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Duke Team Uncovers RNAi-based Drug-resistance Mechanism in Pathogenic Fungus


NEW YORK (GenomeWeb) – While RNAi's role as an antiviral defense mechanism in plants and invertebrates is well established, new data out of Duke University shows that the gene-silencing system can also be used, at least by a certain pathogenic fungus, to develop drug resistance.

More notably, this effect is reversible, allowing the fungus — Mucor circinelloides — to revert to its previous state once the pharmacologic threat is removed. Known as an epimutation, this kind of transient behavior is not only likely to be used by M. circinelloides for other purposes, but may be also present in other species, according to Duke's Joseph Heitman, who led the research.

"I think it quite likely that other examples of this will emerge," he told Gene Silencing News this week, adding that it's no surprise they haven't been identified yet given how genetics studies have traditionally been carried out.

"One of the dogmas [of the field] is that whenever you do any screens, the first thing you do is … you only work with [experimental isolates] that are stable and well behaved," he said. "That would weed out a lot of unusual mechanistic processes like this."

Indeed, Heitman characterized his lab's discovery, which was described in a paper appearing in Nature, as "serendipitous."

As part of ongoing research into the mechanisms of action of key antifungal agents including tacrolimus (FK506) and rapamycin, one of Heitman's graduate students was analyzing M. circinelloides that was resistant to the two drugs.

"We thought they would all have very standard mutations in the known drug targets," he said. And while a screen did eventually yield bona fide Mendelian mutants, the scientists also noticed drug-resistant isolates with no mutations. Further, these isolates were found to quickly revert to a wild-type state when passed through drug-free media.

Even more perplexing was the discovery that expression of the gene fkbA, which confers FK506 resistance when mutated, was completely lost in drug-resistant isolates that were grown in media containing FK506.

"In contrast, mRNA and protein levels were reduced but detectable in some resistant isolates when grown in drug-free media and were restored to wild-type levels in revertant isolates that became FK506-sensitive following growth in drug-free media," the team wrote in Nature.

Additionally, small RNAs complementary to fkbA were detected in the drug-resistant isolates, and because the RNAi pathway is known to exist in M. circinelloides Heitman and his colleagues speculated that an RNAi effect was at work.

This theory was borne out when the researchers repeated their screening experiments using M. circinelloides mutants in which RNAi had been inactivated. As expected, drug-resistant variants arose, but with classical Mendelian mutations in target genes.

"None of them are of this unstable RNAi-mediate epimutant class," Heitman said.

Further investigation revealed that the gene silencing observed involves the generation of a dsRNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA, the investigators wrote in their paper. Additionally, several components of the canonical RNAi pathway — namely Dicer, Argonaute, and RNA-dependent RNA polymerase 2 (RdRp 2) — were shown to be necessary for fkbA silencing.

Interestingly, the scientists found that RdRp 1, rather than promoting epimutational silencing, actually suppresses it, with mutants lacking the enzyme showing an elevated rate of target gene inhibition and failing to revert to wild-type upon passage through drug-free media. They hypothesize that in M. circinelloides, RdRp 1 may be promoting assembly of exosome machinery on specific mRNA targets and "thereby avoiding activation of RNAi under normal conditions."

Heitman and his colleagues put forth two possible models for their observations. In the first, small RNAs are produced "constitutively and stochastically at low levels against the entire genome or some designated loci, allowing adaptation to environments through an RNAi-based pathway. In the second model, some mechanism activates RNAi under adverse or novel physiological conditions, facilitating genomic and phenotypic plasticity," they wrote. "Either could explain the broad range of environments in which M. circinelloides grows, and the limited antifungal drug susceptibility."

In either case, the work uncovers a novel epigenetic RNAi-based mechanism, giving M. circinelloides two methods of phenotypic variation, one stable and one temporary, the researchers concluded in Nature. "This plasticity evokes a broader phenotypic repertoire including the ability to reverse epimutations when selective pressures are relaxed."

It also provides a new area of investigation into antimicrobial resistance and, potentially, a point of intervention for overcoming such resistance in human pathogens, Heitman said.

To extend these findings, he said that his lab is investigating if this RNAi effect occurs in M. circinelloides against other genes and under different conditions to help determine whether it is a generalizable phenomenon.

"We've also been looking in Cryptococcus, which is another human pathogen, and we've just started doing some studies in Neurospora," Heitman said. "So we're looking hard for other examples in other fungi known to have active RNAi pathways."