Researchers at the University of Toronto have developed a cell-based yeast-phenotypic assay that, when combined with a large-scale inhibitor screen, identified small molecule inhibitors that suppress the toxicity caused by the heterologous expression of selected Pseudomonas aeruginosa open-reading frames.
The research also enabled the investigators to identify what they said is the first small molecule inhibitor of Exoenzyme S, or ExoS, a toxin that is involved in Type III secretion.
The researchers also found that this inhibitor, called exosin, and two of its analogues had a protective effect against Pseudomonas infection in vivo. The investigators concluded that their cell-based approach could augment traditional drug screening approaches and facilitate the discovery of new antimicrobial compounds.
This approach consists of three consecutive steps, said Igor Stagljar, corresponding author on the paper and an associate professor at the Terrence Donnelly Center for Cellular and Biomolecular Research at the University of Toronto.
“The first step was a rapid and uniform parallel cloning assay, based on well-established Gateway technology,” Stagljar said. The researchers then transferred Pseudomonas ORFs that inhibited yeast growth into a yeast expression vector.
Stagljar said that the second step involved a growth selection method to select Pseudomonas genes that cause the growth inhibition phenotype in yeast. The third step was a “chemical genomics approach in which the yeast strains expressing the lethal Pseudomonas ORFs were used to identify compounds that reverse the mortality caused by the heterologous expression of a particular Pseudomonas ORF.”
The researchers were able to show that exosin modulates ExoS ADP-ribosyltransferase activity in vitro, which suggests that its inhibition of ExoS is direct. They were also able to demonstrate that several yeast homologues of the known human ExoS targets were ADP-ribosylated by ExoS.
“I think a lot of drug companies are a little bit skeptical about using yeast in their drug discovery programs.”
Finally, the investigators identified Brain Modulosignalin Homolog 1p, a yeast homologue of the human Factor Activating Exoenzyme S, as an ExoS cofactor. This finding meant that parts of the bacterial toxin’s mechanism of action are conserved from yeast to human.
This cell-based approach in yeast is something that Stagljar said he can commercialize or offer as a service to drug makers.
“I think a lot of drug companies are a little bit skeptical about using yeast in their drug discovery programs,” he said. However, he said he believes that after this and two other publications that have shown that yeast can be used as an efficient model for drug discovery, pharmaceutical companies will be interested in this approach.
“I can see us being contacted by different drug companies if they want us to do screens for them, or if they are interested in using this approach in-house,” Stagljar said.
Stagljar said he has patented this approach at University of Toronto. The abstract of the patent, Canadian patent number 2479999, is available here.
Stagljar, who moved to Canada several years ago from Switzerland, said that he began this work when he was an assistant professor at the University of Zurich. He said that he chose to use yeast because he did his PhD training in yeast.
“Yeast have approximately 6,200 genes, and approximately 30 percent of these genes have human homologs,” he said.
Stagljar went on to say that yeast is the simplest eukaryotic organism, and “we thought that because yeast is a microorganism, they would be amenable to high-throughput studies in which we screen for open reading frames from pathogens produced by P. aeruginosa, that when overexpressed, kill yeast and can cause a growth inhibition phenotype.”
Many P. aeruginosa genes that make them lethal to yeast also make them lethal to human cells, so targets must be functionally conserved in both yeast and humans, Stagljar said.
“So we thought, ‘If we can overexpress a gene in Pseudomonas and kill yeast, then this can be used as a platform for drug discovery.’”
In other words, said Stagljar, the researchers could screen for small molecules or compounds that would block the Pseudomonas target, so it does not cause a negative or growth inhibition phenotype in yeast.
In terms of continuing this work, Stagljar pointed out that “there are at least 11 other proteins from Pseudomonas that we could screen using the same approach, which is what we plan to do.”
Stagljar said that he and his team also want to extend this approach to other bacterial pathogens such as Salmonella and Bacillus anthracis.
He added that “one particular platform that we want to implement here very soon is a screen for inhibitors of selected Plasmodium falciparum genes.”