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ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals and individuals with underlying pulmonary disorders. P. aeruginosa virulence is controlled by quorum sensing (QS), a bacterial cell-cell communication mechanism that underpins transitions between individual and group behaviors. In P. aeruginosa , the PqsE enzyme and the QS receptor RhlR directly interact to control the expression of genes involved in virulence. Here, we show that three surface-exposed arginine residues on PqsE comprise the site required for interaction with RhlR. We show that a noninteracting PqsE variant [PqsE(NI)] possesses catalytic activity, but is incapable of promoting virulence phenotypes, indicating that interaction with RhlR, and not catalysis, drives these PqsE-dependent behaviors. Biochemical characterization of the PqsE-RhlR interaction coupled with RNA-seq analyses demonstrates that the PqsE-RhlR complex increases the affinity of RhlR for DNA, enabling enhanced expression of genes encoding key virulence factors. These findings provide the mechanism for PqsE-dependent regulation of RhlR and identify a unique regulatory feature of P. aeruginosa QS and its connection to virulence. IMPORTANCE Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI). QS is required for virulence inmore »the human pathogen Pseudomonas aeruginosa , which can cause fatal infections in patients with underlying pulmonary disorders. In this study, we determine the molecular basis for the physical interaction between two virulence-driving QS components, PqsE and RhlR. We find that the ability of PqsE to bind RhlR correlates with virulence factor production. Since current antimicrobial therapies exacerbate the growing antibiotic resistance problem because they target bacterial growth, we suggest that the PqsE-RhlR interface discovered here represents a new candidate for targeting with small molecule inhibition. Therapeutics that disrupt the PqsE-RhlR interaction should suppress virulence. Targeting bacterial behaviors such as QS, rather than bacterial growth, represents an attractive alternative for exploration because such therapies could potentially minimize the development of resistance.« less

ABSTRACT Streptococcus sanguinis is a common cause of infective endocarditis (IE). Efforts by research groups are aimed at identifying and characterizing virulence factors that contribute to the ability of this organism to cause IE. This Gram-positive pathogen causes heart infection by gaining access to the bloodstream, adhering to host extracellular matrix protein and/or platelets, colonizing the aortic endothelium, and incorporating itself into the aortic vegetation. While many virulence factors have been reported to contribute to the ability of S. sanguinis to cause IE, it is noteworthy that type IV pili (T4P) have not been described to be a virulence factor in this organism, although S. sanguinis strains typically encode these pili. Type IV pili are molecular machines that are capable of mediating diverse virulence functions and surface motility. T4P have been shown to mediate twitching motility in some strains of S. sanguinis , although in most strains it has been difficult to detect twitching motility. While we found that T4P are dispensable for direct in vitro platelet binding and aggregation phenotypes, we show that they are critical to the development of platelet-dependent biofilms representative of the cardiac vegetation. We also observed that T4P are required for in vitro invasion ofmore »S. sanguinis into human aortic endothelial cells, which indicates that S. sanguinis may use T4P to take advantage of an intracellular niche during infection. Importantly, we show that T4P of S. sanguinis are critical to disease progression (vegetation development) in a native valve IE rabbit model. The results presented here expand our understanding of IE caused by S. sanguinis and identify T4P as an important virulence factor for this pathogen. IMPORTANCE This work provides evidence that type IV pili produced by Streptococcus sanguinis SK36 are critical to the ability of these bacteria to attach to and colonize the aortic heart valve (endocarditis). We found that an S. sanguinis type IV pili mutant strain was defective in causing platelet-dependent aggregation in a 24-h infection assay but not in a 1-h platelet aggregation assay, suggesting that the type IV pili act at later stages of vegetation development. In a rabbit model of disease, a T4P mutant strain does not develop mature vegetations that form on the heart, indicating that this virulence factor is critical to disease and could be a target for IE therapy.« less