The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates effector toxins into host cells, impeding the host's rapid innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics in P. aeruginosa infections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structureactivity relationships, including striking stereoselectivity, in its inhibition of P. aeruginosa T3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS gene pscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles of pscF, together with its chaperone and cochaperone genes pscE and pscG, to a ⌬pscF P. aeruginosa strain demonstrated that each of the single-codon mutations in pscF is necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF.

is a leading cause of intra-abdominal infections, wound infections, and community-acquired folliculitis, each of which may involve macro- or microabscess formation. The rising incidence of multidrug resistance among isolates has increased both the economic burden and the morbidity and mortality associated with disease and necessitates a search for novel therapeutics. Previous work from our group detailed novel phenoxyacetamide inhibitors that block type III secretion and injection into host cells In this study, we used a mouse model of abscess formation to test the efficacy of these compounds against the type III secretion system (T3SS). Bacteria used the T3SS to intoxicate infiltrating neutrophils to establish abscesses. Despite this antagonism, sufficient numbers of functioning neutrophils remained for proper containment of the abscesses, as neutrophil depletion resulted in an increased abscess size, the formation of dermonecrotic lesions on the skin, and the dissemination of to internal organs. Consistent with the specificity of the T3SS-neutrophil interaction, bacteria lacking a functional T3SS were fully capable of causing abscesses in a neutropenic host. Phenoxyacetamide inhibitors attenuated abscess formation and aided in the immune clearance of the bacteria. Finally, a strain resistant to the phenoxyacetamide compound was fully capable of causing abscess formation even in the presence of the T3SS inhibitors. Together, our results further define the role of type III secretion in murine abscess formation and demonstrate the efficacy of phenoxyacetamide inhibitors in infection.

The increasing prevalence of drug-resistant bacterial infections is driving the discovery and development not only of new antibiotics, but also of inhibitors of virulence factors that are crucial for in vivo pathogenicity. One such virulence factor is the type III secretion system (T3SS), which plays a critical role in the establishment and dissemination of Pseudomonas aeruginosa infections. We have recently described the discovery and characterization of a series of inhibitors of P. aeruginosa T3SS based on a phenoxyacetamide scaffold. To better characterize the factors involved in potent T3SS inhibition, we have conducted a systematic exploration of this structure, revealing several highly responsive structure-activity relationships indicative of interaction with a specific target. Most of the structural features contributing to potency were additive, and combination of those features produced optimized inhibitors with IC50 values <1 µM.

bNovel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC 50 ] > 80 M). The most potent SABA analogs had MICs of 0.5 to 7.0 M (0.2 to 3.0 g/ml) against an efflux-deficient Escherichia coli (⌬tolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII. Pseudomonas aeruginosa is a highly virulent, persistent human pathogen with both acquired and intrinsic drug resistances. It is the most common cause of nosocomial pneumonia, causing 15% to 20% of hospital-acquired pneumonias (1), and up to 75% of patients in intensive care units are colonized with this pathogen (2). P. aeruginosa is also becoming a major cause of communityacquired pneumonia in severely ill patients (3). An astounding 30% of clinical isolates from critically ill patients are resistant to three or more drugs, which leads to treatment failure (4). The discovery and development of new classes of antibiotics, which are not subject to existing target-based resistance mechanisms, is an important strategy in combating drug resistance, and targeting unexploited or underexploited essential bacterial pathways has been a successful strategy for discovering new compound classes (5, 6).This study focused on the fatty acid synthesis type II (FASII) pathway in P. aeruginosa. While most bacteria utilize exogenous fatty acids for phospholipid synthesis (7), the FASII pathway is absolutely essential for bacterial membrane biogenesis in Gramnegative bacteria because the hydroxyl-fatty acid constituents of LPS cannot be obtained from an extracellular source (8). FASII inhibitors also block the production of Gram-negative signaling molecules, including homoserine lactones and hydroxyquinolines, which are important to establish and maintain P. aeruginosa virulence (9). This FASII macromolecular synthesis pathway is conserved and essential in Gram-negative bacteria but is absent from the mammalian cytopl...

The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether WT, constitutive, or PhA-resistant, was dominant over other pscF alleles expressed from non-native loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at non-native loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation as well as carrying out secretion and translocation. Importance. P. aeruginosa effector toxin translocation into host innate immune cells is critical for the establishment and dissemination of P. aeruginosa infections. The medical need for new anti-P. aeruginosa agents is evident by the fact that P. aeruginosa ventilator-associated pneumonia is associated with a high mortality rate (40%-69%) and recurs in >30% of patients, even with standard-of-care antibiotic therapy. The results described here confirm roles for the needle in T3SS secretion, translocation and suggest that it affects regulation, possibly by interaction with T3SS regulatory proteins. Results also support a model of direct interaction of the needle with PhA, and suggest that with further development, members of the PhA series may prove useful as drugs for P. aeruginosa infection.