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Etiological agents of acute, persistent, or relapsing clinical infections are often refractory to antibiotics due to multidrug resistance and/or antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that causes recalcitrant and severe acute chronic and persistent human infections. Here, we target the MvfR-regulated P. aeruginosa quorum sensing (QS) virulence pathway to isolate robust molecules that specifically inhibit infection without affecting bacterial growth or viability to mitigate selective resistance. Using a whole-cell high-throughput screen (HTS) and structure-activity relationship (SAR) analysis, we identify compounds that block the synthesis of both pro-persistence and pro-acute MvfR-dependent signaling molecules. These compounds, which share a benzamide-benzimidazole backbone and are unrelated to previous MvfR-regulon inhibitors, bind the global virulence QS transcriptional regulator, MvfR (PqsR); inhibit the MvfR regulon in multi-drug resistant isolates; are active against P. aeruginosa acute and persistent murine infections; and do not perturb bacterial growth. In addition, they are the first compounds identified to reduce the formation of antibiotic-tolerant persister cells. As such, these molecules provide for the development of next-generation clinical therapeutics to more effectively treat refractory and deleterious bacterial-human infections., Author Summary Antibiotic resistant and tolerant bacterial pathogens are responsible for acute, chronic and persistent human infections recalcitrant to any current treatments. Therefore, there is an urgent need to identify new antimicrobial drugs that will help circumvent the current antibiotic resistance crisis. Bacterial pathogens often develop resistance to antibiotic drugs that target bacterial growth or viability. In contrast, strategies that specifically target virulence pathways non-essential for growth could limit selective resistance, and thus are candidates for the development of next-generation antimicrobial therapeutics. In this study we target the bacterial communication system MvfR (PqsR), which is known to control virulence of the opportunistic bacterial pathogen Pseudomonas aeruginosa. We identified and improved upon new small molecules that effectively silence the MvfR communication system, and as a result block P. aeruginosa virulence both in vitro and in vivo. Moreover, these new compounds are the first known to restrict the ability of bacteria to form antibiotic-tolerant cells and consequently proved to be very effective at preventing persistent infection in a mammalian infection model. Because of their ability to simultaneously block acute and persistent infections, these new molecules may provide a very strong basis for the development of next generation antimicrobials.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections., Author Summary P. aeruginosa causes acute as well as chronic infections in humans. In this paper we report the identification of a P. aeruginosa small molecule, 2-AA, that modulates this pathogen's virulence to promote chronic infections. We show that the synthesis of 2-AA, responsible for the grape-like odor of P. aeruginosa cultures and of wound infections, is controlled by the multiple virulence factor regulator (MvfR) important for virulence in acute infections. 2-AA reduces the production of MvfR-regulated acute virulence factors, and attenuates acute virulence by negatively fine-tuning the MvfR regulon activity. Moreover, we show that 2-AA adapts P. aeruginosa for chronic infections by promoting mutations in a key acute virulence gene (lasR) and by prolonging bacterial survival. The findings presented here reveal the function of a new MvfR-regulated molecule, and highlight MvfR's importance as a highly promising target for the development of inhibitors that can simultaneously halt acute and chronic infections caused by P. aeruginosa, and possibly by other pathogenic bacteria. This study uncovers insights that paradigmatically pave the way for the search of 2-AA-like small volatile molecules that promote pathogen adaptation and establishment of chronic infections caused by foreboding human pathogens.

Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens., Author Summary Current treatments of human bacterial infections depend on antibiotics, whose long-term effectiveness is limited as they select for multidrug-resistant pathogens. An alternative approach that is likely to limit the development of bacterial super pathogens is to selectively disrupt bacterial virulence mechanisms without affecting bacterial viability. Quorum sensing (QS), a highly regulated bacterial communication system, is a promising candidate target because it is used by numerous pathogens to stimulate and coordinate the expression of many virulence determinants, and its disruption does not affect bacterial cell viability. We have identified three compounds that efficiently inhibited the synthesis of molecules required for the activation of the human opportunistic pathogen Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway that controls the expression of key virulence genes. We showed that prevention of MvfR pathway activation disrupted MvfR-dependent gene expression, and limited P. aeruginosa infection in mice, without perturbing bacterial viability. In addition, the compounds identified limited the ability of a number of bacterial pathogens to tolerate salt, a widespread bacterial function, and possibly other functions relevant to pathogenesis. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens.