马尔尼菲青霉在d1hA基因的克隆及基因功能的初步研究
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摘要
马尔尼菲青霉菌是一类两相条件致病真菌,在25℃培养呈现腐生菌丝态,无致病性,37℃早现酵母态,有致病性。尽管它具有非常重要的医学研究价值以及独特的温度调控的两相转换机制,但对马尔尼菲青霉的研究由于基因组信息和有效操作方法的缺乏而受到局限。在我们的研究中,一个3936bp的新基因dlhA被成功克隆到。dlhA和在皮生芽孢菌中发现的起全局调控作用的基因drk1具有高度保守性。我们构建了RNA干扰表达盒,表达盒内具有目的基因编码序列430bp的反向重复序列并被gus基因隔开。整个干扰载体借助根癌农杆菌体系成功转化马尔尼菲青霉,并在一个可控的木糖诱导启动子xylP调控RNA干扰。研究发现dlhA基因沉默的转化子形态学发生了明显的变化,而且丧失了产孢能力。实验结果表明dlhA在不同温度下调节不同的信号途径,对于研究的极性生长,可能的两相转换机制以及作为药物靶点的筛选都具有重要的意义。
Penicillium marneffei is a thermally dimorphic opportunistic human pathogen with a saprophytic filamentous hyphal form at 25°C and a pathogenic unicellular yeast form at 37°C. Despite its medical importance and its unusual thermal dimorphic character, a large part of P.marneffei still remains unknown and relative molecular researches have been limited due to the lack of genomic information and effective genetic tools. We cloned a 3936 gene dlhA highly conserved to the histidine kinase gene drk1, which has the globe regulation in morphogenesis, cell wall composition, sporulation and expression of virulence factors in Blastomyces dermatitidis. One RNAi expression cassette was designed with inverted repeats of coding sequence of the targeted gene separated by a segment of gus~ gene. Constructs were transferred by Agrobacterium -mediated transformation and under the control of promoter xylP, RNAi can be regulated in a controllable and reversible manner. dlhA-silenced transformants exhibited severe morphological defects and incapability of conidiogenesis compared with their parent strains. These evidences imply that dlhA plays a role regulating the expression of downstream effectors, which are necessary exploring the propable mechanisms of correct polarized growth, dimorphic switch, and further research for drug design.
Penicillium marneffei is a thermally dimorphic opportunistic human pathogen with a saprophytic filamentous hyphal form at 25°C and a pathogenic unicellular yeast form at 37°C. Despite its medical importance and its unusual thermal dimorphic character, a large part of P.marneffei still remains unknown and relative molecular researches have been limited due to the lack of genomic information and effective genetic tools. We cloned a 3936 gene dlhA highly conserved to the histidine kinase gene drk1, which has the globe regulation in morphogenesis, cell wall composition, sporulation and expression of virulence factors in Blastomyces dermatitidis. One RNAi expression cassette was designed with inverted repeats of coding sequence of the targeted gene separated by a segment of gus~ gene. Constructs were transferred by Agrobacterium -mediated transformation and under the control of promoter xylP, RNAi can be regulated in a controllable and reversible manner. dlhA-silenced transformants exhibited severe morphological defects and incapability of conidiogenesis compared with their parent strains. These evidences imply that dlhA plays a role regulating the expression of downstream effectors, which are necessary exploring the propable mechanisms of correct polarized growth, dimorphic switch, and further research for drug design.
引文
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[36]Appleby JL, Parkinson JS, Bourret RB. Signal transduction via the multi-step phosphorelay: not necessarily a road less traveled[J]. Cell, 1996, 86:845-848.
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[38]Yamada-Okabe T, Mio T, Ono N, et al. Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida a!bicans[J]. J Bacteriol, 1999, 181:7243-7247.
[39]Alex LA, Borkovich KA, Simon MI. Hyphal development in Neurospora crassa:involvement of a two-component histidine kinase[J]. Proc Natl Acad Sci U S A,1996,93:3416-3421.
[40]Cui W, Beever RE, Parkes SL, et al. An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botryotinia fuckeliana (Botrytis cinerea) [J].Fungal Genet Biol,2002, 36:187-198.
[41]Virginia M, Appleyard CL, McPheat WL, et al. A novel 'two-component' protein containing histidine kinases and response regulator domains required for sporulation in Aspergillus nidulans[J]. Curr Genet, 2000, 37: 364-372.
