Ⅲ型分泌系统抑制剂对铜绿假单胞菌PAO1毒性因子的影响

【目的】进一步研究Ⅲ型分泌系统(Type Ⅲ secretion system,TTSS)抑制剂对条件致病菌Pseudomonas aeruginosa PAO1的TTSS相关蛋白、鞭毛和纤毛等主要毒性因子的影响,评估TTSS抑制剂的防治效果及潜在风险。【方法】构建TTSS效应蛋白合成基因exo Y和exo T转录报告质粒p AT-exo Y、p AT-exo T,并将其转入菌株PAO1中。菌株PAO1(p AT-exo Y)、PAO1(p AT-exo T)与TTSS抑制剂共同培养后,检测exo Y和exo T的表达。通过SDS-PAGE检测TTSS抑制剂对鞭毛结构蛋白Fli C的影响。将PAO1单菌落穿刺接种于含有TTSS抑制剂的1%琼脂糖平板,观察细菌纤毛介导的蹭行运动(Twitching motility)。【结果】转录报告实验结果表明4个TTSS抑制剂可显著抑制exo Y和exo T的转录;化合物TS52、TS53和TS94虽不影响胞内TTSS针状顶端结构蛋白Pcr V的产量,但可抑制Pcr V蛋白的胞外运输。化合物TS53可降低鞭毛结构蛋白Fli C的产生。另外,化合物TS52、TS53和TS88可降低菌株PAO1的蹭行运动能力,但TS94可提高菌株PAO1的这种运动能力。【结论】TTSS抑制剂除通过抑制TTSS表达外,还可能通过影响其它毒性因子如鞭毛的合成、IV型分泌系统介导的蹭行运动等方式影响菌株PAO1致病性。     

肉桂酸衍生物对铜绿假单胞菌PAO1 III型分泌系统的影响

【目的】铜绿假单胞菌是引起医院获得性感染最常见的条件致病菌,而III型分泌系统(Type III secretion system,TTSS)是其致病的主要因子之一。本文从合成的21个肉桂酸衍生物中筛选影响TTSS效应子(Effector)产生的化合物,并初步研究其作用机制。【方法】将TTSS效应子合成基因exoS的转录报告质粒pAT-exoS转入菌株PAO1中,获得PAO1(pAT-exoS)。待筛选的化合物与PAO1(pAT-exoS)菌株共培养6 h后,检测exoS基因的表达,从中筛选影响exoS基因表达的化合物。【结果】筛选结果表明：21个化合物中,3个化合物抑制exoS基因表达,2个化合物则促进exoS基因表达。此外,化合物TS128、TS143和TS160对菌株生长有明显的抑制作用。Western blot实验进一步证实筛选得到的化合物TS108、TS128和TS165可抑制ExoS的产生;化合物TS139和TS143则促进ExoS的产生。为进一步研究抑制剂的作用机理,过量表达TTSS主要的调控因子exsA基因可部分消除抑制剂TS108和TS165的抑制效果;而rsmZ rsmY双基因突变体PAO6421中添加抑制剂TS108和TS165并不能显著抑制exoS基因的表达,同样,抑制剂TS108和TS165也不影响受Gac/Rsm信号传导系统调控的群体感应信号分子的产生。【结论】抑制剂TS108和TS165的作用机制可能主要是影响esxA基因,从而影响exoS基因表达及蛋白产量。     

肉桂酸衍生物对铜绿假单胞菌PAO1 Ⅲ型分泌系统的影响

【目的】铜绿假单胞菌是引起医院获得性感染最常见的条件致病菌,而Ⅲ型分泌系统(Type Ⅲ secretion system,TTSS)是其致病的主要因子之一。本文从合成的21个肉桂酸衍生物中筛选影响TTSS效应子(Effector)产生的化合物,并初步研究其作用机制。【方法】将TTSS效应子合成基因exoS的转录报告质粒pAT-exoS转入菌株PAO1中,获得PAO1(pAT-exoS)。待筛选的化合物与PAO1(pAT-exoS)菌株共培养6 h后,检测exoS基因的表达,从中筛选影响exoS基因表达的化合物。【结果】筛选结果表明:21个化合物中,3个化合物抑制exoS基因表达,2个化合物则促进exoS基因表达。此外,化合物TS128、TS143和TS160对菌株生长有明显的抑制作用。Western blot实验进一步证实筛选得到的化合物TS108、TS128和TS165可抑制ExoS的产生;化合物TS139和TS143则促进ExoS的产生。为进一步研究抑制剂的作用机理,过量表达TTSS主要的调控因子exsA基因可部分消除抑制剂TS108和TS165的抑制效果;而rsmZ rsmY双基因突变体PAO6421中添加抑制剂TS108和TS165并不能显著抑制exoS基因的表达,同样,抑制剂TS108和TS165也不影响受Gac/Rsm信号传导系统调控的群体感应信号分子的产生。【结论】抑制剂TS108和TS165的作用机制可能主要是影响esxA基因,从而影响exoS基因表达及蛋白产量。     

