摘要
铜绿假单胞菌(绿脓杆菌,Pseudomonas aeruginosa,PA)是一种条件致病菌,其很多致病性都与毒性因子的分泌有关,其中吩嗪类复合物是很重要的毒性因子,包括:吩嗪-1-羧酸(phenazine-1-carboxylic acid,PCA)、绿脓菌素(pyocyanin)等。PCA等吩嗪类复合物在细胞内可作为电子载体,增加感染细胞内的氧化压力,使感染的目标细胞中毒死亡,绿脓菌素与囊肿性纤维化病人(CF)的肺部感染有关。PCA为绿脓菌素合成的中间体,而phzA1B1C1D1G1(phzA1)操纵子参与了PCA的合成过程。已有的研究报道表明次抑制浓度的抗生素可以作为信号分子调节致病基因的表达。本实验室前期研究表明:次抑制浓度(subinhibitory concentrations)的四环素可以调节phzA1操纵子的表达;在次抑制浓度四环素条件下,以发光杆菌的荧光素酶基因操纵子luxCDABE为报道基因,对含有phzA1操纵子报道载体的转座突变库进行了筛选。本实验对筛选出的其中三株突变体PAM0487、PAM0487R、PAM0207进行了研究。
在三株突变体PAM0487、PAM0487R、PAM0207中,phzA1操纵子的表达降低。通过随机PCR、基因测序及基因比对确定了突变点在染色体上的位置。发现其中的两株突变体PAM0487、PAM0487R的突变点均位于PA0487基因启动子区内,PA0487基因为假定钼元素的转运调节基因。PAM0207位于PA0207基因启动子区内。
通过测定绿脓菌素在野生型PAO1、PAM0487、PAM0487R中的生成量,发现在突变体PAM0487和PAM0487R中绿脓菌素的生成量降低,证实PA0487基因对phzA1操纵子在转录水平的正向调节作用表现在表型上。在不同浓度的四环素条件下,phzA1操纵子表达受四环素调节的现象在突变体PAM0487R中消失,说明PA0487是四环素对phzA1操纵子调节途径中的一员。由于铜绿假单胞菌中吩嗪类复合物的合成受群体感应系统(quorum-sensing,QS)的调控,本实验研究了PA0487基因与QS系统的关系,发现PA0487对群体感应系统相关基因的表达有调节作用。
将Gm~r-lacZ基因插入野生型PAO1的PA0207基因中间构建了突变体PAK0207,在突变体PAK0207中phzA1操纵子的表达无明显变化,表明对phzA1具有调节作用的基因并不是PA0207。
Pseudomonas aeruginosa is an opportunistic pathogen. A number of secretable virulence factors contribute to its pathogenicity, including phenazines. P. aeruginosa releases at least four phenazine compounds. They are 1-hydroxy-phenazine (1-OH-PHZ)、PCA、phenazine-1-carboxamide (PCN) and pyocyanin. Pyocyanin is an important phenazine compound, and it is suggested to be involved in lung infecion of cystic fibrosis patients. Quorum sensing network controls the production of phenazines.
A novel regulator of phzA1 operon in Pseudomoas aeruginosa had been identified by screening a transponson mutagenesis library in the presence of subinhibitory concentrations of tetracycline (Tet). Three mutants that exhibited changed expression of the phzA1 operon were obtained. The transposon insertion sites were determined by arbitrary PCR and subsequent PCR product sequencing. Two sites found to be at the promoter region of gene PA0487, and one at the promoter region of gene PA0207. PA0487 was required for the expression of phzA1 operon, and in the knockout mutant some of the genes involved in quorum sensing were down-regulated. The activation of phzA1 by subinhibitory concentrations of Tet was abolished in PAM0487, probably due to that PA0487 is involved in subinhibitory antibiotic regulation pathway. In conclusion, our results indicate that PA0487 is involved in regulating the expression of phzA1, possibly through the quorum sensing system.
引文
1.Duan,K.,et al.,Modulation of Pseudomonas aeruginosa gene expression by host microflora through interspecies communication.Mol Microbiol[J],2003.50(5):p.1477-91.
2.Leduc,D.,et al.,The Pseudomonas aeruginosa LasB metalloproteinase regulates the human urokinase-type plasminogen activator receptor through domain-specific endoproteolysis.Infect Immun[J],2007.75(8):p.3848-58.
3.Dietrich,L.E.,et al.,The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa.Mol Microbiol[J],2006.61(5):p.1308-21.
4.Hassett,D.J.,et al.,Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase:demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria.J Bacteriol[J],1993.175(23):p.7658-65.
