Multi-tiered actions of Legionella effectors to modulate host Rab10 dynamics.

Rab GTPases are representative targets of manipulation by intracellular bacterial pathogens for hijacking membrane trafficking. Legionella pneumophila recruits many Rab GTPases to its vacuole and exploits their activities. Here, we found that infection-associated regulation of Rab10 dynamics involves ubiquitin signaling cascades mediated by the SidE and SidC families of Legionella ubiquitin ligases. Phosphoribosyl-ubiquitination of Rab10 catalyzed by the SidE ligases is crucial for its recruitment to the bacterial vacuole. SdcB, the previously uncharacterized SidC-family effector, resides on the vacuole and contributes to retention of Rab10 at the late stages of infection. We further identified MavC as a negative regulator of SdcB. By the transglutaminase activity, MavC crosslinks ubiquitin to SdcB and suppresses its function, resulting in elimination of Rab10 from the vacuole. These results demonstrate that the orchestrated actions of many L. pneumophila effectors fine-tune the dynamics of Rab10 during infection.

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA.

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.

Synthetic engineering and biological containment of bacteriophages.

The serious threats posed by drug-resistant bacterial infections and recent developments in synthetic biology have fueled a growing interest in genetically engineered phages with therapeutic potential. To date, many investigations on engineered phages have been limited to proof of concept or fundamental studies using phages with relatively small genomes or commercially available "phage display kits". Moreover, safeguards supporting efficient translation for practical use have not been implemented. Here, we developed a cell-free phage engineering and rebooting platform. We successfully assembled natural, designer, and chemically synthesized genomes and rebooted functional phages infecting gram-negative bacteria and acid-fast mycobacteria. Furthermore, we demonstrated the creation of biologically contained phages for the treatment of bacterial infections. These synthetic biocontained phages exhibited similar properties to those of a parent phage against lethal sepsis in vivo. This efficient, flexible, and rational approach will serve to accelerate phage biology studies and can be used for many practical applications, including phage therapy.

Tackling recalcitrant Pseudomonas aeruginosa infections in critical illness via anti-virulence monotherapy.

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.

Reversible modification of mitochondrial ADP/ATP translocases by paired Legionella effector proteins.

Adenosine diphosphate (ADP) ribosylation is a reversible posttranslational modification involved in the regulation of numerous cellular processes. Prototype ADP ribosyltransferases (ARTs) from many pathogenic bacteria are known to function as toxins, while other bacterial ARTs have just recently emerged. Recent studies have shown that bacteria also possess enzymes that function as poly-ADP ribose (ADPr) glycohydrolases (PARGs), which reverse poly-ADP ribosylation. However, how bacteria manipulate host target proteins by coordinated reactions of ARTs and ADPr hydrolases (ARHs) remains elusive. The intracellular bacterial pathogen Legionella pneumophila, the causative agent of Legionnaires' disease, transports a large array of effector proteins via the Dot/Icm type IV secretion system to host cells. The effector proteins, which mostly function as enzymes, modulate host cellular processes for the bacteria's benefit. In this study, we identified a pair of L. pneumophila effector proteins, Lpg0080 and Lpg0081, which function as an ART and an ARH, respectively. The two proteins were shown to coordinately modulate mitochondrial ADP/adenosine triphosphate (ATP) translocases (ANTs) by their enzymatic activities to conjugate ADPr to, and remove it from, a key arginine residue. The crystal structures of Lpg0081 and the Lpg0081:ADPr complex indicated that Lpg0081 is a macroD-type ARH with a noncanonical macrodomain, whose folding topology is strikingly distinct from that of the canonical macrodomain that is ubiquitously found in eukaryotic PARGs and ARHs. Our results illustrate that L. pneumophila has acquired an effector pair that coordinately manipulate mitochondrial activity via reversible chemical modification of ANTs.

Requirement of phosphatidic acid binding for distribution of the bacterial protein Lpg1137 targeting syntaxin 17.

The gram-negative bacterium, Legionella pneumophila is known to manipulate the host cellular functions. L. pneumophila secretes bacterial proteins called Legionella effectors into the host cytosol that are necessary for these manipulations. The Legionella effector Lpg1137 was identified as a serine protease responsible for the degradation of syntaxin 17 (Stx17). However, how Lpg1137 specifically recognizes and degrades Stx17 remained unknown. Given that Stx17 is localized in the ER, mitochondria-associated membrane (MAM), and mitochondria, Lpg1137 likely distributes to these compartments to recognize Stx17. Here, we show that the C-terminal region of Lpg1137 binds to phosphatidic acid (PA), a MAM and mitochondria-enriched phospholipid, and that this binding is required for the correct intracellular distribution of Lpg1137. Two basic residues in the C-terminal region of Lpg1137 are required for PA binding and their mutation causes mislocalization of Lpg1137. This mutant also fails to degrade Stx17 while retaining protease activity. Taken together, our data reveal that Lpg1137 utilizes PA for its distribution to the membranous compartments in which Stx17 is localized.

