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Analysis of the Aspergillus fumigatus proteomic response to amphotericin B (AmB) reveals involvement of the Rta1-like protein RtaA in resistance

ABSTRACT: The filamentous fungus Aspergillus fumigatus is an opportunistic human pathogen, which can cause mycoses and allergies in susceptible individuals. With regard to therapy, the polyene drug amphotericin B (AmB) is still frequently used to treat Aspergillus fumigatus infections due to the fact that it remains effective against azole-resistant Aspergillus strains. AmB binds to ergosterol in the fungal membrane, but its mode of action and the fungal resistance mechanisms involved have not been completely elucidated yet. To get further insights into the drug mechanisms of AmB, we investigated the proteomic profile of A. fumigatus in response to AmB and its liposomal formulation by LC-MS/MS analysis. A significant increase (≥ 2-fold) in abundance of 202 different proteins in response to AmB and 193 in case of liposomal AmB was observed, while the level of 70 and 83 proteins decreased, respectively. In particular, the level of proteins anchored to the membrane, involved in catabolic processes, aromatic acid degradation, or secondary metabolism increased prominently. Of particular note was the more than 300-fold increase of a RTA1 domain-containing protein after AmB treatment. Fungal RTA-like proteins represent lipid-translocating exporters, which are characterized by multiple transmembrane regions and often confer resistance to toxic chemicals. Indeed, the deletion of the corresponding gene in A. fumigatus led to an increased susceptibility to AmB and other antifungal polyenes such as nystatin. Particularly worthy of mention is that some of the most significantly increased proteins included enzymes involved in the biosynthesis of secondary metabolites such as the prenylated polyphenol fumicycline as well as xanthocillin and hexadehydro-astechrome, which both form complexes with transition metals.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Neosartorya Fumigata (aspergillus Fumigatus)

SUBMITTER: Thomas Krüger

LAB HEAD: Axel A. Brakhage

PROVIDER: PXD042190 | Pride | 2025-01-06

REPOSITORIES: pride

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Project description:Aspergillus fumigatus can cause Invasive Pulmonary Aspergillosis (IPA), the most severe form of Aspergillus-related infections. Fungicidal azoles and fungistatic caspofungin (CAS) are the first- and second-line therapies, respectively, used to treat IPA. However, azole-resistance is on the rise and new therapeutic alternatives need to be implemented. Recently, we showed that treatment of A. fumigatus with CAS or micafungin, but not voriconazole, induces production of the oxylipin 5,8-diHODE by the fungal oxygenase PpoA. Here we investigated the influence of ppo genes, that encode fatty acid oxygenases responsible for oxylipin biosynthesis, on CAS tolerance. The ppo mutants showed a very complex pattern of CAS susceptibility. PpoA and PpoC influence on CAS tolerance is mediated by MpkA phosphorylation and protein kinase A (PKA) activity. RNAseq transcriptional profiling and label-free quantitative proteomics (spectral counts) of the ppoA, and ppoC mutants showed that differentially expressed genes and proteins are related to secondary metabolites and carbohydrate metabolism. We tested several mutants of these differentially expressed genes encoding transcription factors and signal transduction proteins and some of them are more susceptible to CAS. We also characterized two clinical isolates, CNM7555 and IFM41607, that have decreased and increased susceptibility to CAS. CNM7555 does not have increased oxylipin production in the presence of CAS while the oxylipin induction upon CAS exposure is increased in the IFM41607, suggesting oxylipins are not the single mechanism involved in CAS tolerance in these isolates. Upon CAS exposure, CNM7555 has increased MpkA phosphorylation and PKA activity than IFM41607. Modulation of secondary metabolite production and carbohydrate metabolism is also essential in these clinical isolates for acquiring CAS tolerance. Our results reveal different aspects and genetic determinants involved in A. fumigatus CAS tolerance.

Project description:Aspergillus species are a leading cause of invasive fungal infections. As resistance to first-line therapy involving triazole antifungal agents is rising, second-line therapy with echinocandins is expanding. Resistance to echinocandins is well-established to result from amino acid substitutions in the echinocandin drug target β-(1,3)-D-glucan synthase encoded by the fks1 gene. Recently, we identified several high MIC clinical isolates of A. fumigatus from patients failing echinocandin therapy that did not contain any mutation in the fks1 gene, indicating that echinocandin resistance in these isolates results from an undefined mechanism. To explore possible new mechanisms of resistance, we used a lab-derived strain, RG101, with a nearly identical susceptibility phenotype, as a model system. This strain does not contain fks1 mutations but showed prominent resistance to the echinocandin class drug caspofungin (CAS), while remaining sensitive to other echinocandins, azoles and polyenes. Glucan synthase isolated from RG101 was fully sensitive to drug. Yet, exposure to CAS during its growth yielded a modified enzyme that was insensitive (4-log orders) in kinetic inhibition assays to CAS, as well as other echinocandins. This induction of cross-resistance by CAS was also observed in clinical isolates. To determine the nature of a presumptive posttranslational modification (PTM) of the enzyme, we analyzed whole enzyme PTMs, including the known hot-spot regions, for methylation, acetylation and phosphorylation. While we did not identify any PTMs linked to resistance, analysis of the lipid microenvironment of CAS-induced resistant enzyme revealed a prominent increase in the abundance of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to sensitive enzyme in in vitro kinetic inhibition assays recapitulated the CAS insensitivity of the cellular-derived enzyme. To further examine induction of drug-induced resistance, we used an in vitro assay to demonstrate that CAS, but not other echinocandin class drugs, prominently induced the production of mitochondrial-derived Reactive Oxygen Species (ROS) in A. fumigatus. RNASeq evaluation of whole cells confirmed a ROS signature in cells treated with CAS. Dampening the formation of ROS by antimycin A or thiourea eliminated the induction of drug resistance by CAS. We conclude that CAS-induced formation of ROS promotes a cellular stress response that alters the composition of plasma membrane lipids surrounding glucan synthase, changing its enzymatic properties to make it insensitive to echinocandins. This stress-induced response constitutes a novel mechanism of echinocandin resistance in Aspergillus, with implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.

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Analysis of the Aspergillus fumigatus proteomic response to amphotericin B (AmB) reveals involvement of the Rta1-like protein RtaA in resistance

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