Bhagirath Anjali Y, Somayajula Deepti, Li Yanqi, Duan Kangmin
Department of Oral Biology, Rady Faculty of Health Sciences, College of Dentistry, Biology of Breathing Group, Children's Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, MB, R3E 0W2, Canada.
J Membr Biol. 2018 Feb;251(1):35-49. doi: 10.1007/s00232-017-9994-6. Epub 2017 Oct 23.
Pseudomonas aeruginosa is an ubiquitous organism which is able to infect and colonize many types of hosts including humans. Colonization of P. aeruginosa in chronic infections leads to the formation of biofilms, which are difficult to eradicate. P. aeruginosa is capable of regulating its virulence factors in response to external environment triggers and its signaling mechanism involves two-component regulatory systems and small molecules such as bis-(3'-5')-cyclic dimeric guanosine monophosphate. PA1611-RetS-GacS/A-RsmA/Y/Z is a key regulatory pathway in P. aeruginosa that controls several virulence factors and biofilm formation. We have previously identified a conserved cytoplasmic membrane protein cmpX (PA1775), as a regulator for PA1611 expression. In this study, we demonstrate that cmpX regulates virulence, and controls biofilm formation in P. aeruginosa as well as provide evidence showing that cmpX affects Gac/Rsm pathway, possibly by modulating intra-cellular c-di-GMP levels. A cmpX knockout showed significantly decreased promoter activity of exoS (PA1362) and increased activity of small RNA, RsmY. As compared to the wild-type PAO1, cmpX mutant had elevated intracellular c-di-GMP level as measured indirectly by cdrA (PA4625) activity, as well as increased expression of wspR (PA3702), a c-di-GMP synthase. The transcription of the major outer membrane porin gene oprF (PA1777), and sigma factor sigX (PA1776) was also significantly decreased in the cmpX mutant. Biolog phenotype microarray experiments further indicated that the cmpX knockout mutant had increased sensitivity to membrane detergents and antibiotics such as lauryl sulfobetaine, tobramycin, and vancomycin. These results point to a significant role of cmpX in P. aeruginosa virulence and colonization.
铜绿假单胞菌是一种广泛存在的微生物，能够感染并定植于包括人类在内的多种宿主。铜绿假单胞菌在慢性感染中的定植会导致生物膜的形成，而生物膜难以根除。铜绿假单胞菌能够响应外部环境触发因素来调节其毒力因子，其信号传导机制涉及双组分调节系统和小分子，如双（3'-5'）-环二聚鸟苷单磷酸。PA1611-RetS-GacS/A-RsmA/Y/Z是铜绿假单胞菌中控制多种毒力因子和生物膜形成的关键调节途径。我们之前已鉴定出一种保守的细胞质膜蛋白cmpX（PA1775），作为PA1611表达的调节因子。在本研究中，我们证明cmpX调节毒力，控制铜绿假单胞菌中的生物膜形成，并提供证据表明cmpX可能通过调节细胞内c-di-GMP水平来影响Gac/Rsm途径。cmpX基因敲除显示外毒素S（PA1362）的启动子活性显著降低，而小RNA RsmY的活性增加。与野生型PAO1相比，通过cdrA（PA4625）活性间接测量，cmpX突变体细胞内c-di-GMP水平升高，同时c-di-GMP合酶wspR（PA3702）的表达也增加。在cmpX突变体中，主要外膜孔蛋白基因oprF（PA1777）和σ因子sigX（PA1776）的转录也显著降低。Biolog表型微阵列实验进一步表明，cmpX基因敲除突变体对膜洗涤剂和抗生素如十二烷基磺基甜菜碱、妥布霉素和万古霉素的敏感性增加。这些结果表明cmpX在铜绿假单胞菌的毒力和定植中起着重要作用。
