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。     

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide.

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.     

Inactivation of Pseudomonas aeruginosa biofilm by dense phase carbon dioxide

Dense phase carbon dioxide (DPCD) is one of the most promising techniques available to control microorganisms as a non-thermal disinfection method. However, no study on the efficiency of biofilm disinfection using DPCD has been reported. The efficiency of DPCD in inactivating Pseudomonas aeruginosa biofilm, which is known to have high antimicrobial resistance, was thus investigated. P. aeruginosa biofilm, which was not immersed in water but was completely wet, was found to be more effectively inactivated by DPCD treatment, achieving a 6-log reduction within 7 min. The inactivation efficiency increased modestly with increasing pressure and temperature. This study also reports that the water-unimmersed condition is one of the most important operating parameters in achieving efficient biofilm control by DPCD treatment. In addition, observations by confocal laser scanning microscopy revealed that DPCD treatment not only inactivated biofilm cells on the glass coupons but also caused detachment of the biofilm following weakening of its structure as a result of the DPCD treatment; this is an added benefit of DPCD treatment.     

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。     

Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.     

Effects of ginseng on Pseudomonas aeruginosa motility and biofilm formation

Biofilm-associated chronic Pseudomonas aeruginosa lung infections in patients with cystic fibrosis are virtually impossible to eradicate with antibiotics because biofilm-growing bacteria are highly tolerant to antibiotics and host defense mechanisms. Previously, we found that ginseng treatments protected animal models from developing chronic lung infection by P. aeruginosa. In the present study, the effects of ginseng on the formation of P. aeruginosa biofilms were further investigated in vitro and in vivo. Ginseng aqueous extract at concentrations of 0.5-2.0% did not inhibit the growth of P. aeruginosa, but significantly prevented P. aeruginosa from forming biofilm. Exposure to 0.5% ginseng aqueous extract for 24 h destroyed most 7-day-old mature biofilms formed by both mucoid and nonmucoid P. aeruginosa strains. Ginseng treatment enhanced swimming and twitching motility, but reduced swarming of P. aeruginosa at concentrations as low as 0.25%. Oral administration of ginseng extracts in mice promoted phagocytosis of P. aeruginosa PAO1 by airway phagocytes, but did not affect phagocytosis of a PAO1-filM mutant. Our study suggests that ginseng treatment may help to eradicate the biofilm-associated chronic infections caused by P. aeruginosa.     

HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa

HD-GYP is a protein domain involved in the hydrolysis of the bacterial second messenger cyclic-di-GMP. The genome of the human pathogen Pseudomonas aeruginosa PAO1 encodes two proteins (PA4108, PA4781) with an HD-GYP domain and a third protein, PA2572, which contains a domain with variant key residues (YN-GYP). Here we have investigated the role of these proteins in biofilm formation, virulence factor synthesis and virulence of P. aeruginosa . Mutation of PA4108 and PA4781 led to an increase in the level of cyclic-di-GMP in P. aeruginosa , consistent with the predicted activity of the encoded proteins as cyclic-di-GMP phosphodiesterases. Mutation of both genes led to reduced swarming motility but had differing effects on production of the virulence factors pyocyanin, pyoverdin and ExoS. Mutation of PA2572 had no effect on cyclic-di-GMP levels and did not influence swarming motility. However, PA2572 had a negative influence on swarming that was cryptic and was revealed only after removal of an uncharacterized C-terminal domain. Mutation of PA4108 , PA4781 and PA2572 had distinct effects on biofilm formation and architecture of P. aeruginosa. All three proteins contributed to virulence of P. aeruginosa to larvae of the Greater Wax moth Galleria mellonella.     

The mechanism of formation of Pseudomonas aeruginosa biofilm,a type of structured population

The paper is an attempt to analyze and generalize molecular and cell biology data on the formation of polysaccharide matrix--based biofilms. The conception of biofilms as structured populations sharing the characteristics of uni- and multicellular organisms and population is proposed.     

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。     

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa

Biofilms are communities of surface-attached microbial cells that resist environmental stresses. In this study, we found that low concentrations of ethanol increase biofilm formation in Pseudomonas aeruginosa PAO1 but not in a mutant of it lacking both Psl and Pel exopolysaccharides. Low concentrations of ethanol also increased pellicle formation at the air–liquid interface.     

PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

Pyoverdine and PQS mediated subpopulation interactions involved in Pseudomonas aeruginosa biofilm formation

Using flow chamber‐grown Pseudomonas aeruginosa biofilms as model system, we show in the present study that formation of heterogeneous biofilms may occur through mechanisms that involve complex subpopulation interactions. One example of this phenomenon is expression of the iron‐siderophore pyoverdine in one subpopulation being necessary for development of another subpopulation that does not itself express the pyoverdine synthesis genes. Another example is quorum sensing‐controlled DNA release in one subpopulation being necessary for development of another subpopulation that does not itself express the quorum‐sensing genes.     

一氧化碳释放分子的作用机制

一氧化碳释放分子(carbon monoxide releasing molecules,CORMs)是一种羰基化合物,能够模拟内源性CO的生理及病理过程,具有多种生物学效能.CORMs化合物不断改良,极大地推进了CORMs生物学活性的研究,目前CORMs已被认为是一种能够可控地释放少量CO的新型药物.本文主要就有关C...