Cell Calcium. 2017 Jan;61:32-43. doi: 10.1016/j.ceca.2016.11.004.

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

Sharmily Khanam, Manita Guragain, Dirk L. Lenaburg, Ryan Kubat, and Marianna A. Patrauchan*

Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK.

*Corresponding author: Marianna A. Patrauchan

Dept. of Microbiology and Molecular Genetics
Oklahoma State University, 307 LSE
Stillwater, OK, 74075
Tel: (405) 744-8148
Fax: (405) 744-6790
E-mail: m.patrauchan@okstate.edu 


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.

Key words: antibiotic resistance, RND transporters, calcium transporters, virulence, promoter activity, calcium efflux, qRT-PCR.

Running title: Calcium and RND in P. aeruginosa





Pseudomonas aeruginosa is one of the major human pathogens causing severe and life threatening infections. At present, several types of antibiotics including aminoglycosides are considered to be an effective choice for treating P. aeruginosa infections (1-3). However, the increasing resistance of the pathogen to most available antimicrobials represents a serious threat and requires a new knowledge of the mechanisms of resistance and their regulation in response to host factors. Here we studied Ca2+-dependent antibiotic resistance in P. aeruginosa and its regulation by elevated extracellular Ca2+ and the transient changes in the intracellular levels of Ca2+ ([Ca2+in]), which are generated in response to sudden addition of extracellular Ca2+. Since Ca2+ is a major second messenger in a human body, understanding its regulatory effects on antibiotic resistance of human pathogens is of high importance. The main experiments and results are outlined in Fig. 1.

First, this study showed that Ca2+ at the concentration commonly detected in the lungs of cystic fibrosis (CF) patients (4, 5), increases P. aeruginosa resistance to several antibiotics (Fig. 1). The most significant increase was detected for tobramycin and polymyxin B, both used to treat P. aeruginosa infections. To identify the molecular mechanisms responsible for Ca2+-induced tobramycin resistance in P. aeruginosa, we compared membrane proteomes of cells grown at 5 mM versus no added CaCl2 by using a semi-quantitative LC-MS/MS-based spectrum (peptide) counting approach. This allowed confident identification of several proteins representing RND transporters, including MexB, MexV, and CzcA, whose abundance was affected during growth at elevated Ca2+.

To test whether RND transporters play role in Ca2+-induced resistance to tobramycin, we measured the minimal inhibitory concentrations (MIC) of this antibiotic in the transposon insertion mutants deficient in each of the twelve RND genes encoded in the PAO1 genome. The disruption of mexB, mexY, muxC, mexE, mexJ and czcB reduced Ca2+-induced tobramycin resistance at least twofold (Fig. 1), suggesting that theses six RND transporters are involved in Ca2+-induced tobramycin resistance.


Figure 1. A diagram representing the main experiments and the results of the study.


To determine whether Ca2+-dependent involvement of multiple RND systems in tobramycin resistance is mediated by Ca2+ effect on the transcription of RND genes, we used RT-qPCR and promoter activity approaches. This analysis revealed that growth at elevated Ca2+ affected the expression of four RND genes by at least two fold. Transcripts of mexV were twofold more abundant at elevated Ca2+, whereas transcription of mexX, muxC, and mexM was reduced in response to 5 mM Ca2+. Since this transcriptional profile did not correlate with the involvement of mexB, mexY, muxC, mexE, mexJ, czcB, and mexX in Ca2+ regulated tobramycin resistance, we hypothesized that Ca2+ regulation is growth-phase-dependent. To test this hypothesis, we monitored the temporal effect of Ca2+ on promoter activities of the six RND transporters by using lux-based reporter system. The results confirmed that promoter activities of five RND transporters were transiently increased by Ca2+ in a growth-phase-dependent manner with multiple spikes of activity observed mostly during transitions between different growth phases. The most significant effect of Ca2+ was observed for mexAB-oprM promoter, whose activity increased 7 fold after 2 h of growth during the transition to early-log phase.

We also studied whether Ca2+ regulatory effect on the transcription of RND transporters is mediated by changes in [Ca2+in]. Earlier we established that addition of extracellular Ca2+ causes transient changes in the intracellular levels of the ion, and suggested that this response likely mediates Ca2+ regulation in P. aeruginosa (6). Several putative Ca2+ transporters were identified and shown to be required for maintaining Ca2+in homeostasis. They include P-type ATPase PA2435, ion exchanger PA2092, and mechanosensitive channel PA4614 (6). To test whether Ca2+in homeostasis is involved in regulating the transcriptional changes, we measured the activity of mexAB-oprM promoter in the mutants with disrupted PA2435, PA2092, or PA4614, and therefore disturbed Ca2+in homeostasis. The most significant reduction of Ca2+ induction of PmexAB-oprM activity was detected in PA2435:Tn5 mutant, indicating that Ca2+in homeostasis regulated by PA2435 mediates Ca2+ regulation of PmexAB-oprM activity.