5.Girard,G,et al.,Pip,a novel activator of phenazine biosynthesis in Pseudomonas chlororaphis PCL1391.J Bacteriol[J],2006.188(23):p.8283-93.
6.Carty,N.L.,et al.,PtxR modulates the expression of QS-controlled virulence factors in the Pseudomonas aeruginosa strain PAO1.Mol Microbiol[J],2006.61(3):p.782-94.
7.肖辉,张睢扬,铜绿假单胞菌与机体相互作用在呼吸道感染中的研究进展.Int J Respir[J],2006.26(10):p.765-770.
8.Sadikot,R.T.,et al.,Pathogen-host interactions in Pseudomonas aeruginosa pneumonia.Am J Respir Crit Care Med[J],2005.171(11):p.1209-23.
9.Ohlsen,K.,et al.,Effects of subinhibitory concentrations of antibiotics on alpha-toxin(hla)gene expression of methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates.Antimicrob Agents Chemother[J],1998.42(11):p.2817-23.
10.胡晓梅,饶贤才,黄建军,金晓琳,胡福泉,铜绿假单胞菌外毒素A表达载体的构建及可溶性表达.Med J Chin PLA[J],2004.29(2):p.156-158.
11.胡晓梅,胡福泉,铜绿假单胞菌外毒素A基因表达的研究进展.微生物学免疫学进展[J],2004.32(3):p.73-75.
12.杨本升,刘玉斌,苟仕金,动物微生物学[M],1995:p.523-531.
13.陈以旺,张咏梅,铜绿假单胞菌生物膜耐药性研究进展.海峡药学[J],2004.16(2):p.10-12.
14.Stewart,P.S.and J.W.Costerton,Antibiotic resistance of bacteria in biofilms.Lancet[J],2001.358(9276):p.135-8.
15.Brooun,A.,S.Liu,and K.Lewis,A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms.Antimicrob Agents Chemother[J],2000.44(3):p.640-6.
16.Spoering,A.L.and K.Lewis,Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials.J Bacteriol[J],2001.183(23):p.6746-51.
17.徐怀英,朱瑞良,铜绿假单胞菌致病作用及防治.黑龙江畜牧兽医[J],2000 11.
18.卢东升,杨学海,卢红光,防治绿脓杆菌感染研究进展.生物技术[J],1992.2(6):p.6-10.
19.Chadwick,D.,Whelan,J.,Secondary Metabolites:Their Function and Evolution.Ciba Foundation Symposium[J],1992.171.
20.Grace Y.,H.H.,Julian D.,The truth about antibiotics.Med.Microbiol[J],2006.296:p.163-170.
21.Herold,S.,et al.,Global expression of prophage genes in Escherichia coli O157:H7 strain EDL933 in response to norfloxacin.Antimicrob Agents Chemother[J],2005.49(3):p.931-44.
22.Matar,G.M.and E.Rahal,Inhibition of the transcription of the Escherichia coli O157:H7genes coding for shiga-like toxins and intimin,and its potential use in the treatment of human infection with the bacterium.Ann Trop Med Parasitol[J],2003.97(3):p.281-7.
23.Horii,T.,et al.,Effects of mupirocin at subinhibitory concentrations on flagella formation in Pseudomonas aeruginosa and Proteus mirabilis.J Antimicrob Chemother[J],2003.51(5):p.1175-9.
24.Linares,J.F.,et al.,Antibiotics as intermicrobial signaling agents instead of weapons.Proc Natl Acad Sci U S A[J],2006.103(51):p.19484-9.
25.Bader,M.W.,et al.,Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides.Mol Microbiol[J],2003.50(1):p.219-30.
26.Shaw,K.J.,et al.,Comparison of the changes in global gene expression of Escherichia coli induced by four bactericidal agents.J Mol Microbiol Biotechnol[J],2003.5(2):p.105-22.
27.Hastings,J.W.and E.P.Greenberg,Quorum sensing:the explanation of a curious phenomenon reveals a common characteristic of bacteria.J Bacteriol[J],1999.181(9):p.2667-8.
28.Salrnond,G.P.,et al.,The bacterial 'enigma':cracking the code of cell-cell communication.Mol Microbiol[J],1995.16(4):p.615-24.
29.Whitehead,N.A.,et al.,Quorum-sensing in Gram-negative bacteria.FEMS Microbiol Rev[J],2001.25(4):p.365-404.
30.Shiner,E.K.,K.P.Rumbaugh,and S.C.Williams,Inter-kingdom signaling:deciphering the language of acyl homoserine lactones.FEMS Microbiol Rev[J],2005.29(5):p.935-47.
31.Parsek,M.R.and E.P.Greenberg,Acyl-homoserine lactone quorum sensing in gram-negative bacteria:a signaling mechanism involved in associations with higher organisms.Proc Natl Acad Sci U S A[J],2000.97(16):p.8789-93.
