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。     

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。     

群体感应对铜绿假单胞菌生物被膜形成的调控

生物被膜是一种与浮游细胞相对应的生长方式,由细菌和自身分泌的包外基质组成。铜绿假单胞菌是研究这一生长方式的模式生物。在过去十年,对铜绿假单胞菌生物被膜的研究已取得显著进展。群体感应(QS)的细胞沟通机制在铜绿假单胞菌生物被膜形成中发挥着重要作用。介绍生物被膜的特点,并重点讨论了QS和生物被膜之间的关系。     

铜绿假单胞菌群体感应系统研究进展

群体感应系统是一种细胞密度依赖的基因表达系统,其广泛存在于细菌性病原体中,是细菌细胞通讯方式的一种。群体感应系统可利用细菌释放的信号分子不断监控周围细菌的密度。当细菌密度达到阈值时,群体感应系统网络将启动,参与调控生物被膜、细菌毒力等特定基因的表达,从而使临床抗感染治疗失败。而通过抑制群体感应系统,可一定程度上治疗铜绿假单胞菌引起的感染。本文通过查阅近年国内外相关文献,对铜绿假单胞菌群体感应系统研究进展进行总结,为临床铜绿假单胞菌治疗提供新的方向,即群体感应系统抑制剂有可能成为治疗铜绿假单胞菌感染的新策略。     

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。     

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...     

绿原酸通过增强RsmA的表达抑制铜绿假单胞菌生物被膜的形成

铜绿假单胞菌是常见的人类条件致病菌,其生物被膜的形成会增强菌体的耐药性。已有文献报道绿原酸可抑制铜绿假单胞菌生物被膜的形成,本研究在此基础上主要探究了其对全局性次级代谢调控系统Gac-Rsm表达的影响。结果显示,绿原酸可抑制铜绿假单胞菌生物被膜形成的能力,降低胞外总多糖合成量,但关键胞外多糖psl的合成酶基因pslA转录未受影响,还可增强Gac-Rsm系统中关键调控因子RsmA的表达水平,降低细胞内关键信使分子环二鸟苷酸(cyclic dimeric guanosine monophosphate,c-di-GMP)水平。结果表明,绿原酸可通过增强RsmA的表达来抑制铜绿假单胞菌生物被膜的形成。     

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。     

铜绿假单胞菌与机体免疫的相互关系

铜绿假单胞菌是导致许多急性和慢性感染的重要细菌,在机械通气或某些免疫缺陷的患者中常常导致严重的感染。在其所造成的感染中,随着感染的持续发展,铜绿假单胞菌极易形成生物膜。生物膜是一个具有结构性、协调性和功能性的高度组织群体,生物膜细菌的生物学特性与浮游菌显著不同,其环境适应能力更强,不仅可以防止抗生素的杀灭,还能逃避宿主免疫,使得铜绿假单胞菌相关感染的临床治疗变得更加棘手。本文主要综述铜绿假单胞菌如何逃避宿主免疫和机体在铜绿似单胞菌感染时的免疫反应及在免疫方面的治疗进展。     

靶向破坏铜绿假单胞菌生物被膜的定向蛋白投放技术

【目的】随着合成生物学的发展,通过在细菌体内设计合成复杂、多功能的基因线路进行靶向治疗已经取得巨大进展。虽然这种使用细菌作为治疗传递系统,选择性地在体内释放有效治疗成分的方式具有极大优势,但是如何使细菌在代谢负荷增加较低的情况下有效地分泌功能蛋白并发挥作用依旧是一个难题。【方法】针对这一难题,本研究提供了一种新的策略,即以细菌中广泛存在的蛋白类杀菌素和丝状噬菌体等相关编码基因作为生物模块,通过对铜绿假单胞菌的这些内源生物模块的重新编排和组装,构建了一种能在特定条件下裂解并投放功能蛋白的工程菌。为了评价工程菌中构建的生物模块能否工作,本研究选择胞外多糖水解酶PelA和PslG作为工程菌投放的功能蛋白,以此构建了工程菌PAO1102。通过对铜绿假单胞菌生物被膜的破坏实验、抑制形成实验以及抗生素耐药性实验,检验PAO1102对铜绿假单胞菌生物被膜的破坏和预防效果。【结果】与对照组相比,工程菌PAO1102的处理可以显著破坏已形成的生物被膜并抑制生物被膜的形成,同时还可显著增强生物被膜中的细菌对妥布霉素的敏感性,且这些功能主要通过外界Pf4丝状噬菌体侵染并使工程菌裂解而释放功能蛋白这一途径实现的。【结论】本研究所构建的工程菌可以作为一种微生物工具,用于靶向破坏铜绿假单胞菌生物被膜。在后续的研究中可根据不同的需求,在工程菌中表达不同的功能基因并实现功能蛋白的定向投放,从而执行不同的生物学功能。     

Multifunctional membrane vesicles in Pseudomonas aeruginosa

Gram‐negative bacteria secrete small particles called membrane vesicles (MVs) into the extracellular milieu. While MVs have important roles in delivering toxins from pathogenic bacteria to eukaryotic cells, these vesicles also play ecological roles necessary for survival in various environmental conditions. Pseudomonas aeruginosa, which lives in soil, ocean, plant, animal and human environments, has become a model organism for studying these small extracellular particles. Such studies have increased our understanding of the function and biogenesis of bacterial MVs. Pseudomonas aeruginosa MVs possess versatile components and chemical substances with unique structures. These characteristics allow MVs to play their multifunctional biological roles, including microbial interaction, maintenance of biofilm structure and host infection. This review summarizes the comprehensive biochemical and physiochemical properties of MVs derived from P. aeruginosa. These studies will help us understand their biological roles of MVs not only in pathogenicity but also in microbial ecology. Also, the mechanisms of MV production, as currently understood, are discussed.     

