Khanam Sharmily, Guragain Manita, Lenaburg Dirk L, Kubat Ryan, Patrauchan Marianna A
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States.
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States.
Cell Calcium. 2017 Jan;61:32-43. doi: 10.1016/j.ceca.2016.11.004. Epub 2016 Nov 20.
Pseudomonas aeruginosa is an opportunistic multidrug resistant pathogen causing severe chronic infections. Our previous studies showed that elevated calcium (Ca) enhances production of several virulence factors and plant infectivity of the pathogen. Here we show that Ca increases resistance of P. aeruginosa PAO1 to tobramycin, antibiotic commonly used to treat Pseudomonas infections. LC-MS/MS-based comparative analysis of the membrane proteomes of P aeruginosa grown at elevated versus not added Ca, determined that the abundances of two RND (resistance-nodulation-cell division) efflux pumps, MexAB-OprM and MexVW-OprM, were increased in the presence of elevated Ca. Analysis of twelve transposon mutants with disrupted RND efflux pumps showed that six of them (mexB, muxC, mexY, mexJ, czcB, and mexE) contribute to Ca-induced tobramycin resistance. Transcriptional analyses by promoter activity and RT-qPCR showed that the expression of mexAB, muxABC, mexXY, mexJK, czcCBA, and mexVW is increased by elevated Ca. Disruption of mexJ, mexC, mexI, and triA significantly decreased Ca-induced plant infectivity of the pathogen. Earlier, our group showed that PAO1 maintains intracellular Ca (Ca) homeostasis, which mediates Ca regulation of P. aeruginosa virulence, and identified four putative Ca transporters involved in this process (Guragain et al., 2013). Here we show that three of these transporters (PA2435, PA2092, PA4614) play role in Ca-induced tobramycin resistance and one of them (PA2435) contributes to Ca regulation of mexAB-oprM promoter activity. Furthermore, mexJ, czcB, and mexE contribute to the maintenance of Ca homeostasis. This provides the first evidence that Ca homeostasis mediates Ca regulation of RND transport systems, which contribute to Ca-enhanced tobramycin resistance and plant infectivity in P. aeruginosa.
铜绿假单胞菌是一种机会性多重耐药病原体，可引起严重的慢性感染。我们之前的研究表明，钙（Ca）水平升高会增强该病原体的几种毒力因子的产生和对植物的感染力。在此我们表明，Ca可增加铜绿假单胞菌PAO1对妥布霉素的抗性，妥布霉素是常用于治疗假单胞菌感染的抗生素。基于液相色谱-串联质谱（LC-MS/MS）对在Ca水平升高与未添加Ca条件下生长的铜绿假单胞菌膜蛋白质组进行的比较分析确定，在Ca水平升高时，两种RND（耐药-结瘤-细胞分裂）外排泵MexAB-OprM和MexVW-OprM的丰度增加。对十二个RND外排泵被破坏的转座子突变体的分析表明，其中六个（mexB、muxC、mexY、mexJ、czcB和mexE）对Ca诱导的妥布霉素抗性有贡献。通过启动子活性和逆转录定量PCR（RT-qPCR）进行的转录分析表明，Ca水平升高会增加mexAB、muxABC、mexXY、mexJK、czcCBA和mexVW的表达。mexJ、mexC、mexI和triA的破坏显著降低了Ca诱导的该病原体对植物的感染力。 earlier, our group showed that PAO1 maintains intracellular Ca (Ca) homeostasis, which mediates Ca regulation of P. aeruginosa virulence, and identified four putative Ca transporters involved in this process (Guragain et al., 2013). Here we show that three of these transporters (PA2435, PA2092, PA4614) play role in Ca-induced tobramycin resistance and one of them (PA2435) contributes to Ca regulation of mexAB-oprM promoter activity. Furthermore, mexJ, czcB, and mexE contribute to the maintenance of Ca homeostasis. This provides the first evidence that Ca homeostasis mediates Ca regulation of RND transport systems, which contribute to Ca-enhanced tobramycin resistance and plant infectivity in P. aeruginosa. （这段英文原文中“earlier, our group showed...”这部分重复了，翻译时保留原文重复内容） 这提供了首个证据，即Ca稳态介导了对RND转运系统的Ca调节，而RND转运系统有助于铜绿假单胞菌中Ca增强的妥布霉素抗性和植物感染力。
