Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen.
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.
Arnoldo
Anthony
A
Terrence Donnelly Centre for Cellular and Biomolecular Research, Department of Biochemistry, University of Toronto, Ontario, Canada.
Curak
Jasna
J
Kittanakom
Saranya
S
Chevelev
Igor
I
Lee
Vincent T
VT
Sahebol-Amri
Mehdi
M
Koscik
Becky
B
Ljuma
Lana
L
Roy
Peter J
PJ
Bedalov
Antonio
A
Giaever
Guri
G
Nislow
Corey
C
Merrill
A Rod
AR
Merrill
Rod A
RA
Lory
Stephen
S
Stagljar
Igor
I
D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D016366:Open Reading Frames; D010641:Phenotype; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D014774:Virulence / Q000235:genetics
D000067956:Adenylyl Cyclase Inhibitors; D000900:Anti-Bacterial Agents; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D000262:Adenylyl Cyclases