Calcium induces tobramycin resistance in Pseudomonas aeruginosa by regulating RND efflux pumps.

Pseudomonas aeruginosa is an opportunistic multidrug resistant pathogen causing severe chronic infections. Our previous studies showed that elevated calcium (Ca2+ ) enhances production of several virulence factors and plant infectivity of the pathogen. Here we show that Ca2+ 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 Ca2+ , 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 Ca2+ . Analysis of twelve transposon mutants with disrupted RND efflux pumps showed that six of them (mexB, muxC, mexY, mexJ, czcB, and mexE) contribute to Ca2+ -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 Ca2+ . Disruption of mexJ, mexC, mexI, and triA significantly decreased Ca2+ -induced plant infectivity of the pathogen. Earlier, our group showed that PAO1 maintains intracellular Ca2+ (Ca2+ in ) homeostasis, which mediates Ca2+ regulation of P. aeruginosa virulence, and identified four putative Ca2+ transporters involved in this process (Guragain et al., 2013). Here we show that three of these transporters (PA2435, PA2092, PA4614) play role in Ca2+ -induced tobramycin resistance and one of them (PA2435) contributes to Ca2+ regulation of mexAB-oprM promoter activity. Furthermore, mexJ, czcB, and mexE contribute to the maintenance of Ca2+ in homeostasis. This provides the first evidence that Ca2+ in homeostasis mediates Ca2+ regulation of RND transport systems, which contribute to Ca2+ -enhanced tobramycin resistance and plant infectivity in P. aeruginosa.