Proteomic profiling of the antifungal drug response of Aspergillus fumigatus to voriconazole

Abstract

Antifungal resistance is an emerging problem and one of the reasons for treatment failure of invasive aspergillosis (IA). Voriconazole has become a standard therapeutic for the treatment of this often fatal infection. We studied the differentially expressed proteins as a response of Aspergillus fumigatus to voriconazole by employing the two-dimensional difference gel electrophoresis (DIGE) technique. Due to addition of drug, a total of 135 differentially synthesized proteins were identified by MALDI-TOF/TOF-mass spectrometry. In particular, the level of proteins involved in the general stress response and cell detoxification increased prominently. In contrast, cell metabolism and energy proteins were down-regulated, which suggests the cellular effort to maintain balance in energy utilization while trying to combat the cellular stress exerted by the drug. We detected several so-far uncharacterized proteins which may play a role in stress response and drug metabolism and which could be future targets for antifungal treatment. A mutant strain, which is deleted in the cross-pathway control gene cpcA, was treated with voriconazole to investigate the contribution of the general control of amino acid biosynthesis to drug resistance. We compared the mutant strain’s protein expression profile with the wild-type strain. The absence of CpcA led to an increased resistance to voriconazole and a reduced activation of some general stress response proteins, while the transcript level of the triazole target gene erg11A (cyp51A) remained unchanged. In contrast, the sensitivity of strain ΔcpcA to terbinafine and amphotericin B was slightly increased. These findings imply a role of CpcA in the cellular stress response to azole drugs at the post transcriptional level.

Introduction

Aspergillus fumigatus is one of the most important human fungal pathogens with high incidence of mortality in infected immunocompromised patients. Due to its eukaryotic origin, the number of drugs capable of treating invasive aspergillosis is rather limited. The azole group of antifungal drugs comprises the main therapeutics for aspergillosis patients (Petrikkos and Skiada 2007). However, there has been an increasing tendency in fungal azole resistance (Hoffmann et al., 2000). For this reason, there is a growing interest in investigating the underlying mechanisms of drug resistance for effective treatment of fungal infections (Arendrup et al., 2010). Fungi gain resistance to azole antifungals by developing point mutations in the erg11A (cyp51A) gene encoding the ergosterol biosynthesis target protein, overexpressing the drug target Cyp51A and drug transport proteins or by inducing the internal stress response machinery (Kontoyiannis and Lewis, 2002, Snelders et al., 2008). However, other possible means of resistance development have been described (e.g. gene mutations of transcription factors or mitochondrial respiratory complexes) and are also being investigated (Bromley et al., 2016, Gsaller et al., 2016).

The multifactorial mechanisms of drug resistance are highly amenable to global approaches, such as transcriptome and proteome profiling. The response of A. fumigatus to the clinically important triazole voriconazole has been studied at the transcript level, but not yet at the proteome level (Ferreira et al., 2006). More data are available from the triazole itraconazole (Gautam et al., 2016), and other groups of antifungals such as the polyene Amphotericin B (Gautam et al., 2008) and the cell wall-targeting drug caspofungin (Cagas et al., 2011) or from other pathogenic fungi such as Candida albicans (Hoehamer et al., 2010). Apparently, triazoles induce an increase in the abundance of transcripts and proteins which are involved in ergosterol biosynthesis. This is a known compensatory mechanism due to the fact that azoles block the ergosterol biosynthesis pathway. In addition, the global transcript analysis by Ferreira et al. (2006) has shown an increased level of transcripts coding for heat shock proteins, transcripts of drug transporter genes and calmodulin/cAMP signaling components upon triazole treatment. The study also revealed a decreased mRNA expression of several ergosterol biosynthesis genes, but an upregulation of transporters and transcriptional regulators including CpcA, an activator of the cross-pathway control (CPC) system of amino acid biosynthesis, which counteracts amino acid starvation.

Here, we investigated the response of A. fumigatus to voriconazole, a second generation triazole antifungal medication, which has become the new standard for the treatment of IA (Ghannoum and Kuhn, 2002). We show that proteins overrepresented in response to voriconazole are primarily contributing to combat cell stress. In contrast, proteins involved in cellular energy and primary metabolism were present in lower abundance. Moreover, there were so-far minimally characterized proteins obtained in this study that may be possible candidates for further studying in terms of their novel role in drug resistance in addition to known targets. We studied the potential role of fungal metabolic pathway regulator protein CpcA in mediating antifungal drug resistance at protein level by investigating the change of the proteome of the ΔcpcA strain due to the addition of voriconazole (Busch et al., 2003). The comparative proteomic profiling of wild-type and ΔcpcA strains and their response to voriconazole suggested a possible indirect and profound effect on the fungal stress proteome, since the mutant strain ΔcpcA exhibited decreased sensitivity to voriconazole and itraconazole.