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.