铜绿假单胞菌中III型分泌系统受Rhl和PQS群体感应系统调节

摘要：【目的】研究铜绿假单胞菌中群体感应系统（Quorum sensing, QS）与III型分泌系统（Type III secretion system, T3SS）的关系。【方法】通过基因敲除的方法破坏铜绿假单胞菌QS系统相关基因,将T3SS相关基因exoS、exoY、exoT、exsD-pscA-L启动子-报道子luxCDABE融合体整合到野生型菌株及QS系统突变菌株的染色体组上,通过检测启动子活性,比较这些基因在不同菌株中的表达情况。【结果】研究结果表明,T3SS中的exoS与exoT在pqsR基因突变体中的表达有明显的增强,Rhl系统对这四种基因的表达具有抑制作用,而Las系统存在与否对T3SS基本没有影响。【结论】铜绿假单胞菌中的Rhl系统和奎诺酮信号（Pseudomonas Quinolone Signal, PQS）系统对T3SS相关基因的表达具有重要的调节作用。     

铜绿假单胞菌pfm基因对三型分泌系统效应蛋白的影响

[目的]检测铜绿假单胞菌基因pfm对三型分泌系统效应蛋白的影响.[方法]构建pfm基因互补菌株pfmC.提取野生株PAO1、敲除株Δpfm和互补株pfmC的RNA,利用Real-time PCR从转录水平检测效应蛋白ExoS、ExoT和ExoY转录水平的变化.以ExoS为代表,检测细胞内和分泌到细胞外效应蛋白的含量.收集铜绿假单胞菌PAO1、Δpfm和pfmC菌体内和分泌到细胞外的总蛋白,利用ExoS多克隆抗体进行Western杂交,特异检测ExoS的蛋白水平.[结果]与野生型相比,Δpfm中exoS、exoT和exoY转录水平明显降低,而pfmC中这3个蛋白的转录水平得到回补.Δpfm菌体内和分泌到细胞外的ExoS量均明显低于野生株PAO1,pfmC细胞内和细胞外分泌的ExoS蛋白量均得到恢复.[结论]铜绿假单胞菌基因pfm会影响三型分泌系统效应蛋白的水平.     

铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA0575对表型的影响

【背景】铜绿假单胞菌PAO1中存在与环鸟苷二磷酸(cyclic-di-guanosine monophosphate,c-di-GMP)代谢相关基因PA0575。【目的】探讨铜绿假单胞菌PAO1中环鸟苷二磷酸代谢相关基因PA0575对运动能力及生物膜的影响。【方法】通过PCR对菌株遗传背景进行确认;利用刚果红结合实验及电转PcdrA-gfp质粒间接测量胞内c-di-GMP水平;利用泳动性(swimming)、蜂群泳动(swarming)、蹭行运动(twiching)和生物膜定量实验对细菌进行表型分析,并在运动培养基中添加抗生素研究其对运动能力的影响;针对PA0575基因进行融合蛋白表达载体的构建,并对蛋白进行原核诱导表达。【结果】3株突变体菌株的转座子插入突变位点不一致,胞内c-di-GMP水平检测结果显示,PA0575-1菌株的c-di-GMP含量高于野生型PAO1菌株(P0.05),PA0575-2、PA0575-3菌株胞内c-di-GMP水平与野生型PAO1菌株无差异(P0.05)。运动能力检测实验中,与野生型PAO1菌株相比,PA0575-1菌株泳动性增强(P0.05);PA0575-2、PA0575-3菌株的泳动性、蜂群运动均增强(P0.05);该基因不同位点的突变均导致氯霉素对菌株的运动能力产生抑制作用。生物膜定量结果显示,与野生型PAO1菌株相比,细菌培养18 h后PA0575-1的生物膜含量降低(P0.05),PA0575-2、PA0575-3菌株的生物膜含量升高。最后成功构建了PA0575基因不同结构域的8个表达载体,并获得了异源表达蛋白。【结论】PA0575基因降低铜绿假单胞菌胞内c-di-GMP的水平,影响表型的同时也抑制了氯霉素抗性基因的表达。以上研究为PA0575基因对表型的影响奠定了基础。     

铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA2072的生物学分析

【背景】铜绿假单胞菌为革兰氏阴性杆菌,是医院感染的常见条件致病菌之一。广泛存在于细菌中的第二信使分子环鸟苷二磷酸(cyclic-di-guanosine monophosphate,c-di-GMP)对细菌生理生化功能具有重要的调节作用。铜绿假单胞菌PAO1中存在参与c-di-GMP代谢的基因PA2072。【目的】探讨铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA2072的生物学功能。【方法】运用PCR及分子克隆技术构建PA2072基因及各结构域的自杀载体,运用基因敲除方法获取PA2072基因的3个突变株;利用泳动性(swimming)、蜂群运动(swarming)、蹭行运动(twitching)和生物膜定量实验对细菌进行初步的表型分析,进一步通过刚果红染色法对菌株进行分析。【结果】成功构建PA2072基因敲除突变菌株及回补菌株;生物膜定量结果发现基因PA2072的敲除会影响细菌生物膜的形成,PA2072蛋白的不同结构域对生物膜的合成也起到了重要作用;细菌运动能力检测中发现PA2072相关基因的敲除对细菌运动能力也有一定影响。刚果红平板检测结果显示,与野生型PAO1菌株相比,P...     

苏云金芽胞杆菌bkd基因簇的转录调控

【目的】通过分析苏云金芽胞杆菌bkd基因簇的转录调控和bkdR突变体的表型特征,明确bkdR所在基因簇的转录调控机制和对Cry蛋白产量的影响。【方法】通过生物信息学方法分析bkdR所在基因簇的结构,RT-PCR分析基因簇的转录单元,采用同源重组技术敲除苏云金芽胞杆菌HD73菌株的bkdR基因,利用启动子融合lacZ的方法分析启动子的转录活性。利用总蛋白定量确定Cry1Ac蛋白产量。【结果】bkd基因簇由8个基因组成,其中ptb-bkdB7个基因组成1个转录单元。ptb基因的启动子转录活性在sigL和bkdR突变体中均明显降低。bkdR基因的缺失对菌体生长、芽胞形成率和Cry1Ac蛋白产量无影响,但使运动能力减弱。【结论】bkd操纵子受Sigma 54控制,并由BkdR激活,bkdR基因的缺失对Cry蛋白产量无影响,对菌株的运动能力有影响。     

单增李斯特菌二硫键形成蛋白DsbG介导酸耐受及鞭毛运动性调控北大核心

【目的】本研究旨在通过构建单增李斯特菌(Listeria monocytogenes)二硫键形成蛋白编码基因dsbG缺失株和回补株,探究该蛋白在酸耐受和鞭毛介导的运动性中发挥的生物学功能。【方法】利用同源重组方法构建单增李斯特菌dsbG缺失株和回补株,比较野生株和突变株在不同pH梯度培养基条件下的生长速率和存活率;通过实时荧光定量PCR方法,比较致死酸应激条件下野生株和缺失株酸耐受基因转录水平。通过半固体培养基、荧光定量PCR方法和鞭毛负染色透射电镜观察,比较野生株和突变株的运动能力、鞭毛相关基因转录水平变化和鞭毛形态差异。【结果】与野生株相比,dsbG缺失株的生长能力和速率差异不显著;在pH 3.5(盐酸和柠檬酸)培养条件下,存活率显著降低;精氨酸合成途径基因argD和argF转录水平分别下调2.4和3.7倍。同时,dsbG缺失株的运动能力减弱,且鞭毛形成相关的flaA、flgB和flgD等基因转录水平显著下调(分别为29.7、6.7和6.9倍),鞭毛形成能力减弱。【结论】本研究首次证实了单增李斯特菌二硫键形成蛋白DsbG能感应低pH应激,并形成耐受;证实了DsbG通过调控鞭毛相关基因的转录进而影响细菌的鞭毛形成和运动性。本研究有助于深入了解二硫键形成蛋白家族介导单增李斯特菌环境适应的分子机制,为食源性致病菌的防控提供理论基础。     

