Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus

Abstract

ABSTRACTMechanistic studies on gliotoxin biosynthesis and self-protection inAspergillus fumigatus, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that anA. fumigatusΔgliAstrain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of theglicluster, as disablingglicluster activation, viagliZdeletion, attenuates the sensitivity of anA. fumigatusΔgliTstrain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxinbis-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the ΔgliTstrain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significantS-adenosylmethionine (SAM) depletion andS-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity ofgliT-deficientA. fumigatusto gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in theA. fumigatusΔgliKstrain, but not the ΔgliGstrain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared to gliotoxin-naive, fungi to facilitate their cellular presence.