铜绿假单胞菌铁摄取与生物被膜形成研究进展

生物被膜是单细胞微生物通过其分泌的胞外多聚基质粘附于介质表面并将其自身包绕其中而成的膜样微生物细胞聚集物。生物被膜的形成使细菌具有更强的适应外界环境的能力,也是导致微生物产生耐药性及慢性感染性疾病难以治疗的重要原因之一。铜绿假单胞菌在肺部的定殖是肺囊性纤维化病患者发病和死亡主要原因,其造成的感染通常与形成抗生素抗性极强的生物被膜有关。铜绿假单胞菌生物被膜的形成受控于多种复杂的细菌调控体系之下,包括群体感应系统及参与调节胞外多聚基质合成的双组分调控系统等。此外,为了利用低浓度的环境铁来维持生存并完成各种生理功能,铜绿假单胞菌进化出了一系列铁摄取系统,这些系统对其毒力因子的释放和生物被膜的形成又起着重要的调控作用。本文主要对铜绿假单胞菌生物被膜的形成与调控机制及其铁摄取系统进行了综述,为进一步了解及清除铜绿假单胞菌引发的问题提供途径与思路。     

假单胞菌生物被膜核心胞外多糖生物合成系统研究进展

胞外多糖是假单胞菌生物被膜的重要组成部分,能增强菌体对外界环境、抗菌剂和宿主防御的耐受性.假单胞菌能产生3种与生物被膜形成密切相关的核心胞外多糖:褐藻胶、Psl和Pel,它们在细菌细胞中的合成和转运分别依赖对应的褐藻胶、Psl和Pel生物合成系统.因此,本综述系统全面地总结了假单胞菌3种胞外多糖生物合成系统结构生物学的...     

铜绿假单胞菌全局调控因子RsmA的缺失影响2个吩嗪基因簇的表达

[目的]为了研究铜绿假单胞菌全局调控因子RsmA对两个吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制.[方法]采用基因缺失和抗性基因(gentamycin resistance cassette,aacC1)插入相结合的策略构建了rsmA基因缺失突变株PA-RG ;通过构建互补表达载体和过表达载体,进一步确认RsmA对绿脓菌素的调控作用 ;采用电转化方法将构建的翻译融合表达载体pMEZ1(phz1'-'lacZ)和pMEZ2(phz2'-'lacZ)分别导入铜绿假单胞菌突变株PA-RG和野生株PAO1,采用Miller法测定融合β-半乳糖苷酶活性.[结果]在GA培养基中,互补分析和过表达分析表明,RsmA抑制绿脓菌素的合成.此外,pMEZ1在突变株PA-RG中的表达增强,为野生株的2-3倍 ;而pMEZ2在突变株PA-RG中的表达降低,野生株是突变株的2倍.[结论]由此初步判定,铜绿假单胞菌全局调控因子RsmA对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上RsmA负调控phz1,正调控phz2.     

铜绿假单胞菌铁摄取调节子Fur诱导表达突变株构建及表型分析

铁摄取调节子 (Ferric uptake regulator,Fur) 是细菌控制细胞内铁平衡的一类重要的调节子。铜绿假单胞菌Pseudomonas aeruginosa的fur为必需基因,不能直接敲除。文中通过构建诱导型缺失突变株Δfur/attB::PBAD-fur,来研究该基因对铜绿假单胞菌的生长、生物被膜形成、运动能力和抗氧应激能力等方面的影响。结果表明,当Fur低表达时,铜绿假单胞菌在高铁和低铁环境中出现了生长阻滞的现象;低表达Fur的铜绿假单胞菌抵抗H2O2的能力降低,形成生物被膜的能力减弱,游动、颤动 (Twitching) 和丛集 (Swarming) 运动能力也出现了减弱的现象。Fur的表达直接影响铜绿假单胞菌荧光嗜铁素的产量。在铜绿假单胞菌体内表达来自格瑞菲斯瓦尔德磁螺菌Fur超级家族中的蛋白,可以部分恢复铜绿假单胞菌荧光嗜铁素的产量。由此说明,Fur对铜绿假单胞菌的生长、生物被膜形成、抗氧应激能力和运动能力方面都起着至关重要的作用。本研究为铜绿假单胞菌的防治提供理论指导。     