青枯菌Rsc1285参与III型分泌系统及致病力的调控

摘要:【目的】研究青枯菌Rsc1285参与调控其III型分泌系统(Type III secretion system,T3SS)及致病力的途径。【方法】通过基因敲除、基因互补等研究Rsc1285对T3SS基因表达和致病力的影响。【结果】青枯菌rsc1285基因缺失突变体对寄主西红柿植株的致病力明显减弱,其hrpB、T3SS等基因表达水平较野生型明显降低,但hrpG、prhG的表达不受影响。【结论】青枯菌通过一个全新的途径利用Rsc1285调控hrpB及T3SS的转录表达并决定其致病力。     

FliC,a Flagellin Protein,Is Essential for the Growth and Virulence of Fish Pathogen Edwardsiella tarda

Edwardsiella tarda is a flagellated Gram-negative bacterium which causes edwardsiellosis in fish. FliC, as a flagellar filament structural protein, is hypothesized to be involved in the pathogenesis of infection. In this study, a fliC in-frame deletion mutant of a virulent isolate of E. tarda was constructed through double crossover allelic exchange by means of the suicide vector pRE112, and its virulence-associated phenotypes and pathogenicity were tested. It was found that the deletion of fliC significantly decreased the diameter of flagella filaments. In addition, the mutant showed reduced pathogenicity to fish by increasing the LD₅₀ value for 100-fold compared to the wild-type strain, as well as showed impaired bacterial growth, reduced motility, decreased biofilm formation and reduced levels of virulence-associated protein secretion involved in the type III secretion system (TTSS). The phenotypic characteristics of the fliC deletion mutant uncovered in this investigation suggest that fliC plays an essential role in normal flagellum function, bacterial growth, protein secretion by TTSS and bacterial virulence.     

Novel proteins that modulate type IV pilus retraction dynamics in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses type IV pili to colonize various materials and for surface-associated twitching motility. We previously identified five phylogenetically distinct alleles of pilA in P. aeruginosa, four of which occur in genetic cassettes with specific accessory genes (J. V. Kus, E. Tullis, D. G. Cvitkovitch, and L. L. Burrows, Microbiology 150:1315-1326, 2004). Each of the five pilin alleles, with and without its associated pilin accessory gene, was used to complement a group II PAO1 pilA mutant. Expression of group I or IV pilA genes restored twitching motility to the same extent as the PAO1 group II pilin. In contrast, poor twitching resulted from complementation with group III or group V pilA genes but increased significantly when the cognate tfpY or tfpZ accessory genes were cointroduced. The enhanced motility was linked to an increase in recoverable surface pili and not to alterations in total pilin pools. Expression of the group III or V pilins in a PAO1 pilA-pilT double mutant yielded large amounts of surface pili, regardless of the presence of the accessory genes. Therefore, poor piliation in the absence of the TfpY and TfpZ accessory proteins results from a net increase in PilT-mediated retraction. Similar phenotypes were observed for tfpY single and tfpY-pilT double knockout mutants of group III strain PA14. A PilA_V-TfpY chimera produced few surface pili, showing that the accessory proteins are specific for their cognate pilin. The genetic linkage between specific pilin and accessory genes may be evolutionarily conserved because the accessory proteins increase pilus expression on the cell surface, thereby enhancing function.     

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

Biofilm formation by Pseudomonas aeruginosa wild type,flagella and type IV pili mutants

Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.     

Evolutionary and functional diversity of the Pseudomonas type IVa pilin island

In Pseudomonas aeruginosa, most proteins involved in type IVa pilus (T4aP) biogenesis are highly conserved except for the major pilin PilA and the minor pilins involved in pilus assembly. Here we show that each of the five major pilin alleles is associated with a specific set of minor pilins, and unrelated strains with the same major pilin type have identical minor pilin genes. The sequences of the minor pilin genes of strains with group III and V pilins are identical, suggesting that these groups diverged recently through further evolution of the major pilin cluster. Both gene clusters are localized on a single ‘pilin island’ containing putative tRNA recombinational hotspots, and a similar organization of pilin genes was identified in other Pseudomonas species. To address the biological significance of group‐specific differences, cross‐complementation studies using group II (PAO1) and group III (PA14) minor pilins were performed. Heterologous minor pilins complemented twitching motility to various extents except in the case of PilX, which was non‐functional in non‐native backgrounds. A recombinant PA14 strain expressing the PAO1 minor pilins regained motility only upon co‐introduction of the PA14 pilX gene. Comparison of PilX and PilQ secretin sequences from group II, III and V genomes revealed discrete regions of sequence that co‐varied between groups. Our data suggest that changes in PilX sequence have led to compensatory changes in the PilQ secretin monomer such that heterologous PilX proteins are no longer able to promote opening of the secretin to allow pili to appear on the cell surface.