Recent advances in structural studies of the Legionella pneumophila Dot/Icm type IV secretion system.

The intracellular bacterial pathogen Legionella pneumophila utilizes the Dot/Icm type IV secretion system to translocate approximately 300 effector proteins to establish a replicative niche known as the Legionella-containing vacuole. The Dot/Icm system is classified as a type IVB secretion system, which is evolutionarily closely related to the I-type conjugation systems and is distinct from type IVA secretion systems, such as the Agrobacterium VirB/D4 system. Although both type IVA and IVB systems directly transport nucleic acids or proteins into the cytosol of recipient cells, the components and architecture of type IVB systems are much more complex than those of type IVA systems. Taking full advantage of rapidly developing cryo-electron microscopy techniques, the structural details of the transport apparatus and coupling complexes in the Dot/Icm system have been clarified in the past few years. In this review, we summarize recent progress in the structural studies of the L. pneumophila type IVB secretion system and the insights gained into the mechanisms of substrate recognition and transport.

Protocol for imaging proteins associated with Legionella-containing vacuoles in host cells.

The intracellular bacterial pathogen Legionella pneumophila exploits host cellular systems using approximately 300 effector proteins to establish a replicative niche known as the Legionella-containing vacuole (LCV). During infection, both host and bacterial proteins interactively function on the LCVs. Here, we describe a detailed step-by-step protocol to visualize proteins associated with LCVs in host cells. This protocol can aid in analyzing whether a protein of interest influences the subcellular localization of LCV-associated proteins during infection. For complete details on the use and execution of this protocol, please refer to Kitao et al. (2020).

Isolation and Characterization of a Novel Phage SaGU1 that Infects Staphylococcus aureus Clinical Isolates from Patients with Atopic Dermatitis.

The bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus.

Staphylococcal Phage in Combination with Staphylococcus Epidermidis as a Potential Treatment for Staphylococcus Aureus-Associated Atopic Dermatitis and Suppressor of Phage-Resistant Mutants.

Atopic dermatitis is accompanied by the abnormal overgrowth of Staphylococcus aureus, a common cause of skin infections and an opportunistic pathogen. Although administration of antibiotics is effective against S. aureus, the resulting reduction in healthy microbiota and the emergence of drug-resistant bacteria are of concern. We propose that phage therapy can be an effective strategy to treat atopic dermatitis without perturbing the microbiota structure. In this study, we examined whether the S. aureus phage SaGU1 could be a tool to counteract the atopic exacerbation induced by S. aureus using an atopic mouse model. Administration of SaGU1 to the back skin of mice reduced both S. aureus counts and the disease exacerbation caused by S. aureus. Furthermore, the S. aureus-mediated exacerbation of atopic dermatitis with respect to IgE plasma concentration and histopathological findings was ameliorated by the application of SaGU1. We also found that Staphylococcus epidermidis, a typical epidermal symbiont in healthy skin, significantly attenuated the emergence of SaGU1-resistant S. aureus under co-culture with S. aureus and S. epidermidis in liquid culture infection experiments. Our results suggest that phage therapy using SaGU1 could be a promising clinical treatment for atopic dermatitis.

Divergence of Legionella Effectors Reversing Conventional and Unconventional Ubiquitination.

The intracellular bacterial pathogen Legionella pneumophila employs bacteria-derived effector proteins in a variety of functions to exploit host cellular systems. The ubiquitination machinery constitutes a crucial eukaryotic system for the regulation of numerous cellular processes, and is a representative target for effector-mediated bacterial manipulation. L. pneumophila transports over 300 effector proteins into host cells through its Dot/Icm type IV secretion system. Among these, several effector proteins have been found to function as ubiquitin ligases, including unprecedented enzymes that catalyze ubiquitination through unconventional mechanisms. Recent studies have identified many L. pneumophila effector proteins that can interfere with ubiquitination. These effectors include proteins that are distantly related to the ovarian tumor protein superfamily described as deubiquitinases (DUBs), which regulate important signaling cascades in human cells. Intriguingly, L. pneumophila DUBs are not limited to enzymes that exhibit canonical DUB activity. Some L. pneumophila DUBs can catalyze the cleavage of the unconventional linkage between ubiquitin and substrates. Furthermore, novel mechanisms have been found that adversely affect the function of specific ubiquitin ligases; for instance, effector-mediated posttranslational modifications of ubiquitin ligases result in the inhibition of their activity. In the context of L. pneumophila infection, the existence of enzymes that reverse ubiquitination primarily relates to a fine tuning of biogenesis and remodeling of the Legionella-containing vacuole as a replicative niche. The complexity of the effector arrays reflects sophisticated strategies that bacteria have adopted to adapt their host environment and enable their survival in host cells. This review summarizes the current state of knowledge on the divergent mechanisms of the L. pneumophila effectors that can reverse ubiquitination, which is mediated by other effectors as well as the host ubiquitin machinery.