铜绿假单胞菌生物被膜对巨噬细胞的免疫逃逸作用及机制

目的探讨铜绿假单胞菌生物被膜对巨噬细胞的免疫逃逸作用以及相关机制。方法用PMA刺激THP-1细胞获得巨噬细胞模型。用6孔板建膜法获得铜绿假单胞菌生物被膜菌。分别用铜绿假单胞菌的生物被膜菌和浮游菌感染巨噬细胞,观察巨噬细胞形态的变化,并检测巨噬细胞的细胞毒性和吞噬功能的变化。进一步用ELISA试剂盒检测感染的巨噬细胞培养上清中炎症细胞因子IL-1β的变化。结果成功构建巨噬细胞模型和铜绿假单胞菌生物被膜菌模型。与感染了浮游菌的细胞相比,感染了生物被膜菌的巨噬细胞形态变化小,释放的LDH降低,吞噬功能减弱,IL-1β的表达量减少。结论铜绿假单胞菌生物被膜菌可以逃逸巨噬细胞的免疫防御作用,其机制可能与降低巨噬细胞的炎症反应有关。     

铜绿假单胞菌群体感应信号分子PQS的功能多样性研究进展

铜绿假单胞菌(Pseudomonas aeruginosa)是一种革兰氏阴性条件致病菌,可对免疫功能低下或损伤的患者造成持续性感染。铜绿假单胞菌能成功感染离不开其自身产生的毒力因子,而这些毒力因子大多数都受群体感应系统(quorum sensing,QS)调控。铜绿假单胞菌有4个QS系统,分别为las系统、rhl系统、pqs系统和iqs系统。2-庚基-3-羟基-4-喹诺酮(Pseudomonas quinolone signal,PQS)作为铜绿假单胞菌pqs系统的信号分子,不仅能够调控许多毒力因子的表达,也能够影响一些微生物和宿主的多种生理过程。本文总结了PQS多种生物学功能,如介导QS系统、调控生物被膜形成、介导外膜囊泡产生及铁摄取、调节宿主免疫活性、介导细胞毒性作用,以及提供种群保护等。本文旨在突出铜绿假单胞菌PQS的功能多样性,并为PQS新功能研究和抗菌药物的研发提供指导。     

铜绿假单胞菌生物被膜组成及其受群体感应系统和c-di-GMP调控的研究进展

作为人类条件性感染的前三大病原菌之一的铜绿假单胞菌,是一种革兰氏阴性细菌,对免疫功能低下和囊性纤维化患者可以造成严重和持续性感染。造成这种持续感染的原因主要是由于细菌接收外界信号后,在自身调控网络的协同作用下,会依附于固体表面,并产生胞外多糖、基质蛋白和胞外DNA等大分子物质形成高度结构化的膜状复合物将自身包裹形成生物被膜群体结构。生物被膜可以有效帮助细菌定殖、提高细菌对抗菌物质和宿主免疫反应的抵抗能力、促进群落细菌的细胞-细胞之间的信号交流等,是临床治疗中病原菌慢性感染和反复感染最重要的原因之一。本篇综述重点介绍了铜绿假单胞菌生物被膜的各组成成分及其在生物被膜形成中的重要功能,并进一步阐述了群体感应系统(las、rhl、pqs与iqs)和c-di-GMP对铜绿假单胞菌生物被膜形成的调控作用。通过本篇综述可以更清晰地了解细菌生物被膜形成和调控的过程,为开发新的治疗生物被膜感染策略提供帮助。     

