Calcium induces tobramycin resistance in Pseudomonas aeruginosa by regulating RND efflux pumps
Graphical abstract
Introduction
Pseudomonas aeruginosa causes severe infections in lung airways of cystic fibrosis (CF) patients, in burn wounds, as well as in intensive care patients and patients with indwelling medical devices, catheters and shunts [1], [2]. P. aeruginosa is also one of the leading causes of infective endocarditis [3], [4]. The high morbidity and mortality of Pseudomonas infections is mainly attributed to the combination of multifactorial virulence, outstanding antimicrobial resistance, and physiological adaptability of this organism [5], [6]. Besides its ability to undergo genetic alterations, P. aeruginosa possesses multiple mechanisms of intrinsic and adaptive resistance, that together make it resistant to most antimicrobials available for treatments. Efflux mediated antibiotic resistance in P. aeruginosa has been recognized as one of the major determinants of its intrinsic resistance [5], [7]. Among five families of efflux pumps, resistance nodulation division (RND) family of transporters has drawn the most attention in this regard. It is mainly due to the fact that RND transporters effectively pump out a broad range of toxic substances, including antimicrobial drugs [7], [8]. So far, 12 efflux pumps have been identified in P. aeruginosa PAO1 genome: MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexPQ-OprM, MexXY-OprM, MexVW-OprM, MexMN-OprJ, MexJK-OprM, TriABC-OpmB, MuxABC-OpmB, MexGHI-OpmD, and CzcCBA-OpmY [9].
RND efflux pumps are comprised of three components including inner membrane component (RND), periplasmic membrane fusion protein (MFP) and outer membrane porin, thus spanning both inner and outer membranes. Their role in P. aeruginosa physiology is not limited to efflux, and includes growth control [10], biofilm formation [11], oxidative [12] and nitrosative stress responses [13], as well as transport of signaling molecules involved in cell–cell communication [10], [14], [15]. Furthermore, RND efflux pumps play role in host colonization by modulating such mechanisms of pathogen invasion as pyocyanin production and cell motility [14], [16], [17], [18].
Calcium (Ca2+) is an essential messenger regulating a great number of vital eukaryotic processes [19], [20]. Imbalance in Ca2+ homeostasis is associated with many human diseases including those associated with bacterial infections, for example, infective endocarditis and CF [21], [22], [23]. There is an elevated level of Ca2+ in mitral annulus of endocarditis patients [24], as well as in pulmonary fluids of CF patients [25], [26]. Thus, Ca2+ likely serves as a host factor triggering physiological adjustments in the invading bacterial pathogens. In agreement, our earlier studies showed that elevated Ca2+ enhances P. aeruginosa biofilm formation, production of several virulence factors, including pyocyanin, extracellular proteases, and alginate [27], [28]. Furthermore, Ca2+ and Mg2+ modulate antibiotic resistance in P. aeruginosa to gentamycin [29], tetracycline, carbenicillin, polymyxin B [30], [31], and chloramphenicol [32]. Whereas several resistance mechanisms regulated by low Mg2+ have been characterized [33], [34], very little is known about the underlying mechanisms of Ca2+ regulation. The roles of cations in P. aeruginosa antimicrobial resistance have been mainly attributed to reduced cell membrane permeability, which consequently reduces the uptake of cationic antibiotics like polycationic polypeptides and aminoglycosides [35], [36]. It has been also suggested that P. aeruginosa can utilize the outer membrane protein OprH (H1), also cationic in nature, to stabilize the membrane integrity and to reduce the uptake of cationic antibiotics when deficient in magnesium [37]. Finally, the multidrug efflux pump MexXY-OprM has been shown to be required for the antagonistic effect of Ca2+ and Mg2+ on aminoglycosides resistance in P. aeruginosa [38].
Earlier we showed that P. aeruginosa maintains intracellular Ca2+ homeostasis, and the level of intracellular Ca2+ concentration ([Ca2+in]) is responsive to changes in extracellular Ca2+ [39] as well as to membrane permeabilizers (not published). Furthermore, we identified several putative Ca2+ transporters playing role in maintaining Ca2+in homeostasis, whose disruption disturbed Ca2+- induced swarming [39]. Here we hypothesize that Ca2+-dependent increase of antibiotic resistance in P. aeruginosa is regulated by the transient changes in [Ca2+in], which are generated in response to sudden addition of extracellular Ca2+. This novel perspective is important for understanding the mechanisms of adaptive antibiotic resistance in bacterial pathogens.
This study showed that tobramycin resistance is significantly increased in P. aeruginosa grown at elevated Ca2+. To characterize the mechanisms of this induction, we applied a global proteomic approach and identified several RND transporters, whose abundance was affected during growth at elevated Ca2+. Analysis of the corresponding transposon mutants determined that six RND transporters are involved in Ca2+-induced tobramycin resistance. We also determined that Ca2+ affects the transcription of several RND transporters, and this effect is mediated by changes in [Ca2+in]. Finally, we identified the role of RND transporters in maintaining Ca2+in homeostasis and Ca2+-induced plant infectivity in P. aeruginosa. Overall, this is the first report of the regulatory relationship between [Ca2+in] homeostasis and Ca2+-induced antibiotic resistance.
Section snippets
Bacterial strains, plasmids, and media
P. aeruginosa strain PAO1, the non-mucoid sequenced strain was used in the study [40]. Biofilm minimal medium (BMM) [27] contained (per liter): 9.0 mM sodium glutamate, 50 mM glycerol, 0.02 mM MgSO4, 0.15 mM NaH2PO4, 0.34 mM K2HPO4, 145 mM NaCl, 20 μl trace metals, and 1 ml vitamin solution. Trace metal solution (per liter of 0.83 M HCl): 5.0 g CuSO4·5H2O, 5.0 g ZnSO4·7H2O, 5.0 g FeSO4·7H2O, 2.0 g MnCl2·4H2O). Vitamins solution (per liter): 0.5 g thiamine, 1 mg biotin. pH of the medium was adjusted to 7.0.
Ca2+ enhances resistance of P. aeruginosa PAO1 to tobramycin
To determine whether Ca2+ affects tobramycin resistance in P. aeruginosa PAO1, we measured the minimal inhibitory concentration (MIC) of this aminoglycoside antibiotic that is commonly used to treat P. aeruginosa infections. For this, PAO1 was grown in BMM at high (5 mM) and low (not added) concentrations of CaCl2, and the MIC of the antibiotic was determined by using both conventional serial dilution approach and E-strips from BioMerieux. Resistance to tobramycin was increased almost 10 fold
Discussion
Pseudomonas is one of the leading causes of severe and life threatening infections in patients with compromised immune system, CF patients, patients with burn wounds, chronic obstructive pulmonary diseases, endocarditis, etc. At present, several types of antibiotics including aminoglycosides are considered to be an effective choice for treating Pseudomonas infections [61], [62], [63]. However, the increasing resistance of P. aeruginosa to most available antimicrobials represents a serious
Supplementary references
Following references cited in supplementary files:
[94], [99].
Acknowledgments
We thank Dr. Steve Hartson and Janet Rogers at the OSU Proteomic Facility for performing MS-based protein identification and analyses. We thank the RT-qPCR core facility at the OSU Department of Botany for support and instrumentation. We also thank Drs. Peter Hoyt and Hong Hwang at the Biochemistry and Molecular Biology Array and Bioinformatics core facility for their help with RNA analyses. We would like to thank Dr. Kangmin Duan at University of Manitoba, Winnipeg, Canada and Dr. Meng Meng
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