Legionella Manipulates Non-canonical SNARE Pairing Using a Bacterial Deubiquitinase.

The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legionella-containing vacuole (LCV). On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L. pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB.

[Molecular biological studies toward controlling infectious diseases caused by multidrug-resistant Pseudomonas aeruginosa].

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes serious acute, persistent, and relapsing infections. Recent year, the effectiveness of antibiotics for eliminating P. aeruginosa infections has been further complicated by the emergence of multidrug-resistant strains. Thus, new approaches for the rapid detection and novel antimicrobial drug discovery are urgently needed to control such intractable infections caused by the pathogen. Also, we do need deep understanding of the drug resistance mechanisms to overcome this issue. Here I describe a brief review on my biological studies toward controlling infectious diseases caused by multidrug-resistant P. aeruginosa.

Emerging insights into bacterial deubiquitinases.

Bacterial pathogens utilize eukaryotic cellular systems in various ways for their own benefits. To counteract host immune responses and survive in cells, bacteria modify host signaling pathways. For this aim, they have evolved virulence secretion systems. Bacteria-encoded effector proteins delivered via these secretion systems are the key players in bacterial pathogenesis. Ubiquitination is a post-translational modification that governs eukaryotic cellular systems. Recent studies have revealed that many bacterial effector proteins target the host ubiquitin system, often acting as ubiquitin-modulating enzymes such as ubiquitin ligases and deubiquitinases. Emerging lines of evidence have unveiled the diversity of bacterial deubiquitinases and have provided insights into the bacterial strategy to exploit the host ubiquitin system.

LotA, a Legionella deubiquitinase, has dual catalytic activity and contributes to intracellular growth.

The intracellular bacterial pathogen, Legionella pneumophila, establishes the replicative niche as a result of the actions of a large array of effector proteins delivered via the Legionella Type 4 secretion system. Many effector proteins are expected to be involved in biogenesis and regulation of the Legionella-containing vacuole (LCV) that is highly decorated with ubiquitin. Here, we identified a Legionella deubiquitinase, designated LotA, by carrying out a genome analysis to find proteins resembling the eukaryotic ovarian tumour superfamily of cysteine proteases. LotA exhibits a dual ability to cleave ubiquitin chains that is dependent on 2 distinctive catalytic cysteine residues in the eukaryotic ovarian tumour domains. One cysteine dominantly contributes to the removal of ubiquitin from the LCVs by its polyubiquitin cleavage activity. The other specifically cleaves conjugated Lys6-linked ubiquitin. After delivered by the Type 4 secretion system, LotA localises on the LCVs via its PI(3)P-binding domain. The lipid-binding ability of LotA is crucial for ubiquitin removal from the vacuoles. We further analysed the functional interaction of the protein with the recently reported noncanonical ubiquitin ligases of L. pneumophila, revealing that the effector proteins are involved in coordinated regulation that contributes to bacterial growth in the host cells.

Molecular Insights into Function and Competitive Inhibition of Pseudomonas aeruginosa Multiple Virulence Factor Regulator.

New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and persistent infections. Recently identified non-ligand-based benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth. Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability. Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of MvfR regulation.IMPORTANCEPseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes serious acute, persistent, and relapsing infections. New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by this pathogen. The Pseudomonas aeruginosa quorum sensing transcription factor MvfR regulates functions important in both acute and persistent infections. We used recently identified inhibitors of MvfR to perform structural studies and reveal important insights that would benefit the optimization of anti-MvfR compounds. Altogether, the results reported here provide critical detailed mechanistic insights into the function of MvfR domains that may benefit the optimization of the chemical, pharmacological, and safety properties of MvfR antagonist series.

Polypharmacology Approaches against the Pseudomonas aeruginosa MvfR Regulon and Their Application in Blocking Virulence and Antibiotic Tolerance.

