase PknF [J]. FEMS Microbiol Lett, 2008, 278(1): 121-127.

[35] Singh A, Singh Y, Pine R, Shi L, Chandra R, Drlica K. Protein kinase I of Mycobacterium tuberculosis: cellular localization and expression during infection of macrophage-like cells [J]. Tuberculosis (Edinb), 2006, 86(1): 28-33.

[36] Gopalaswamy R, Narayanan PR, Narayanan S. Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv [J]. Protein Expr Purif, 2004, 36(1): 82-89.

[37] Gopalaswamy R, Narayanan S, Chen B, Jacobs WR, Av-Gay Y. The serine/threonine protein kinase PknI controls the growth of Mycobacterium tuberculosis upon infection [J]. FEMS Microbiol Lett, 2009, 295(1): 23-29.

[38] Arora G, Sajid A, Gupta M, Bhaduri A, Kumar P, Basu-Modak S, Singh Y. Understanding the role of PknJ in Mycobacterium tuberculosis: biochemical characterization and identification of novel substrate pyruvate kinase A [J]. PLoS One, 2010, 5(5): e10772.

[39] Jang J, Stella A, Boudou F, Levillain F, Darthuy E, Vaubourgeix J, Wang C, Bardou F, Puzo G, Gilleron M, Burlet-Schiltz O, Monsarrat B, Brodin P, Gicquel B, Neyrolles O. Functional characterization of the Mycobacterium tuberculosis serine/threonine kinase PknJ [J]. Microbiology, 2010, 156(Pt 6): 1619-1631.

[40] Walburger A, Koul A, Ferrari G, Nguyen L, Prescianotto-Baschong C, Huygen K, Klebl B, Thompson C, Bacher G, Pieters J. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages [J]. Science, 2004, 304(5678): 1800-1804.

[41] Cowley S, Ko M, Pick N, Chow R, Downing KJ, Gordhan BG, Betts JC, Mizrahi V, Smith DA, Stokes RW, Av-Gay Y. The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo [J]. Mol Microbiol, 2004, 52(6): 1691-1702.

[42] Nguyen L, Walburger A, Houben E, Koul A, Muller S, Morbitzer M, Klebl B, Ferrari G, Pieters J. Role of protein kinase G in growth and glutamine metabolism of Mycobacterium bovis BCG [J]. J Bacteriol, 2005, 187(16): 5852-5856.

[43] Wolff KA, Nguyen HT, Cartabuke RH, Singh A, Ogwang S, Nguyen L. Protein kinase G is required for intrinsic antibiotic resistance in mycobacteria [J]. Antimicrob Agents Chemother, 2009, 53(8): 3515-3519.

[44] Chaurasiya SK, Srivastava KK. Downregulation of protein kinase C-alpha enhances intracellular survival of Mycobacteria: role of PknG [J]. BMC Microbiol, 2009, 9: 271.

[45] Kumar P, Kumar D, Parikh A, Rananaware D, Gupta M, Singh Y, Nandicoori VK. The Mycobacterium tuberculosis protein kinase K modulates activation of transcription from the promoter of mycobacterial monooxygenase operon through phosphorylation of the transcriptional regulator VirS [J]. J Biol Chem, 2009, 284(17): 11090-11099.

[46] Singh A, Gupta R, Vishwakarma RA, Narayanan PR, Paramasivan CN, Ramanathan VD, Tyagi AK. Requirement of the mymA operon for appropriate cell wall ultrastructure and persistence of Mycobacterium tuberculosis in the spleens of guinea pigs [J]. J Bacteriol, 2005, 187(12): 4173-4186.

[47] Meena LS, Rajni. Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv [J]. FEBS J, 2010, 277(11): 2416-2427.

[48] Tian C, Jian-Ping X. Roles of PE_PGRS family in Mycobacterium tuberculosis pathogenesis and novel measures against tuberculosis [J]. Microb Pathog, 2010, 49(6): 311-314.

[49] Ehrt S, Schnappinger D. Mycobacterial survival strategies in the phagosome: defence against host stresses [J]. Cell Microbiol, 2009, 11(8): 1170-1178.