[42]Pott GB, Miller TK, Bartlett JA, et al. The isolation of FOS-1, a gene encoding a putative two-component histidine kinase from Aspergillus fumigatus[J]. Fungal Genet Biol, 2000, 31:55-67.
[43]Nemecek JC, Wuthrich M, Klein BS. Global control of dimorphism and virulence in fungi[J]. Science, 2006, 312:583-588.
[44]Zadra I, ABT B, Parson W, et al. xylP promoter-based expression system and its use for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE[J]. Applied and Environmental Microbiology, 2000, 66:4810-4816.
[45]Todd RB, Greenhalgh JR, Hynes MJ, et al. TupA, the Penicillium marneffei Tup1p homologue, represses both yeast and spore development. Mol Microbiol, 2003,48:85-94.
[46] Jungnitz H, West NP, Walker MJ, et al. A second two-component regulatory system of Bordetella bronchiseptica required for bacterial resistance to oxidative stress,production of acid phosphatase, and in vivo persistence[J]. Infect Immun, 1998,66:4640-4650.
[47]Nierman WC, Pain A, Anderson MJ, et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigates[J]. Nature, 2005, 438(7071):l 151-1156.
[48]Wurgler-Murphy, SM, Saito H. Two-component signal transducers and MAP cascades[J]. Trends Biochem Sci, 1997,22:172-176.
[49]Tayior BL, Zhulin IB. PAS domains: internal sensors of oxygen, redox potential, and light[J]. Microbiol Mol Biol Rev, 1999, 63:479-506.
[50]Zhang Y, Lamm R, Pillonel C, et al. Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue[J]. Appl Environ Microbiol, 2002, 68:532-538.
[51]Beijersbergen. Agrobacterium mediated transformation of moulds, in particular those belonging to the genus Aspergillus[P]. USA: 6255115, 2001:
[52]Boyce KJ, Andrianopoulos A. A p21-activated kinase is required for conidial germination in Penicillium marneffei[J]. PLoS Pathog, 2007, 3(11):e162.
[53]Mosch HU, Roberts RL, Fink GR. Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae[J]. Proc Natl Acad Sci U S A, 1996, 93:5352-5356.
[54]Colombo S, Ma P, Cauwenberg L, et al. Involvement of distinct G-proteins, Gpa2 and Ras, in glucose and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae[J].EMBO J, 1998, 17:3326-3341.
[55]Zheng Y, Cerione R, Bender A. Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3[J]. J Biol Chem, 1994,269:2369-2372.
[56]Leberer E, Wu C, Leeuw T, et al. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase[J]. EMBO J, 1997, 16:83-97.
[57]Wu C, Whiteway M, Thomas DY, et al. Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae[J]. J Biol Chem, 1995,270:15984-15992.
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[62]Leberer E, Wu C, Leeuw T, et al. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase[J]. EMBO J, 1997, 16:83-97.
[63]Adams AE, Johnson DI, Longnecker RM, et al. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae[J]. J Cell Biol, 1990, 111:131-142.
[2]Di Salvo AF,Fickling AM,Ajello L.Infection causad by Penicillium marniffei description of first natuarai infection in man[J].Am J Cim Pathol,1973,60(2):259-263.
[3]邓卓霖.进行性播散性马尔尼菲青霉病[J].广西医学院学报,1984,1:124.
[4]Drouher E.Penicilliosis due to P.Marneffei:A new emerging system mycosis in AIDS patients travelling or living in Southeast Asia[J].J Mycol Med,1993:3(4):195
[5]Cooper CR,Jr.,McGinnis MR.Pathology of Penicillium marneffei:an emerging acquired immunodeficiency syndrome related pathogen[J].Arch Pathol Lab Med,1997,121(8):798-804.
[6]Andrianopoulos.Control of morphogenesis in the human fungal pathogen penicillium marneffei[J].Int J Med Microbiol,2002,292(526):331-347.
[7]廖晚珍.马尔尼菲青霉菌与马尔尼菲青菌病[J].江西医学检验,2001,19(5):295-297.
[8]Singh PN,Ranjana K,Singh YI,et al.Indigenous disseminated Penicillium marneffei infection in the state of Manipur,India:report of four autochthonous cases[J].J Clin Microbiol,1999,37(8):2699-2702.
