Non-interchangeable functions of efflux transporters of Pseudomonas aeruginosa in survival under infection-associated stress

Abstract

Pseudomonas aeruginosa is a challenging opportunistic pathogen due to its intrinsic and acquired mechanisms of antibiotic resistance. A large repertoire of efflux transporters actively expels antibiotics, toxins, and metabolites from cells and enables growth of P. aeruginosa in diverse environments. In this study, we analyzed the roles of representative efflux pumps from the Resistance-Nodulation-Division (RND), Major Facilitator Superfamily (MFS), and Small Multidrug Resistance (SMR) families of proteins in the susceptibility of P. aeruginosa to antibiotics and bacterial growth under stresses imposed by human hosts during bacterial infections: an elevated temperature, osmotic stress, low iron, bile salts, and acidic pH. We selected five RND pumps MexAB-OprM, MexEF-OprN, MexCD-OprJ, MuxABC-OpmB, and TriABC-OpmH that differ in their substrate specificities and expression profiles, two MFS efflux pumps PA3136-3137 and PA5158-5160 renamed here into MfsAB and MfsCD-OpmG, respectively, and an SMR efflux transporter PA1540-1541 (MdtJI). We found that the most promiscuous RND pumps such as MexEF-OprN and MexAB-OprM are integrated into diverse survival mechanisms and enable P. aeruginosa growth under various stresses. MuxABC-OpmB and TriABC-OpmH pumps with narrower substrate spectra are beneficial only in the presence of the iron chelator 2,2′-dipyridyl and bile salts, respectively. MFS pumps do not contribute to antibiotic efflux but play orthogonal roles in acidic pH, low iron, and in the presence of bile salts. In contrast, MdtJI protects against polycationic antibiotics but does not contribute to survival under stress. Thus, efflux pumps play specific, non-interchangeable functions in P. aeruginosa cell physiology and bacterial survival under stresses. IMPORTANCE The role of multidrug efflux pumps in the intrinsic and clinical levels of antibiotic resistance in Pseudomonas aeruginosa and other gram-negative bacteria is well-established. Their functions in bacterial physiology, however, remain unclear. The P. aeruginosa genome comprises an arsenal of efflux pumps from different protein families, the substrate specificities of which are typically assessed by measuring their impact on susceptibility to antibiotics. In this study, we analyzed how deletions and overproductions of efflux pumps affect P. aeruginosa growth under human-infection-induced stresses. Our results show that the physiological functions of multidrug efflux pumps are non-redundant and essential for the survival of this important human pathogen under stress.