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.

Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility.

Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca(2+)) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca(2+), we characterized Ca(2+) homeostasis in P. aeruginosa PAO1 cells. By using Ca(2+)-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca(2+) ([Ca(2+)]in) is 0.14±0.05µM. In response to external Ca(2+), the [Ca(2+)]in quickly increased at least 13-fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca(2+) modulated this response. Treatment with inhibitors known to affect Ca(2+) channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca(2+)]in response, suggesting the importance of the corresponding mechanisms in Ca(2+) homeostasis. To identify Ca(2+) transporters maintaining this homeostasis, bioinformatic and LC-MS/MS-based membrane proteomic analyses were used. [Ca(2+)]in homeostasis was monitored for seven Ca(2+)-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca(2+) homeostasis. The lack of PA3920 and vanadate treatment abolished Ca(2+)-induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca(2+).