Since at least one putative Ca2+ transporter (PA2435), required for maintaining Ca2+in homeostasis, plays role in Ca2+ regulation of mexAB-oprM transcription, we also tested the role of four earlier identified putative Ca2+ transporters (PA2435, PA2092, PA3920, and PA4614) in Ca2+-induced tobramycin resistance. No significant changes in the tobramycin MIC were detected when the mutants were grown at no added Ca2+. However, when mutants with disrupted PA2435, PA2092, or PA4614 were grown at 5 mM Ca2+, the MIC was reduced by almost twofold (from 3.5 µg/ml in PAO1 to 1.75-2.0 µg/ml in the mutants). Considering that in response to extracellular Ca2+, these mutants increase [Ca2+]in to the level of the wild type, but are not able to bring it back to the basal level (6), we proposed that this failure of generating a temporally transient elevation of [Ca2+]in reduces their responses to Ca2+ regulation and decreases the level of Ca2+-induced tobramycin resistance. These observations support the hypothesis that Ca2+in mediates Ca2+ regulation of tobramycin resistance.

Since CzcCBA-OpmY RND system was shown to translocate ions (7), we tested whether the RND pumps involved in Ca2+-induced tobramycin resistance play role in transporting Ca2+ and maintaining its intracellular concentration. For this, we monitored [Ca2+in] in the transposon mutants with disrupted mexB, mexY, mexJ, muxC, mexE or czcB by using a recombinant Ca2+-binding luminescence protein, aequorin. Each strain producing aequorin was cultured without Ca2+ or in the presence of 5 mM CaCl2 and challenged with 1 mM CaCl2. When no CaCl2 was added during growth, disruption of mexE significantly affected the Ca2+in profile in PAO1. When grown at 5 mM Ca2+, disruption of czcB and mexE abolished the ability of PAO1 to maintain [Ca2+in] level. Disruption of mexJ reduced the response to Ca2+. These observations suggest that at least three RND pumps, MexJK, CzcCBA, and MexEF contribute to the maintenance of Ca2+in homeostasis.

Our earlier studies showed that elevated Ca2+ induces the production of secreted virulence factors and plant infectivity in P. aeruginosa (8). Several RND systems exemplified by MuxABC-OpmB and MexGHI-OpmD were shown to contribute to P. aeruginosa virulence (9, 10). Considering the above and the presented here findings that Ca2+ regulates the expression of multiple RNDs and that at least three RNDs contribute to maintaining Ca2+in homeostasis, we tested whether any of the 12 RND transporters play role in Ca2+-induced virulence of the pathogen. For this, we used lettuce leaves (Lactuca sativa) as an infection model and measured the disease area in the midribs of the leaves infected with PAO1 or RND transposon mutants grown at different Ca2+ levels. Four mutants with disrupted mexC, mexI, mexJ, or triA, when grown at elevated Ca2+, reduced their ability to cause disease by at least twofold in comparison to PAO1, but showed no significant difference in disease development when grown without added Ca2+. This indicates that the RND systems contribute to Ca2+-induced virulence of the pathogen.

To summarize, elevation of extracellular Ca2+ causes a transient increase in [Ca2+]in, which regulates the transcription of several efflux pumps resulting in Ca2+-induced tobramycin resistance and host infectivity. This is the first report of the regulatory relationship between [Ca2+in] homeostasis and Ca2+-induced antibiotic resistance illustrating a novel mechanism regulating P. aeruginosa adaptive resistance in response to elevated Ca2+.



1. Young DC, Zobell JT, Stockmann C, Waters CD, Ampofo K, Sherwin CMT, et al. Optimization of Anti-Pseudomonal Antibiotics for Cystic Fibrosis Pulmonary Exacerbations: V. Aminoglycosides. Pediatric Pulmonology. 2013;48(11):1047-61.
2. Chuchalin A, Amelina E, Bianco F. Tobramycin for inhalation in cystic fibrosis: Beyond respiratory improvements. Pulmonary Pharmacology & Therapeutics. 2009;22(6):526-32.
3. Zobell JT, Young DC, Waters CD, Ampofo K, Stockmann C, Sherwin CMT, et al. Optimization of anti-pseudomonal antibiotics for cystic fibrosis pulmonary exacerbations: VI. Executive summary. Pediatric Pulmonology. 2013;48(6):525-37.
4. Halmerbauer G, Arri S, Schierl M, Strauch E, Koller DY. The relationship of eosinophil granule proteins to ions in the sputum of patients with cystic fibrosis. Clinical and Experimental Allergy. 2000;30(12):1771-6.
5. Lorin MI, Gaerlan PF, Mandel ID, Denning CR. Composition of nasal secretion in patients with cystic fibrosis. The Journal of laboratory and clinical medicine. 1976;88(1):114-7.
6. Guragain M, Lenaburg DL, Moore FS, Reutlinger I, Patrauchan MA. Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility. Cell Calcium. 2013;54(5):350-61.
7. Goldberg M, Pribyl T, Juhnke S, Nies DH. Energetics and topology of CzcA, a cation/proton antiporter of the resistance-nodulation-cell division protein family. Journal of Biological Chemistry. 1999;274(37):26065-70.
8. Sarkisova S, Patrauchan MA, Berglund D, Nivens DE, Franklin MJ. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. Journal of Bacteriology. 2005;187(13):4327-37.
9. Yang L, Chen L, Shen L, Surette M, Duan K. Inactivation of MuxABC-OpmB Transporter System in Pseudomonas aeruginosa Leads to Increased Ampicillin and Carbenicillin Resistance and Decreased Virulence. Journal of Microbiology. 2011;49(1):107-14.
10. Aendekerk S, Diggle SP, Song Z, Hoiby N, Cornelis P, Williams P, et al. The MexGHI-OpmD multidrug efflux pump controls growth, antibiotic susceptibility and virulence in Pseudomonas aeruginosa via 4-quinolone-dependent cell-to-cell communication. Microbiology-Sgm. 2005;151:1113-25.