Abstract
Fungal infections affect a broad spectrum of the population, including premature
babies, the elderly and individuals with a range of disease- or medically-induced comorbidities.
Fungal pathogens such as Candida albicans and Aspergillus fumigatus
cause a variety of conditions, from minor infections to life threatening disease. Some
fungi, including C. albicans and Candida glabrata can be commensal living in
harmony with the superficial microflora, but these organisms can behave as
opportunistic pathogens when individuals become immunodeficient due to comorbidities
or become immunocompromised due to AIDS or through medical
intervention. Fungal infections have become recognised in recent years as a growing
health burden that currently causes around 1.5 million deaths per year worldwide.
The azole antifungal drugs (imidazoles and triazoles) are used widely to treat fungal
infections and as antifungal prophylaxis. The azole drugs target the fungal enzyme
lanosterol 14α-demethylase (Erg11p, CYP51). This monospanning bitopic membrane
protein belongs to the CYP51 class in the cytochrome P450 superfamily of enzymes
and is involved in the rate-limiting step of ergosterol biosynthesis. Ergosterol is the
fungal equivalent of cholesterol and is required for fungal cell growth. Fungal
pathogens have evolved several mechanisms of resistance that diminish the action of
the azole drugs. The emergence of resistant fungal strains due to mutations in CYP51
can limit therapeutic options and make treatment of fungal infections increasingly
problematic. The need for better drugs that overcome resistance is becoming
increasingly urgent. The present project builds on the research of Monk et al. who
successfully crystallised and obtained the first high-resolution X-ray structures of a
fungal CYP51. The aim of this project is to investigate the effect of CYP51 mutations
on enzyme structure and function, including different types of triazole drug, by using
Saccharomyces cerevisiae Erg11p as a model for the homologous enzymes in
pathogenic fungi.
A S. cerevisiae overexpression system was used to hyper-express the wild type and
the mutant ScErg11p enzymes in order to obtain sufficient quantities of protein for
structure-function studies. The C. albicans CYP51 mutations Y132F/H, K143R,
G464S and the double mutation Y132F G464S (Y140F/H, K151R, G464S and Y140F
G464S S. cerevisiae numbering), as well as the CYP51A G54E/R/W mutations of A.
fumigatus (G73E/R/W S. cerevisiae numbering) have been reproduced in a C-terminal
hexahistidine-tagged version of S. cerevisiae Erg11p (ScErg11p6×His). In addition,
the innate resistance of A. fumigatus CYP51A to fluconazole (FLC) was investigated
using the S. cerevisiae Erg11p T322I mutant. Microdilution assays were used to
determine triazole susceptibilities of these strains. ScErg11p6×His mutant and wild
type enzymes were purified from crude membranes by solubilisation with the
detergent n-decyl-β-D-maltoside followed by affinity and size exclusion
chromatography. Spectral analysis of the purified protein was used to determine
dissociation constants for triazole drugs. Purified preparations of the enzyme were
also used to obtain crystals for X-ray crystallographic analysis. High-resolution (1.98
– 2.35 Å) X-ray crystal structures were obtained for mutant enzymes in complex with
triazole drugs and without added ligand, as well as for the wild type enzyme in
complex with FLC.
Microdilution assays revealed that strains overexpressing ScErg11p6×His Y140F/H
or Y140F G464S had reduced susceptibility to the short-tailed triazoles FLC and
voriconazole but not the long-tailed triazole itraconazole. Strains overexpressing
ScErg11p6×His G464S, T322I and K151R mutants had triazole susceptibility patterns
similar to the wild type enzyme overexpressing strain but the G73E/R/W mutants
showed increased susceptibility to all triazoles tested. Binding studies revealed that
the triazole binding was tight for all the mutant enzymes.
The high-resolution (2.05 Å) structure of wild type ScErg11p6×His in complex with
FLC revealed a water-mediated hydrogen bonding network between residue Y140 and
the hydroxyl group of the drug. The crystal structures of the ScErg11p6×His
Y140F/H mutants showed that these mutations disrupted the key water-mediated
hydrogen-bonding network seen in the wild type enzyme complex. The disruption of
these interactions is proposed to weaken the interactions between the drug and the
mutant enzyme leading to resistance. These observations explain reduced
susceptibility to FLC and voriconazole and the retention of susceptibility to
itraconazole of these mutants.
The X-ray crystal structures of the ScErg11p6×His G73E/W mutants in complex with
itraconazole showed that the drug bound in different conformations compared to the
wild type enzyme structure. The piperazine ring of the itraconazole molecule acts as a
hinge, which can adopt different conformations. The crystal structures indicated
potential π-anion interactions between the tail of the itraconazole and the E73 residue
and π stacking interactions between W73 and the tail of itraconazole. The bending of
the drug molecule was found to accommodate each mutation. The conformation of
itraconazole bound to the G73W mutant had not been seen previously. These extra
interactions between the drug and the site of the mutation in part explain the increased
susceptibility of G73E/W mutant strains to itraconazole.
The structure of ScErg11p6×His G464S revealed that the mutated residue had
replaced polar interactions between a water molecule and the propionate group of the
heme. No obvious tilting of the heme was observed in this mutant. The
ScErg11p6×His T322I and G73W mutant structures without added ligand revealed
some density in the active site and some movement of the carbonyl group of helix I
residue G314, previously seen in the wild type ScErg11p6×His structure in complex
with lanosterol. Residue G314 may be involved in catalysis by potentially stabilising
oxygen bound heme iron intermediates.
In summary, this work provides insight into the molecular interactions between the
triazole drugs and the CYP51 enzyme. The high-resolution structure of the wild type
enzyme in complex with FLC has allowed us to identify the potential basis for
resistance of the Y140F/H mutants, which we confirmed by recreating those
mutations in our S. cerevisiae system. In addition, other mutations reproduced in our
system reveal that despite a relatively high sequence similarity amongst fungal
CYP51s, our model does not adequately reflect the effect of the same mutations in
pathogenic fungi. This knowledge will aid in the structure-directed design of next generation
azole-based antifungal drugs that can be used to overcome antifungal
resistance.