Scientists from the Duke University School of Medicine say that they discovered that microorganisms can use a temporary silencing of drug targets, known as epimutations, to gain the benefits of drug resistance without the commitment to permanent mutations. Though the new mechanism was discovered in a fungus called Mucor circinelloides, it is likely to be employed by other fungi as well as bacteria, viruses, and other organisms to withstand treatment with various drugs, according to the researchers.
The team published its study (“Antifungal drug resistance evoked via RNAi-dependent epimutations”) in Nature.
“This mechanism gives the organism more flexibility,” said Joseph Heitman, M.D., Ph.D., senior study author and professor and chair of molecular genetics and microbiology at Duke University School of Medicine. “A classic, Mendelian mutation is a more permanent binding decision. These epimutations are reversible. If conditions change, it is easier to revert to the way things were.”
The epimutations are so transient, in fact, that the researchers almost disregarded them. Cecelia Wall, a graduate student, had been looking for mutations that would make the human fungal pathogen M. circinelloides resistant to the antifungal drug FK506 (also known as tacrolimus). This pathogen causes the rare but lethal fungal infection mucormycosis, an emerging infectious disease that predominantly affects individuals with weakened immune systems.
As is typical for most drug resistance experiments, Wall first grew the pathogen in Petri dishes containing the antifungal drug. She found that the few organisms that survived treatment looked different, being smaller and less diffuse than their parent fungi. Wall then isolated those fungi and sequenced the gene FKBP12, which is the target of FK506, to look for mutations that would confer drug resistance.
However, she couldn't detect any mutations in about a third of the isolates. What's more, Wall found that many of the mutants kept “disappearing,” looking less like mutants and more like their parents after she took the drug away.
“This is an example of something you might find in the laboratory and just throw away,” said Silvia Calo, Ph.D., lead study author and postdoctoral fellow in the Heitman and Cardenas labs. “You look for mutants in one gene and when you don't find a mutation in some of the isolates, you decide not to work on those anymore and instead focus on others. But we wanted to know what was going on.”
The researchers began to wonder whether RNA interference (RNAi) could be the cause of this unstable drug resistance. RNAi uses bits of RNA to silence specific genes. Though RNAi doesn't exist in every organism, the researchers knew it was active in M. circinelloides. So Dr. Calo looked for the presence of small RNAs in the drug resistant isolates. She didn't find small RNAs in the isolates that contained mutations in FKBP12, but she did find them in those lacking mutations.
Importantly, Dr. Calo found that these small RNAs only silenced the FKBP12 gene and not any other loci in the genome. The results demonstrate that M. circinelloides can develop drug resistance two different ways, either stably through permanent mutations or transiently through reversible epimutations.
“This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes,” wrote the investigators.
The researchers think these epimutations could be employed in a variety of situations, enabling an organism to adapt to an unfavorable environment and then adapt again when conditions improve. Though they have only shown epimutations in two species of M. circinelloides, they have already been approached by a number of other researchers who are interested in investigating similar unstable behavior in other organisms like Aspergillus and Neurospora.