乳酸锌和氟化亚锡对铜绿假单胞菌、鲍曼不动杆菌和变异链球菌生物被膜的抑制作用

乳酸锌(Zn lactate·3H_2O)和氟化亚锡(SnF_2)常作为牙膏中的活性物质添加剂用来预防龋齿及口腔生物被膜的形成。文中评估了Zn lactate·3H_2O和SnF_2对铜绿假单胞菌、鲍曼不动杆菌和变异链球菌生物被膜的作用。对铜绿假单胞菌PAO1生物被膜的抑制实验证实乳酸锌和氟化亚锡都具有抑制其生物被膜的功能,联用效果尤佳。乳酸锌通过干扰胞外多糖基质网的形成起作用,而氟化亚锡则可以明显降低生物被膜的生物量。更为重要的是,工作浓度的两种化合物联用几乎可以完全抑制3种实验菌株生物被膜的形成。     

多重耐药铜绿假单胞菌群体感应系统与主动外排基因表达的研究

目的研究临床多重耐药铜绿假单胞菌群体感应(QS)系统与主动外排泵MexAB-OprM系统基因表达水平与抗生素耐药关系。方法收集苏州市立医院和上海市江湾医院2011年2月至6月间临床标本中分离的铜绿假单胞菌,定量分析细菌生物被膜形成能力;MIC法检测细菌抗生素耐药性,用多重聚合酶链反应(PCR)扩增群体感应系统lasI、lasR及主动外排泵系统mexA基因,实时定量逆转录RT-PCR检测lasI、lasR和mexA基因的相对表达量。结果临床样本分离出84株铜绿假单胞菌,其中产生物被膜菌58株,占比69%;多重耐药菌共24株,占比28.6%;多重耐药菌株中产生物被膜有11株,占45.8%;多重耐药菌中mexA基因表达上调有18株,占75%;lasI基因表达上调有8株,占33.3%。结论多重耐药菌株的生物被膜形成率显著低于非多重耐药组,多重耐药铜绿假单胞菌的主动外排泵MexAB-OprM系统基因表达出现显著上调,生物被膜菌的lasI基因表达显著上调而lasR基因的表达无明显变化。     

铜绿假单胞菌群体感应抑制剂研究进展

铜绿假单胞菌是一种常见的机会致病菌,可在人群中引起严重的急性和慢性感染,是病人在医院期间发生感染的第三大致病菌。铜绿假单胞菌多种毒力因子的分泌以及生物被膜的形成都是受一种被称为群体感应(Quorum Sensing,QS)的胞间信号传导系统调控的。QS使细菌能够根据细胞密度变化进行基因表达的调控。通过抑制QS来治疗铜绿假单胞菌感染是一个很有前景的发展方向。本文将就近年来铜绿假单胞菌群体感应抑制剂的研究进展进行综述。     

Expression of the Pho regulon negatively regulates biofilm formation by Pseudomonas aureofaciens PA147-2

We report the isolation of insertional mutations to the pstC and pstA genes of the phosphate-specific transport (pst) operon that results in loss of biofilm formation by Pseudomonas aureofaciens PA147-2. Consistent with the known roles of the Pst system in Escherichia coli and Pseudomonas aeruginosa, both P. aureofaciens pst mutants were demonstrated to have defects in inorganic phosphate (P(i)) transport and repression of Pho regulon expression. Subsequently, biofilm formation by the wild type was shown to require a threshold concentration of extracellular P(i). The two-component regulatory pair PhoR/PhoB is responsible for upregulation of Pho regulon expression in response to P(i)-limiting environments. By generating phoR mutants that were unable to express the Pho regulon, we were able to restore biofilm formation by P. aureofaciens in P(i)-limiting conditions. This result suggests that gene(s) within the Pho regulon act to regulate biofilm formation negatively in low-P(i) environments, and that phoR mutations uncouple PA147-2 from such regulatory constraints. Furthermore, the inability of pst mutants to repress Pho regulon expression accounts for their inability to form biofilms in non-limiting P(i) environments. Preliminary evidence suggests that the Pst system is also required for antifungal activity by PA147-2. During phenotypic analysis of pst mutants, we also uncovered novelties in relation to P(i) assimilation and Pho regulon control in P. aureofaciens.     

Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.     

Two-component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression

Pseudomonas aeruginosa is responsible for chronic and acute infections in humans. Chronic infections are associated with production of fimbriae and the formation of a biofilm. The two-component system Roc1 is named after its role in the regulation of cup genes, which encode components of a machinery allowing assembly of fimbriae. A non-characterized gene cluster, roc2, encodes components homologous to the Roc1 system. We show that cross-regulation occurs between the Roc1 and Roc2 signalling pathways. We demonstrate that the sensors RocS2 and RocS1 converge on the response regulator RocA1 to control cupC gene expression. This control is independent of the response regulator RocA2. Instead, we show that these sensors act via the RocA2 response regulator to repress the mexAB-oprM genes. These genes encode a multidrug efflux pump and are upregulated in the rocA2 mutant, which is less susceptible to antibiotics. It has been reported that in cystic fibrosis lungs, in which P. aeruginosa adopts the biofilm lifestyle, most isolates have an inactive MexAB-OprM pump. The concomitant RocS2-dependent upregulation of cupC genes (biofilm formation) and downregulation of mexAB-oprM genes (antibiotic resistance) is in agreement with this observation. It suggests that the Roc systems may sense the environment in the cystic fibrosis lung.     

Opr86 is essential for viability and is a potential candidate for a protective antigen against biofilm formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is one of the most refractory to therapy when it forms biofilms in the airways of cystic fibrosis patients. To date, studies regarding the production of an immunogenic and protective antigen to inhibit biofilm formation by P. aeruginosa have been superficial. The previously uncharacterized outer membrane protein (OMP) Opr86 (PA3648) of P. aeruginosa is a member of the Omp85 family, of which homologs have been found in all gram-negative bacteria. Here we verify the availability of Opr86 as a protective antigen to inhibit biofilm formation by P. aeruginosa PAO1 and several other isolates. A mutant was constructed in which Opr86 expression could be switched on or off through a tac promoter-controlled opr86 gene. The result, consistent with previous Omp85 studies, showed that Opr86 is essential for viability and plays a role in OMP assembly. Depletion of Opr86 resulted in streptococci-like morphological changes and liberation of excess membrane vesicles. A polyclonal antibody against Opr86 which showed reactivity to PAO1 cells was obtained. The antibody inhibited biofilm formation by PAO1 and the other clinical strains tested. Closer examination of early attachment revealed that cells treated with the antibody were unable to attach to the surface. Our data suggest that Opr86 is a critical OMP and a potential candidate as a protective antigen against biofilm formation by P. aeruginosa.     

Hybrid sensor kinase PA1611 in Pseudomonas aeruginosa regulates transitions between acute and chronic infection through direct interaction with RetS

Pseudomonas aeruginosa causes serious acute and chronic infections in humans. Major differences exist in disease pathogenesis, clinical treatment and outcomes between acute and chronic infections. P. aeruginosa acute infection characteristically involves the type III secretion systems (T3SS) while chronic infection is often associated with the formation of biofilms, a major cause of difficulties to eradicate chronic infections. The choice between acute and chronic infection or the switch between them by P. aeruginosa is controlled by regulatory pathways that control major virulence factors and genes associated with biofilm formation. In this study, we characterized a hybrid sensor kinase PA1611 that controls the expression of genes associated with acute and chronic infections in P. aeruginosa PAO1. Expression of PA1611 completely repressed T3SS and swarming motility while it promoted biofilm formation. The protein PA1611 regulates two small RNAs (sRNAs), rsmY and rsmZ which in turn control RsmA. Independent of phosphate relay, PA1611 interacts directly with RetS in vivo. The positive effect of RetS on factors associated with acute infection could presumably be restrained by PA1611 when chronic infection conditions are present. This RetS–PA1611 interaction, together with the known RetS–GacS interaction, may control disease progression and the lifestyle choice of P. aeruginosa.