Identification of Small Molecule Inhibitors of Pseudomonas aeruginosa Exoenzyme S Using a Yeast Phenotypic Screen

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.

Newly Discovered and Characterized Antivirulence Compounds Inhibit Bacterial Mono-ADP-Ribosyltransferase Toxins

“…Effective inhibitors of mART (ExoA c ) activity can be screened in this yeast-based system, where such agents abrogate the growth defect phenotype (33). The yeast strains employed for initial screening were W303 (wild type), ERG6 Ϫ (which lacks the ⌬24-sterol C-methyltransferase), MTID:2955 (which lacks two master regulators in the expression of pleiotropic drug response elements), and 2775 and 7034 (deficient in mannosylphosphate transferase); however, no differences between the various yeast strains were observed, and so W303 was used as the tester strain for this study (3). In yeast, generally compounds from both the P and V series that were able to at least partially restore yeast growth (although V23 and V30 are exceptions) were nonpolar, lacked solubility in aqueous buffer, and were not ionizable (log values; see Tables S1 and S2 in the supplemental material), suggesting that the cell wall acts as a physical barrier, particularly to polar molecules.…”

“…Saccharomyces cerevisiae cells (strains W303, ERG6 Ϫ , MTID:2955, 2775, and 7034) expressing the catalytic domain of P. aeruginosa ExoA (ExoA c ) were cultured in the presence of 50 M (each) compound and 1% dimethyl sulfoxide (DMSO) in 96-well plates for 48 h as previously described (3,31).…”

Salicylidene Acylhydrazides and Hydroxyquinolines Act as Inhibitors of Type Three Secretion Systems in Pseudomonas aeruginosa by Distinct Mechanisms

“…Applied to hydroxyquinolines and salicylidene acylhydrazides, these techniques allowed us to position them as a potentially useful addition to our future therapeutic armamentarium, since both show activity against clinical isolates, disregarding their resistance profile to currently used antipseudomonal antibiotics. Compared to previously described inhibitors of iT3SS in P. aeruginosa (see reference 22 for a review), they may offer a broader spectrum of activity than (i) compounds blocking toxins only (51)(52)(53), the activity of which will probably be restricted to toxin-producing strains, and (ii) antibodies for which activity can be variable due to the polymorphism of the targeted antigens (54). The present data, together with the results obtained previously with INP1855 (35), strongly suggest an advantage for hydroxyquinolines over salicylidene acylhydrazides.…”

Database on the taxonomical characterisation and potential toxigenic capacities of microorganisms used for the industrial production of food enzymes and feed additives, which do not have a recommendation for Qualified Presumption of Safety

“…2-aminoacetophenone virulence factor [223] alkylquinoline signaling molecule [224] CFTR inhibitory factor virulence factor [225,226] elastase [227] ETA [228] ExoS virulence factor [228][229][230][231] ExoT virulence factor [228,229] Exotoxin A virulence factor [227,232] ExoU virulence factor [233][234][235][236][237] L-2-amino-4-methoxy-3-butenoic acid [238] lipopolysaccharide (LPS) endotoxin [239,240] Database on potential toxigenic capacities of microorganisms used for industrial production…”