Aspegillus

Fungal Genetics and Biology 48 (2011) 1062–1070

Contents lists available at SciVerse ScienceDirect

Fungal Genetics and Biology
journal homepage: www.elsevier.com/locate/yfgbi

The structure–function relationship of the Aspergillus fumigatus cyp51A L98H conversion by site-directed mutagenesis: The mechanism of L98H azole resistance
Eveline Snelders a,b,⇑, Anna Karawajczyk c, Rob J.A.Verhoeven a,b, Hanka Venselaar c, Gijs Schaftenaar c, Paul E. Verweij a,b, Willem J.G. Melchers a,b
a b c

Radboud University Nijmegen Medical Centre, Department of Medical Microbiology, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Nijmegen Institute for Infection Inflammation and Immunity (N4i), P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Centre for Molecular and Biomolecular Informatics,Nijmegen Centre for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

a r t i c l e

i n f o

a b s t r a c t
Since 1998, the rapid emergence of multi-azole-resistance (MAR) was observed in Aspergillus fumigatus in the Netherlands. Two dominant mutations were found in the cyp51A gene, a 34 bp tandem repeat (TR) in the promoter region combined with a leucine tohistidine substitution at codon 98 (L98H). In this study, we show that molecular dynamics simulations combined with site-directed mutagenesis of amino acid substitutions in the cyp51A gene, correlate to the structure–function relationship of the L98H substitution conferring to MAR in A. fumigatus. Because of a L98H directed change in the flexibility of the loops, that comprise a gate-like structure inthe protein, the capacity of the two ligand entry channels is modified by narrowing the diameter and thereby binding of azoles is obstructed. Moreover, the L98H induced relocation of tyrosine 121 and tyrosine 107 seems to be related to the MAR phenotype, without affecting the biological activity of the CYP51A protein. Site-directed mutagenesis showed that both the 34 bp TR and the L98H mutation arerequired to obtain the MAR phenotype. Furthermore, the amino acid leucine in codon 98 in A. fumigatus is highly conserved and important for maintaining the structure of the CYP51A protein that is essential for azole docking. Ó 2011 Elsevier Inc. All rights reserved.

Article history: Received 13 May 2011 Accepted 18 August 2011 Available online 30 August 2011 Keywords: Aspergillus fumigatusMulti-azole resistance cyp51A Homology modelling Molecular dynamics simulations

1. Introduction Invasive aspergillosis (IA) is a disease with a significant mortality rate, with the opportunistic mould Aspergillus fumigatus as the principle etiological agent (Maschmeyer et al., 2007). It is a feared complication of immunosuppressive therapy mostly in patients with haematological malignancies.Patient survival is directly associated with timely diagnosis and prompt appropriate antifungal therapy. In medicine three groups of antifungal drugs are most used: the triazoles, polyenes and echinocandins. The triazoles are the main antifungal drugs recommended for IA as primary therapy and prophylaxis (Pascual et al., 2008). The use of triazoles in the management of Aspergillus diseases may bethreatened by the emergence of acquired resistance in A. fumigatus. Modifications of the target gene of the triazoles, the cyp51A gene, has been described to be correlated to specific triazole resistance phenotypes. The cyp51A gene encodes for a CYP450-dependent enzyme, the 14a-lanosterol demethylase, which removes the 14a-methyl group from lanosterol. Triazole antifungals inhibit this enzyme
⇑Corresponding author at: Radboud University Nijmegen Medical Centre, Department of Medical Microbiology, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Fax: +31 24 3540216. E-mail address: E.Snelders@mmb.umcn.nl (E. Snelders).
1087-1845/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2011.08.002

by binding with one of the nitrogen atoms of the triazole ring to the...