Genetic Pursuit of Antifungal Drug Mechanisms of Action

Around 1.5 million deaths are caused by fungal pathogens each year. This exceeds mortality estimates for either tuberculosis or malaria. In many cases the mortality rate of invasive fungal infections is higher than 50% despite the availability of several antifungal drugs (Brown et al, 2012). These drugs are blighted by toxicity, limited bioavailability, and low efficacy for some disease types. In addition, resistance caused by prolonged exposure of patients to these agents and the widespread use of azoles in agriculture is emerging and presents an exceptional health risk, especially for immunodeficient patients (Snelders et al, 2012). Industry has largely withdrawn from research into antifungal drugs, drug target discovery and validation. This has resulted in a lack of antifungal agents in commercial development pipelines, therefore its indispensable that academia steps in to develop new therapeutics to treat fungal disease. Novel antifungal drugs with new mechanisms of action (MoA) are urgently needed to overcome these problems. A first step to address this problem is to identify essential pathways or molecular targets that can be effectively inhibited. This project aims to identify novel druggable targets in filamentous fungi by determining hitherto unknown mechanisms of action (MoA) of potent antifungal agents and to assess the potential of resistance development to these agents. Therefore, I will monitor the transcriptional responses of A. fumigatus exposed to known as well as unknown MoA drugs. Transcriptional profiles will be used to differentiate between a drug-caused transcriptional response and a general stress response. Parallel fitness analyses of a genome-scale (350 deletion mutants) A. fumigatus transcription factor (TF) knock out library in response to these drugs will be performed to unveil the transcriptional networks governing drug resistance. This aspect of the project is designed to identify TFs, mutation of which cause altered drug sensitivities and therefore bear a high risk of drug resistance. Transcriptional response(s) of A. fumigatus wild-type and unveiled TF mutants will be compared to identify pathways targeted by respective TFs. Moreover, I aim to elucidate novel drug-specific targets through increasing individual gene dosage with genome-wide coverage by employing the autonomously replicating plasmid pAMA1. pAMA1 has successfully been used to overexpress both individual genes as well as whole genomes in screens to identify genes that support drug resistance (Liu et al, 2004; Osherov et al, 2001). An A. fumigatus genomic library hosted on pAMA1 will be employed to identify specific drug resistance- related factors. In addition, target genes identified within this project will be functionally characterised and their role in virulence and physiology will be investigated. The information obtained from these approaches will be combined to expand our knowledge of drug MoA enabling the identification and validation of new antifungal drug targets.

Major goals of this project involved the identification of novel druggable targets as well as the characterization of antifungal resistance mechanisms to drugs that are currently used for the treatment of aspergillosis. A predominat part of the work involved the molecular investigation of one of the most common resistance mechanisms to the antifungal drug class the azoles. Azoles represent the major antifungal drug class used in the clinical setting and therefore resistance to this medicine raises a huge problem in the treatment of patients that are infected with a resistant isolate. Azoles targeting ergosterol biosynthesis (equivalent to human cholesterol), more specifically the sterol 14a-demethylase Cyp51. The mechanism we investigated is partly based on a DNA duplication within the cyp51A promoter causing increased cyp51A gene expression. Previous studies revealed the regulatory protein SrbA to be an activator of cyp51A gene expression. We found that a duplication of DNA in the promoter of the gene leads to increased binding of SrbA to the promoter, which leads to increased expression of cyp51A and, therefore, elevated resistance. A second Azole resistance mechanism we unraveled in this project, is based on mutation of the so called CCAAT binding complex CBC, a transcriptional regulatory complex comprising three subunits termed HapB/C/E. A proline to leucine substitution at position 88 in the subunit HapE (HapEP88L) resulted in azole resistance in a clinical isolate. We discovered that mutation of the CBC leads to increased expression of multiple genes in the ergosterol biosynthetic pathway. This in turn causes an increased production of sterols in the cell and eventually azole resistance. A further major part of the project involved the elucidation of 5-flucytosine (5FC) tolerance found in A. fumigatus. 5FC is barely used for the treatment of aspergillosis. This compound is hardly active at neutral pH, which resembles the pH of most of our body fluids. We could identify the major protein (FcyB) responsible for 5FC transport into A. fumigatus and found its gene expression downregulated at neutral pH. In contrast, acidic pH activates expression of the gene and therefore enhances 5FC uptake and activity. In this work we also identified two regulatory proteins the CBC and the pH regulator PacC to repress fcyB at neutral pH and, therefore, mediating 5FC resistance on the transcriptional regulatory level.