32.徐凯进,李兰娟,邢卉春,细菌的群体感应系统.中华传染病杂志[J],2006.24(5):p.356-358.
33.郭嘉亮,陈卫民,细菌群体感应信号分子与抑制剂研究进展.Chinese Bulletin of Life Sciences[J],2007.19(2):p.224-231.
34.Fuqua,W.C.,S.C.Winans,and E.P.Greenberg,Quorum sensing in bacteria:the LuxR-LuxI family of cell density-responsive transcriptional regulators.J Bacteriol[J],1994.176(2):p.269-75.
35.Smith,R.S.and B.H.Iglewski,P.aeruginosa quorum-sensing systems and virulence.Curr Opin Microbiol[J],2003.6(1):p.56-60.
36.Diggle,S.P.,et al.,Advancing the quorum in Pseudomonas aeruginosa:MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression.J Bacteriol[J],2002.184(10):p.2576-86.
37.Xiao,G.,et al.,MvfR,a key Pseudomonas aeruginosa pathogenicity LTTR-class regulatory protein,has dual ligands.Mol Microbiol[J],2006.62(6):p.1689-99.
38.Chugani,S.A.,et al.,QscR,a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa.Proc Natl Acad Sci U S A[J],2001.98(5):p.2752-7.
39.Ledgham,F.,et al.,Interactions of the quorum sensing regulator QscR:interaction with itself and the other regulators of Pseudomonas aeruginosa LasR and RhlR.Mol Microbiol[J],2003.48(1):p.199-210.
40.Withers,H.,S.Swift,and P.Williams,Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria.Curr Opin Microbiol[J],2001.4(2):p. 186-93.
41.Thompson,L.S.,et al.,The alternative sigma factor RpoN regulates the quorum sensing gene rhlI in Pseudomonas aeruginosa.FEMS Microbiol Lett[J],2003.220(2):p.187-95.
42.Davey,M.E.,N.C.Caiazza,and G.A.O'Toole,Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAOI.J Bacteriol[J],2003.185(3):p.1027-36.v43.Evans,K.,et al.,Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa.J Bacteriol[J],1998.180(20):p.5443-7.
44.Deziel,E.,et al.,Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines(HAQs)reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication.Proc Natl Acad Sci U S A[J],2004.101(5):p.1339-44.
45.Garske,L.A.,et al.,Sub-inhibitory concentrations of cefiazidime and tobramycin reduce the quorum sensing signals of Pseudomonas aeruginosa.Pathology[J],2004.36(6):p.571-5.
46.Nalca,Y.,et al.,Quorum-sensing antagonistic activities of azithromycin in Pseudomonas aeruginosa PAO1:a global approach.Antimicrob Agents Chemother[J],2006.50(5):p.1680-8.
47.Tateda,K.,Y.Ishii,and K.Yamaguchi,[Quorum-sensing system of Pseudomonas aeruginosa-a novel target for antibacterial agentsl.Nippon Saikingaku Zasshi[J],2004.59(4):p.543-9.
48.Sofer,D.,et al.,'Subinhibitory'erythromycin represses production of Pseudomonas aeruginosa lectins,autoinducer and virulence factors.Chemotherapy[J],1999.45(5):p.335-41.
49.Dong,Y.H.,et al.,Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase.Nature[J],2001.411(6839):p.813-7.
50.Hentzer,M.and M.Givskov,Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections.J Clin Invest[J],2003.112(9):p.1300-7.
51.Goh,E.B.,et al.,Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics.Proc Natl Acad Sci U S A[J],2002.99(26):p.17025-30.
52.Hernandez,M.E.,A.Kappler,and D.K.Newman,Phenazines and other redox-active antibiotics promote microbial mineral reduction.Appl Environ Microbiol[J],2004.70(2):p.921-8.
53.Banin,E.,M.L.Vasil,and E.P.Greenberg,Iron and Pseudomonas aeruginosa biofilm formation.Proc Natl Acad Sci U S A[J],2005.102(31):p.11076-81.
54.Mazzola,M.,et al.,Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.Appl Environ Microbiol[J],1992.58(8):p.2616-24.
55.Wilson,R.,et al.,Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium.Infect Immun[J],1988.56(9):p.2515-7.
56.Wilson,R.,et al.,Pyocyanin and 1-hydroxyphenazine produced by Pseudomonas aeruginosa inhibit the beating of human respiratory cilia in vitro.J Clin Invest[J],1987.79(1):p.221-9.
