title: Calcium induces tobramycin resistance in Pseudomonas aeruginosa by regulating RND efflux pumps.
abstract: 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+inhomeostasis. This provides the first evidence that Ca2+inhomeostasis mediates Ca2+regulation of RND transport systems, which contribute to Ca2+-enhanced tobramycin resistance and plant infectivity in P. aeruginosa.
authors.affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States.
authors.forename: Sharmily
authors.initials: S
authors.lastname: Khanam
authors.affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States.
authors.forename: Manita
authors.initials: M
authors.lastname: Guragain
authors.affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States.
authors.forename: Dirk L
authors.initials: DL
authors.lastname: Lenaburg
authors.affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: Kubat
authors.affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, United States. Electronic address: m.patrauchan@okstate.edu.
authors.forename: Marianna A
authors.initials: MA
authors.lastname: Patrauchan
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D014031:Tobramycin / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D002118:Calcium; D014031:Tobramycin
keywords: Antibiotic resistance; Calcium efflux; Calcium transporters; Promoter activity; RND transporters; Virulence; qRT-PCR