Control of quorum sensing by a Burkholderia pseudomallei multidrug efflux pump

The Burkholderia pseudomallei KHW quorum-sensing systems produced N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxy)-octanoyl-homoserine lactone, N-(3-hydroxy)-decanoyl-homoserine lactone, N-(3-oxo)-decanoyl-homoserine lactone, and N-(3-oxo)-tetradecanoyl-homoserine lactone. The extracellular secretion of these acyl-homoserine lactones is dependent absolutely on the function of the B. pseudomallei BpeAB-OprB efflux pump.     

RND家族外排泵及其与微生物群体感应系统的相互关系

抗生素耐药性一直是细菌病害防治的难题,药物外排泵过量表达是细菌耐药性形成的重要机制之一。在革兰氏阴性细菌中,RND(Resistance-nodulation-cell division)家族外排泵在耐药性中发挥着重要作用,近年来的研究表明,依赖于小分子信号物质进行调控的群体感应系统与RND外排泵家族之间存在紧密的相互作用关系。本文在介绍RND家族外排泵的结构、转运机理和群体感应系统的类型及调控方式的基础上,剖析了群体感应系统对RND外排泵的调控机理以及RND外排泵对群体感应系统信号分子转运的影响。深入研究RND家族外排泵与群体感应系统之间的相互依赖、相互制约关系有利于阐明RND家族外排泵的调控机理,并有可能为克服微生物耐药性问题提供新的思路。     

The mviN homolog in Burkholderia pseudomallei is essential for viability and virulence

The virulence factors of Burkholderia pseudomallei, the causative agent of melioidosis, are not fully understood. We have identified a gene with homology to the Salmonella typhimurium mouse virulence gene, mviN, a member of the mouse virulence factor family. Expression studies with an insertional mutant containing a lux operon demonstrated that the expression of the gene is influenced by free-iron availability in the media and by growth phase. The mutant displayed an increased LD50 value in the hamster infection model and a loss of the ability to invade human lung epithelial cells. The mutant has a slower growth rate than that of the wild type. Both defects were restored to various degrees when complemented in trans with the mviN gene. The mutant contains an insertion at 1229 bp of the 1548 bp gene, resulting in a truncated protein that is presumably responsible for the defects. Deletion mutants of the entire B. pseudomallei mviN gene were obtained only in the presence of the complement vector. This result and the inability of the complemented deletion mutant to lose the plasmid in the absence of antibiotic selection suggest that the gene is essential to B. pseudomallei.     

Burkholderia pseudomallei virulence: definition, stability and association with clonality.

Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.     

Interference of quorum sensing and virulence of the rice pathogen Burkholderia glumae by an engineered endophytic bacterium

Many bacterial species are known to thrive within plants. Among these bacteria, a group referred to as endophytes provide beneficial effects to the host plants by the promotion of plant growth and the suppression of plant pathogens. Among 44 putative endophytic isolates isolated from surface-sterilized rice roots, Burkholderia sp. KJ006 was selected for further study because of a lack of pathogenicity to rice, a broad spectrum of antifungal properties, and the presence of the nifH gene, which is an indicator for nitrogen fixation. In an attempt to control Burkholderia glumae, a casual pathogen of seedling rot and grain rot of rice, an N-acyl-homoserine lactonase (aiiA) gene from Bacillus thuringiensis was introduced into Burkholderia sp. KJ006 given that the major virulence factor of Burkholderia glumae is controlled in a population-dependent manner (quorum sensing). The engineered strain KJ006 (pKPE-aiiA) inhibited production of quorum-sensing signals by Burkholderia glumae in vitro and reduced the disease incidence of rice seedling rot caused by Burkholderia glumae in situ. Our results indicate the possibility that a bacterial endophyte transformed with the aiiA gene can be used as a novel biological control agent against pathogenic Burkholderia glumae that are known to occupy the same ecological niche.     

The impaired quorum sensing response of Pseudomonas aeruginosa MexAB-OprM efflux pump overexpressing mutants is not due to non-physiological efflux of 3-oxo-C12-HSL

Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analysed efflux pump. Herein, we performed an in-depth analysis of the QS-mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB-OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3-oxo-C12-HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This 'apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals.     

Kin selection, quorum sensing and virulence in pathogenic bacteria

Bacterial growth and virulence often depends upon the cooperative release of extracellular factors excreted in response to quorum sensing (QS). We carried out an in vivo selection experiment in mice to examine how QS evolves in response to variation in relatedness (strain diversity), and the consequences for virulence. We started our experiment with two bacterial strains: a wild-type that both produces and responds to QS signal molecules, and a lasR (signal-blind) mutant that does not release extracellular factors in response to signal. We found that: (i) QS leads to greater growth within hosts; (ii) high relatedness favours the QS wild-type; and (iii) low relatedness favours the lasR mutant. Relatedness matters in our experiment because, at relatively low relatedness, the lasR mutant is able to exploit the extracellular factors produced by the cells that respond to QS, and hence increase in frequency. Furthermore, our results suggest that because a higher relatedness favours cooperative QS, and hence leads to higher growth, this will also lead to a higher virulence, giving a relationship between relatedness and virulence that is in the opposite direction to that usually predicted by virulence theory.     

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。     

Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200-kDa antigen of B. pseudomallei cross-reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide-positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain.     

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.     

The role of quorum sensing in the in vivo virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections. The cell-density-dependent signaling mechanisms known as quorum sensing play a role in several of these infections including corneal, lung and burn wound infections. In addition, the quorum-sensing systems contribute to the ability of P. aeruginosa to form biofilms on medically important devices. The quorum-sensing systems accomplish their effect by controlling the production of different virulence factors and by manipulating the host immune response.     

Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter

Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a delta hapR mutant and decreased in a delta luxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between -85 and -58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at -85 and -77. Introducing the -77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin-co-regulated pilus (TCP) and cholera toxin (CT) that occurs in a delta luxO mutant, indicating that HapR function at aphA is critical for density-dependent regulation of virulence genes.