Modelling Co-Infection of the Cystic Fibrosis Lung by Pseudomonas aeruginosa and Burkholderia cenocepacia Reveals Influences on Biofilm Formation and Host Response

The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and those suffering from cystic fibrosis (CF). These two bacteria have been shown to form biofilms in the airways of CF patients that make such infections more difficult to treat. Only recently have scientists begun to appreciate the complicated interplay between microorganisms during polymicrobial infection of the CF airway and the implications they may have for disease prognosis and response to therapy.To gain insight into the possible role that interaction between strains of P. aeruginosa and B. cenocepacia may play during infection, we characterised co-inoculations of in vivo and in vitro infection models. Co-inoculations were examined in an in vitro biofilm model and in a murine model of chronic infection. Assessment of biofilm formation showed that B. cenocepacia positively influenced P. aeruginosa biofilm development by increasing biomass. Interestingly, co-infection experiments in the mouse model revealed that P. aeruginosa did not change its ability to establish chronic infection in the presence of B. cenocepacia but co-infection did appear to increase host inflammatory response.Taken together, these results indicate that the co-infection of P. aeruginosa and B. cenocepacia leads to increased biofilm formation and increased host inflammatory response in the mouse model of chronic infection. These observations suggest that alteration of bacterial behavior due to interspecies interactions may be important for disease progression and persistent infection.     

细菌生物被膜基质的研究进展

细菌生物被膜(biofilm)附着在生物或者非生物表面,由细菌及其分泌的糖、蛋白质和核酸等多种基质组成的细菌群落,是造成病原细菌持续性感染、毒力和耐药性的重要原因之一.细菌的生物被膜基质由复杂的胞外聚合物(extracellular polymeric substances,EPS)构成,影响生物被膜的结构和功能.本文...     

一株肠膜明串珠菌的分离鉴定及其抑菌特性

[背景] 水产病原细菌严重威胁水产动物健康且制约水产养殖业发展,细菌性鱼病的有效防治成为水产养殖领域亟待解决的问题。[目的] 筛选对水产病原细菌有抑制效果的菌株,并研究其抑菌特性及其在水产细菌病害防治中的实际效果。[方法] 通过16S rRNA基因测序、构建系统发育树和生理生化鉴定确定筛选菌株的进化地位,通过乙酸乙酯萃取获得抑菌物质粗提物,通过偶氮酪蛋白法检测菌株胞外蛋白酶活力,采用结晶紫染色法对菌株的生物膜形成能力进行测定,通过浸浴攻毒模型确定所筛菌株对维氏气单胞菌的防治作用。[结果] 从泡菜发酵物中筛选出一株乳酸菌DH,经16S rRNA基因测序、发育树分析和生理生化鉴定确定其为肠膜明串珠菌,该菌分泌的胞外抑菌物质对鼠伤寒沙门氏菌、大肠埃希氏菌、铜绿假单胞菌、杀鲑气单胞菌、希瓦氏菌和维氏气单胞菌表现出抑菌效果,其抑菌物质能被乙酸乙酯萃取并且具有热稳定性。菌株DH能够显著抑制待测菌株的蛋白酶产量和生物膜形成能力,并且对维氏气单胞菌浸浴攻毒有防治作用。[结论] 肠膜明串珠菌DH通过分泌抑菌物质抑制水产病原细菌的生长,能够为细菌性鱼病的防治提供一定的理论和应用潜力。     

Biofilm and Quorum sensing mediated pathogenicity in Pseudomonas aeruginosa

The emergence of multidrug resistance has become an alarming and lifethreatening phenomenon for humans. Various mechanisms are involved in the development of resistance in bacteria towards antimicrobial compounds and immune system. Bacterial biofilm is a complicated, selfdefensive, rigid structure of bacteria crowded together to develop a selfrecessive nature, which enhances the ability to cause infections much easier in the living host. P. aeruginosa biofilm formation is supported by extracellular polymeric substances (EPS) such as exopolysaccharides, extracellular DNA (eDNA), proteins and biomolecules. Published evidences suggest that biofilm formation can also be the result of several other mechanisms such as cell signaling or communication. Bacterial biofilm is also regulated by strong intercellular communication known as Quorum Sensing (QS). It is a cellular communication mechanism involving autoinducers and regulators. In P. aeruginosa, Acyl Homoserine Lactone, the prime signaling molecule, controls approximately 300 genes responsible for various cellular functions, including its pathogenesis. The surrounding environment and metabolism have a specific effect on the biofilm and QS, thus, understanding the involvement of QS in the biofilm developing mechanism is still complicated and complex to understand. Therefore, this review will include basic knowledge of the biofilmforming mechanism and other regulatory factors involved in causing infections and diseases in the host organisms.     

Bacteria can form interconnected microcolonies when a self-excreted product reduces their surface motility: evidence from individual-based model simulations

Recent experimental observations of Pseudomonas aeruginosa, a model bacterium in biofilm research, reveal that, under specific growth conditions, bacterial cells form patterns of interconnected microcolonies. In the present work, we use an individual-based model to assess the involvement of bacteria motility and self-produced extracellular substance in the formation of these patterns. In our simulations, the pattern of interconnected microcolonies appears only when bacteria motility is reduced by excreted extracellular macromolecules. Immotile bacteria form isolated microcolonies and constantly motile bacteria form flat biofilms. Based on experimental data and computer simulations, we suggest a mechanism that could be responsible for these interconnected microcolonies.     

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...