[9]Supparatpinyo K,Khamwan C,Baosoung V,et al.Disseminated Penicillium marneffei infection in Southeast Asia[J].Lancet,1994,344(8915):110-113.
[10]马经野,刘新民.艾滋病并发马尔尼菲青霉病的真菌培养与鉴定[J].中国卫生检验杂志,2002,23(1):30-31.
[11]王莹,马韵.马尔尼菲青霉病[J].中国真菌学杂志,2007,2(4):240-242.
[12]Vanittanakom N,Cooper CR,Fisher MC,et al:Penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects[J].Clinical Microbiology Reviews,2006,19(1):95-110.
[13]Canovas D,Andrianopoulos A.The biology of the thermally dimorphic fungal pathogen Penicillium marneffei[J].New Insights in Medical Mycology, 2007:213-226.
[14]Borneman AR, Hynes MJ, Andrianopoulos A. A basic helix-loop-helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei[J]. Molecular Microbiology, 2002, 44(3):621 -631.
[15]Bomeman AR, Hynes MJ, Andrianopoulos A. An STE12 homolog from the asexual,dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant[J]. Genetics, 2001,157:1003-1014.
[16]Zuber S, Hynes MJ, Andrianopoulos A. G-protein signaling mediates asexual development at 25℃ but has no effect on yeast-like growth at 37℃ in the dimorphic fungus penicillium marneffei[J]. Eukaryot Cell, 2002, 1:440-447.
[17]Zuber S, Hynes MJ, Andrianopoulos A. The G-protein a-subunit GasC plays a major role in germination in the dimorphic fungus Penicillium marneffei[J]. Genetics, 2003,164:487-499.
[18]Boyce KJ, Hynes MJ, Andrianopoulos A. Control of morphogenesis and actin localization by the Penicillium marneffei RAC homolog[J]. J Cell Sci, 2003,116:1249-1260.
[19]Boyce KJ, Hynes MJ, Andrianopoulos A. The CDC42 Homolog of the dimorphic fungus Penicillium marneffei is required for correct cell polarization during growth but not development[J].J Bacteriol, 2001, 183(11):447-3457.
[20]Boyce KJ, Hynes MJ, Andrianopoulos A. The Ras and Rho GTPases genetically interact to co-ordinately regulate cell polarity during development in Penicillium marneffei[J]. Mol Microbiol, 2005, 55(5): 1487-1501.
[21]Mishra NC, Tatum EL. Non-mendelian inheritance of DNA-mediated inositol independence in Neurospora[J]. Proc Natl Acad Sci U S A, 1973, 70:3875-3879.
[22]Ruiz-Diez B. Strategies for the transformation of filamentous fungi[J]. J Appl Microbiol, 2002, 92:189-195.
[23]Miehielse CB, Hooykaas PJ, van den Hondel CA, et al. Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J]. CurrGenet, 2005,48:1-17.
[24]de Groot MJA, Bundock P, Hooykaas PJJ, et al. Agrobaeterium tumefaciens-mediated transformation of filamentous fungi[J]. Nat Biotechnol, 1998,16:839-842.
[25]Christie PJ. Agrobaeterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria[J]. J Bacteriol, 1997,179:3085-3094.
[26]Meister G, Tuschl T. Mechanisms of gene silencing by double stranded RNA[J].Nature, 2004, 431 (7006):343-349.
[27]Bosher JM, Labouesse M. RNA interference: genetic wand and genetic watchdog[J].Nature Cell Biology, 2000, 2:31-36.
[28]Liu H, Cottrell TR, Pierini LM, et al. RNA Interference in the pathogenic fungus Cryptococcus neoformans[J]. Genetics, 2001, 160(2):463-470.
[29]Mouyna I, Henry C, Doering TL, et al. Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus[J]. FEMS Microbiol Lett, 2004,237(2):317-324.
[30]Rappleye CA, Engle JT, Goldman WE. RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1 ,3)-glucan in virulence[J]. Mol Microbiol,2004,53(1):153-165.
[31]McDonald T, Brown D, Keller NP, et al. RNA silencing of mycotoxin production in Aspergillus and Fusarium species[J]. Mol Plant Microbe Interact, 2005, 18(6):539-545.
