Functional interplay between RND efflux pumps and GacS in <i>Pseudomonas aeruginosa</i>

<i>Pseudomonas aeruginosa</i> is an opportunistic human pathogen that is ubiquitous in the environment. The environmental versatility of <i>P. aeruginosa</i> is often attributed to a large arsenal of active efflux pumps, low permeability of the outer membrane, and extensive regulatory networks. In this study, we analyzed putative functional interplay between polyspecific drug efflux pumps of the Resistance-Nodulation-Division (RND) superfamily of proteins and the GacSA two-component regulatory system. RND transporters are the major contributors to antibiotic resistance and survival under various environmental stresses, whereas GacSA is a global regulator and governs critical lifestyles during human colonization. We found that the inactivation of either RND pumps or <i>gacS</i> induces broad, partially overlapping responses in lifestyle (e.g., rhamnolipid biosynthesis, heat shock response, and quorum sensing), virulence (e.g., proteases, toxins, and phenazines secretion systems), and metabolic programs (e.g., electron, iron capture, synthesis/degradation of ketone bodies, and cyanoamino acid metabolism) that are more pronounced during the exponential than the stationary phase. GacSA and RND efflux pumps induced opposite responses in the gene expression of <i>P. aeruginosa</i>, whereas the overexpression of GacS was additive with the deletion of efflux pumps in gene expression and under growth conditions typically associated with human infections such as elevated temperature and iron deprivation. We conclude that RND efflux and GacSA networks partially overlap with each other.IMPORTANCERND efflux pumps are the major contributors to intrinsic and clinical levels of antibiotic resistance of <i>P. aeruginosa,</i> and their expression is regulated by various environmental factors. However, functional relationships between active efflux pumps and regulatory networks involved in virulence and pathogenesis of <i>P. aeruginosa</i> remain unclear. This study showed that the loss of RND-dependent efflux functions or the inactivation of the <i>gacS</i> pathway induces partially overlapping transcriptional and physiological responses, suggesting that these pathways could be complementary to each other in hostile environments.