Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.     

Variation of the mexT gene, a regulator of the MexEF-oprN efflux pump expression in wild-type strains of Pseudomonas aeruginosa

We found three variations of wild-type strains in terms of mexT-mediated regulation of the MexEF-OprN efflux pump, in which overexpression of the pump results in nfxC-type antibiotic resistance in Pseudomonas aeruginosa. Type-I: the mexT gene of the wild-type strain encoded inactive MexT and the nfxC-type mutants derived from this parent had an additional mutation in mexT converting MexT from the inactive to the active form. Type-II: The mexT gene in the wild-type strain had an 8-bp insert producing inactive MexT and the nfxC-type mutants from this parent had a deletion of the 8-bp insert converting inactive MexT to the active form. Type-III: Both the wild-type strain and its nfxC-type derivative produced identical and active MexT. The nfxC mutant from this parent must have an additional mutation. The original nfxC mutant isolated in 1990 might be derived from the Type-I parent strain.     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

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。     

Quorum sensing system lactones do not increase invasiveness of a MexAB-OprM efflux mutant but do play a partial role in Pseudomonas aeruginosa invasion

We studied the quorum sensing (QS) system and the related homoserine lactones (HSLs) observing Pseudomonas aeruginosa invasion using the epithelial cell monolayer penetration assay model. Compared to the PAO1 wild-type, the QS mutants, DeltalasI and DeltarhlI, were compromised in their capacity to invade. The decreased invasiveness of DeltarhlI was restored by adding 100 microM exogenous C(4)-HSL. However, the decreased invasiveness of an efflux mutant, DeltamexAB-oprM, was not restored in the presence of exogenous HSLs. The QS system partially plays a role in P. aeruginosa invasion; however, C(4)-HSL and 3-O-C(12)-HSL are not the essential determinants for invasiveness for P. aeruginosa.     

Mutations in PA2491 (mexS) promote MexT-dependent mexEF-oprN expression and multidrug resistance in a clinical strain of Pseudomonas aeruginosa

Disruption of the PA2491 gene in a mini-Tn5-tet insertion mutant of a clinical isolate of Pseudomonas aeruginosa increased expression of the mexEF-oprN multidrug efflux genes and decreased production of outer membrane protein OprD, concomitant with enhanced resistance to chloramphenicol, quinolones, and imipenem, which was reminiscent of previously described nfxC mutants. PA2491 encodes a probable oxidoreductase previously shown to be positively regulated by the MexT positive regulator of mexEF-oprN expression (T. Kohler, S. F. Epp, L. K. Curty, and J. C. Pechere, J. Bacteriol. 181:6300-6305, 1999). Spontaneous multidrug-resistant mutants of the P. aeruginosa clinical isolate hyperexpressing mexEF-oprN and showing reduced production of OprD were readily selected in vitro, and all of them were shown to carry mutations in PA2491, highlighting the probable significance of such mutations as determinants of MexEF-OprN-mediated multidrug resistance in vivo.     

Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance.

Organic solvent-tolerant mutants of Pseudomonas aeruginosa selected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical of nalB multidrug-resistant mutants. Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems in P. aeruginosa demonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system. Sequence analysis of mexR, the repressor gene of mexAB-oprM efflux operon, identified a nonsense mutation and a point mutation in the mexR genes of two solvent-tolerant mutants. These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.     

Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR

In Pseudomonas aeruginosa many of the clinically relevant resistance mechanisms result from changes in gene expression as exemplified by the Mex drug efflux pumps, the AmpC beta-lactamase and the carbapenem-specific porin OprD. We used quantitative real-time-PCR to analyze the expression of these genes in susceptible and antibiotic-resistant laboratory and clinical strains. In nalB mutants, which overexpress OprM, we observed a four- to eightfold increase in the expression of mexA, mexB, and oprM genes. MexX and mexY genes were induced eight to 12 times in the presence of 2 mg L(-1) tetracycline. The mexC/oprJ and mexE/oprN gene expression levels were increased 30- to 250-fold and 100- to 760-fold in nfxB and nfxC mutants, respectively. We further found that in defined laboratory strains expression levels of ampC and oprD genes paralleled beta-lactamase activity and OprD protein levels, respectively. Our data support the use of quantitative real-time-PCR chain reaction for the analysis of the antimicrobial resistance gene expression in P. aeruginosa.