[32]Ninfa AJ, Magasanik B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli[J]. Proc Natl Acad Sci U S A, 1986; 83(16):5909-13.
[33]Wolanin PM, Thomason PA, Stock JB. Histidine protein kinases: key signal transducers outside the animal kindom[J]. Genome Biol, 2002, 3:REVIEWS3013
[34]Chang C, Stewart RC. The two-component system. Regulation of diverse signaling pathways in prokaryotes and eukaryotes[J]. Plant Physiol, 1998, 117:723-731.
[35]Grebe TW, Stock JB. The histidine protein kinase superfamily[J]. Adv Microb Physiol, 1999,41:139-227.
[36]Appleby JL, Parkinson JS, Bourret RB. Signal transduction via the multi-step phosphorelay: not necessarily a road less traveled[J]. Cell, 1996, 86:845-848.
[37]Posas F, Wurgler-Murphy SM, Maeda T, et al. Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor[J]. Cell, 1996, 86:865-875.
[38]Yamada-Okabe T, Mio T, Ono N, et al. Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida a!bicans[J]. J Bacteriol, 1999, 181:7243-7247.
[39]Alex LA, Borkovich KA, Simon MI. Hyphal development in Neurospora crassa:involvement of a two-component histidine kinase[J]. Proc Natl Acad Sci U S A,1996,93:3416-3421.
[40]Cui W, Beever RE, Parkes SL, et al. An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botryotinia fuckeliana (Botrytis cinerea) [J].Fungal Genet Biol,2002, 36:187-198.
[41]Virginia M, Appleyard CL, McPheat WL, et al. A novel 'two-component' protein containing histidine kinases and response regulator domains required for sporulation in Aspergillus nidulans[J]. Curr Genet, 2000, 37: 364-372.
[42]Pott GB, Miller TK, Bartlett JA, et al. The isolation of FOS-1, a gene encoding a putative two-component histidine kinase from Aspergillus fumigatus[J]. Fungal Genet Biol, 2000, 31:55-67.
[43]Nemecek JC, Wuthrich M, Klein BS. Global control of dimorphism and virulence in fungi[J]. Science, 2006, 312:583-588.
[44]Zadra I, ABT B, Parson W, et al. xylP promoter-based expression system and its use for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE[J]. Applied and Environmental Microbiology, 2000, 66:4810-4816.
[45]Todd RB, Greenhalgh JR, Hynes MJ, et al. TupA, the Penicillium marneffei Tup1p homologue, represses both yeast and spore development. Mol Microbiol, 2003,48:85-94.
[46] Jungnitz H, West NP, Walker MJ, et al. A second two-component regulatory system of Bordetella bronchiseptica required for bacterial resistance to oxidative stress,production of acid phosphatase, and in vivo persistence[J]. Infect Immun, 1998,66:4640-4650.
[47]Nierman WC, Pain A, Anderson MJ, et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigates[J]. Nature, 2005, 438(7071):l 151-1156.
[48]Wurgler-Murphy, SM, Saito H. Two-component signal transducers and MAP cascades[J]. Trends Biochem Sci, 1997,22:172-176.
[49]Tayior BL, Zhulin IB. PAS domains: internal sensors of oxygen, redox potential, and light[J]. Microbiol Mol Biol Rev, 1999, 63:479-506.
[50]Zhang Y, Lamm R, Pillonel C, et al. Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue[J]. Appl Environ Microbiol, 2002, 68:532-538.
[51]Beijersbergen. Agrobacterium mediated transformation of moulds, in particular those belonging to the genus Aspergillus[P]. USA: 6255115, 2001:
[52]Boyce KJ, Andrianopoulos A. A p21-activated kinase is required for conidial germination in Penicillium marneffei[J]. PLoS Pathog, 2007, 3(11):e162.
[53]Mosch HU, Roberts RL, Fink GR. Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae[J]. Proc Natl Acad Sci U S A, 1996, 93:5352-5356.
[54]Colombo S, Ma P, Cauwenberg L, et al. Involvement of distinct G-proteins, Gpa2 and Ras, in glucose and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae[J].EMBO J, 1998, 17:3326-3341.
[55]Zheng Y, Cerione R, Bender A. Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3[J]. J Biol Chem, 1994,269:2369-2372.
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