57.Nakaike,S.,et al.,In vivo activity on murine tumors of a novel antitumor compound,N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]phenazine-6-carboxamide sodium salt(NC-190).Cancer Chemother Pharmacol[J],1989.23(3):p.135-9.
58.Haas,D.,C.Blumer,and C.Keel,Biocontrol ability of fluorescent pseudomonads genetically dissected:importance of positive feedback regulation.Curr Opin Biotechnol[J],2000.11(3):p.290-7.
59.罗鹏,许煜泉,张霞,陈峰,荧光假单胞菌株M18产PCA发酵条件研究.上海农业学报[J],2002.18(2):p.66-71.
60.金颖,胡洪波,张雪洪,许煜泉,假单胞菌产生的抗生素研究.Acta A gricult urae S hanghai[J],2005.21(3):p.106-109.
61.Seveno,N.A.,J.A.Morgan,and E.M.Wellington,Growth of Pseudomonas aureofaciens PGS12 and the Dynamics of HHL and Phenazine Production in Liquid Culture,on Nutrient Agar,and on Plant Roots.Microb Ecol[J],2001.41(4):p.314-324.
62.D.M,T.L.S.W.e.,Current concepts in the use of introduced bacteria for biobgical disease ontrol:mechanisms and an tifungal me-taborites.Plant Microbe Interactions[J],1996.1:p.187-235.
63.Whiteley,M.,K.M.Lee,and E.P.Greenberg,Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa.Proc Natl Acad Sci U S A[J],1999.96(24):p.13904-9.
64.van Rij,E.T.,et al.,Influence of environmental conditions on the production of phenazine-1-carboxamide by Pseudomonas chlororaphis PCL1391.Mol Plant Microbe Interact[J],2004.17(5):p.557-66.
65.Khan,S.R.,et al.,Activation of the phz operon of Pseudomonas fluorescens 2-79 requires the LuxR homolog PhzR,N-(3-OH-Hexanoyl)-L-homoserine lactone produced by the LuxI homolog PhzI,and a cis-acting phz box.J Bacteriol[J],2005.187(18):p.6517-27.
66.Lee,J.H.,Y.Lequette,and E.P.Greenberg,Activity of purified QscR,a Pseudomonas aeruginosa orphan quorum-sensing transcription factor.Mol Microbiol[J],2006.59(2):p.602-9.
67.Kim,E.J.,et al.,Expression of the quorum-sensing regulatory protein LasR is strongly affected by iron and oxygen concentrations in cultures of Pseudomonas aeruginosa irrespective of cell density.Microbiology[J],2005.151(Pt 4):p.1127-38.
68.Chancey,S.T.,D.W.Wood,and L.S.Pierson,3rd,Two-component transcriptional regulation of N-acyl-homoserine lactone production in Pseudomonas aureofaciens.Appl Environ Microbiol[J],1999.65(6):p.2294-9.
69.Chancey,S.T.,et al.,Survival of GacS/GacA mutants of the biological control bacterium Pseudomonas aureofaciens 30-84 in the wheat rhizosphere.Appl Environ Microbiol[J],2002.68(7):p.3308-14.
70.Whistler,C.A.and L.S.Pierson,3rd,Repression of phenazine antibiotic production in Pseudomonas aureofaciens strain 30-84 by RpeA.J Bacteriol[J],2003.185(13):p.3718-25.
71.Holloway,B.W.,V.Krishnapillai,and A.F.Morgan,Chromosomal genetics of Pseudomonas.Microbiol Rev[J],1979.43(1):p.73-102.
72.Brint,J.M.and D.E.Ohman,Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI,another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family.J Bacteriol[J],1995.177(24):p.7155-63.
73.Davies,D.G.,et al.,The involvement of cell-to-cell signals in the development of a bacterial biofilm.Science[J],1998.280(5361):p.295-8.
74.Hoang,T.T.,et al.,Integration-proficient plasmids for Pseudomonas aeruginosa:site-specific integration and use for engineering of reporter and expression strains.Plasmid[J],2000.43(1): p.59-72.
75.Ditta,G.,et al.,Broad host range DNA cloning system for gram-negative bacteria:construction of a gene bank of Rhizobium meliloti.Proc Natl Acad Sci U S A[J],1980.77(12):p.7347-51.
76.Essar,D.W.,et al.,Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa:interchangeability of the two anthranilate synthases and evolutionary implications.J Bacteriol[J],1990.172(2):p.884-900.
77.Friedman,L.and R.Kolter,Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix.J Bacteriol[J],2004.186(14):p.4457-65.
78.Herbert,S.,P.Barry,and R.P.Novick,Subinhibitory clindamycin differentially inhibits transcription of exoprotein genes in Staphylococcus aureus.Infect Immun[J],2001.69(5):p.2996-3003.