Pseudomonas aeruginosa is an important nosocomial pathogen that is frequently recalcitrant to available antibiotics, underlining the urgent need for alternative therapeutic options against this pathogen. Targeting virulence functions is a promising alternative strategy as it is expected to generate less-selective resistance to treatment compared to antibiotics. Capitalizing on our nonligand-based benzamide-benzimidazole (BB) core structure compounds reported to efficiently block the activity of the P. aeruginosa multiple virulence factor regulator MvfR, here we report the first class of inhibitors shown to interfere with PqsBC enzyme activity, responsible for the synthesis of the MvfR activating ligands HHQ and PQS, and the first to target simultaneously MvfR and PqsBC activity. The use of these compounds reveals that inhibiting PqsBC is sufficient to block P. aeruginosa's acute virulence functions, as the synthesis of MvfR ligands is inhibited. Our results show that MvfR remains the best target of this QS pathway, as we show that antagonists of this target block both acute and persistence-related functions. The structural properties of the compounds reported in this study provide several insights that are instrumental for the design of improved MvfR regulon inhibitors against both acute and persistent P. aeruginosa infections. Moreover, the data presented offer the possibility of a polypharmacology approach of simultaneous silencing two targets in the same pathway. Such a combined antivirulence strategy holds promise in increasing therapeutic efficacy and providing alternatives in the event of a single target's resistance development.

Pseudomonas aeruginosa Alginate Overproduction Promotes Coexistence with Staphylococcus aureus in a Model of Cystic Fibrosis Respiratory Infection.

While complex intra- and interspecies microbial community dynamics are apparent during chronic infections and likely alter patient health outcomes, our understanding of these interactions is currently limited. For example, Pseudomonas aeruginosa and Staphylococcus aureus are often found to coinfect the lungs of patients with cystic fibrosis (CF), yet these organisms compete under laboratory conditions. Recent observations that coinfection correlates with decreased health outcomes necessitate we develop a greater understanding of these interbacterial interactions. In this study, we tested the hypothesis that P. aeruginosa and/or S. aureus adopts phenotypes that allow coexistence during infection. We compared competitive interactions of P. aeruginosa and S. aureus isolates from mono- or coinfected CF patients employing in vitro coculture models. P. aeruginosa isolates from monoinfected patients were more competitive toward S. aureus than P. aeruginosa isolates from coinfected patients. We also observed that the least competitive P. aeruginosa isolates possessed a mucoid phenotype. Mucoidy occurs upon constitutive activation of the sigma factor AlgT/U, which regulates synthesis of the polysaccharide alginate and dozens of other secreted factors, including some previously described to kill S. aureus Here, we show that production of alginate in mucoid strains is sufficient to inhibit anti-S. aureus activity independent of activation of the AlgT regulon. Alginate reduces production of siderophores, 2-heptyl-4-hydroxyquinolone-N-oxide (HQNO), and rhamnolipids-each required for efficient killing of S. aureus These studies demonstrate alginate overproduction may be an important factor driving P. aeruginosa coinfection with S. aureusIMPORTANCE Numerous deep-sequencing studies have revealed the microbial communities present during respiratory infections in cystic fibrosis (CF) patients are diverse, complex, and dynamic. We now face the challenge of determining the influence of these community dynamics on patient health outcomes and identifying candidate targets to modulate these interactions. We make progress toward this goal by determining that the polysaccharide alginate produced by mucoid strains of P. aeruginosa is sufficient to inhibit multiple secreted antimicrobial agents produced by this organism. Importantly, these secreted factors are required to outcompete S. aureus, when the microbes are grown in coculture; thus we propose a mechanism whereby mucoid P. aeruginosa can coexist with S. aureus Finally, the approach used here can serve as a platform to investigate the interactions among other CF pathogens.

Evidence for Direct Control of Virulence and Defense Gene Circuits by the Pseudomonas aeruginosa Quorum Sensing Regulator, MvfR.

Pseudomonas aeruginosa defies eradication by antibiotics and is responsible for acute and chronic human infections due to a wide variety of virulence factors. Currently, it is believed that MvfR (PqsR) controls the expression of many of these factors indirectly via the pqs and phnAB operons. Here we provide strong evidence that MvfR may also bind and directly regulate the expression of additional 35 loci across the P. aeruginosa genome, including major regulators and virulence factors, such as the quorum sensing (QS) regulators lasR and rhlR, and genes involved in protein secretion, translation, and response to oxidative stress. We show that these anti-oxidant systems, AhpC-F, AhpB-TrxB2 and Dps, are critical for P. aeruginosa survival to reactive oxygen species and antibiotic tolerance. Considering that MvfR regulated compounds generate reactive oxygen species, this indicates a tightly regulated QS self-defense anti-poisoning system. These findings also challenge the current hierarchical regulation model of P. aeruginosa QS systems by revealing new interconnections between them that suggest a circular model. Moreover, they uncover a novel role for MvfR in self-defense that favors antibiotic tolerance and cell survival, further demonstrating MvfR as a highly desirable anti-virulence target.

Identification of anti-virulence compounds that disrupt quorum-sensing regulated acute and persistent pathogenicity.

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.