Aendekerk Séverine, Diggle Stephen P, Song Zhijun, Høiby Niels, Cornelis Pierre, Williams Paul, Cámara Miguel
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK.
Laboratory of Microbial Interactions, Department of Molecular and Cellular Interactions, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussel, Building E, Room 6.6, Pleinlaan 2, B-1050 Brussels, Belgium.
Microbiology (Reading). 2005 Apr;151(Pt 4):1113-1125. doi: 10.1099/mic.0.27631-0.
In Pseudomonas aeruginosa the production of multiple virulence factors depends on cell-to-cell communication through the integration of N-acylhomoserine lactone (AHL)- and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS)- dependent signalling. Mutation of genes encoding the efflux protein MexI and the porin OpmD from the MexGHI-OpmD pump resulted in the inability to produce N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-c12-hsl) and pqs and a marked reduction in n-butanoyl-L-homoserine lactone levels. Both pump mutants were impaired in growth and exhibited enhanced rather than reduced antibiotic resistance. Provision of exogenous PQS improved growth and restored AHL and virulence factor production as well as antibiotic susceptibility, indicating that the pump mutants retained their capacity to respond to PQS. RT-PCR analysis indicated that expression of the PQS biosynthetic genes, phnA and pqsA, was inhibited when the mutants reached stationary phase, suggesting that the pleiotropic phenotype observed may be due to intracellular accumulation of a toxic PQS precursor. To explore this hypothesis, double mexI phnA (unable to produce anthranilate, the precursor of PQS) and mexI pqsA mutants were constructed; the improved growth of the former suggested that the toxic compound is likely to be anthranilate or a metabolite of it. Mutations in mexI and opmD also resulted in the attenuation of virulence in rat and plant infection models. In plants, addition of PQS restored the virulence of mexI and opmD mutants. Collectively, these results demonstrate an essential function for the MexGHI-OpmD pump in facilitating cell-to-cell communication, antibiotic susceptibility and promoting virulence and growth in P. aeruginosa.
在铜绿假单胞菌中，多种毒力因子的产生依赖于通过整合N-酰基高丝氨酸内酯（AHL）和2-庚基-3-羟基-4（1H）-喹诺酮（PQS）依赖性信号传导进行的细胞间通讯。MexGHI-OpmD泵的外排蛋白MexI和孔蛋白OpmD编码基因的突变导致无法产生N-（3-氧代十二烷酰基）-L-高丝氨酸内酯（3-氧代-c12-hsl）和PQS，并且正丁酰-L-高丝氨酸内酯水平显著降低。两个泵突变体的生长均受损，并且表现出增强而非降低的抗生素抗性。提供外源性PQS可改善生长，并恢复AHL和毒力因子的产生以及抗生素敏感性，表明泵突变体保留了对PQS作出反应的能力。逆转录聚合酶链反应（RT-PCR）分析表明，当突变体进入稳定期时，PQS生物合成基因phnA和pqsA的表达受到抑制，这表明观察到的多效性表型可能是由于有毒PQS前体的细胞内积累所致。为了探究这一假设，构建了双mexI phnA（无法产生邻氨基苯甲酸，PQS的前体）和mexI pqsA突变体；前者生长的改善表明有毒化合物可能是邻氨基苯甲酸或其代谢产物。MexI和opmD中的突变也导致大鼠和植物感染模型中的毒力减弱。在植物中，添加PQS可恢复mexI和opmD突变体的毒力。总体而言，这些结果证明了MexGHI-OpmD泵在促进铜绿假单胞菌的细胞间通讯、抗生素敏感性以及促进毒力和生长方面的重要功能。
