Epigenetic modulators as a new strategies to reduce Aspergillus pathogenicity

Aspergillus spp. are environmental filamentous fungi that cause a wide spectrum of infections in humans, including hypersensitivity reactions, chronic pulmonary infections, and acute life-threatening infections. Although there are different compounds for the prophylaxes and treatment of Aspergillus infections, resistance to these agents is emerging [1]. Aspergillus fumigatus infections occur frequently as breakthrough infections in preterm infants receiving fluconazole prophylaxis. Therefore new therapeutic strategies, such as anti-virulence and anti-resistance drugs, that could be employed in conjunction with antifungal and may help to prolong the life span of these life‐saving drugs, are needed. Epigenetic modifications regulate the phenotypic traits. Recently, epigenetic and posttranslational modifications have also raised concern for the relevance of regulating fungal biology in Aspergillus [2]. Aspergillus spp. produce extracellular proteins that have a significant role in causing pathogenicity to the host. The aim of this work has been to evaluate the activity of selective epigenetic inhibitor compounds on expression of virulence-related factors of A. fumigatus such as exoprotein production and mycelium growth rate (MGR) alone and in combination with fluconazole, to assess whether the epigenetic inhibitors could overcome resistance to fluconazole. JUMONJY inhibitor (IOX-1), bromodomain extra terminal protein inhibitor (JQ-1), DNA methyltransferase-1 inhibitor (MC3343), enhancer of zeste homolog 2 inhibitor (tazemetostat), histone deacetylase, histone deacetylase inhibitors (SAHA, MC 1855, MC 2189, MC 1568, MC1716, MC 1714), acetyltransferases inhibitors (anacardic acid, MC 2580), been synthetized at the laboratories directed by Prof. Mai. The activity was evaluated against A. fumigatus DSM 790 coming from German Collection of Microorganisms (DSMZ, Braunschweig, Germany). The proteins were obtained after 96 h in RPMI media with of without molecules. The proteins were separated by aceton and were measured by the Bradford method using Bradford Reagent and Bovine serum albumin as standard (both from Sigma Aldrich, UK). Gel electrophoresis has been used for separation and analysis of proteins. The minimal inhibitory concentration (MIC) were determined according to the CLSI M38-A2 document [3]. Mycelium growth rate (MGR) assays were performed by averaging the colony diameters (in mm) as measured in two randomly chosen perpendicular directions after 48h and 72h of growth [4]. The inhibitors anacardic acid, SAHA, tazemetostat, MC 2580, MC3343, JQ-1, IOX-1, MC 2189 alone and in association with fluconazole reduce mycelium growth rate.The compounds that showed a inhibition of mycelium growth rate, were tested for the evaluation of proteins production. It was demonstrated that extracellular protein secretion absolutely inhibited by JUMONJY inhibitor and bromodomain extra terminal protein. Enhancer of zeste homolog 2 inhibitor, histone deacetylase inhibitor, lysine-specific demethylase 1 inhibitor and acetyltransferases inhibitors increased level of extracellular proteins. The results of electrophoresis of extracellular show that epigenetic inhibitors modulate extracellular proteins. In particular, epigenetic inhibitors reduce the protein ribotoxin (25 kDa) and thioredoxin reductase (36 kDa ) two important virulence factors. The results have showed that JQ-1 significantly modulated extracellular proteins and reduce mycelium growth rate. Further studies will be conducted to identify the mechanisms involved, with the aim of being able to use active molecules to reduce Aspergillus virulence.