Abstract
Fungal infections afflict the well-being of a broad cross-section of the human population.
These infections can be either superficial or invasive. While superficial infections may be
readily treated, invasive fungal infections (IFIs) can be lethal. Cryptococcus neoformans and
Candida parapsilosis are two emerging opportunistic fungal pathogens that cause IFIs in
humans, especially in individuals with immunocompromised disease such as AIDS or have
undergone surgical condition such as organ transplantation. C. neoformans causes lifethreatening
cryptococcal meningitis in HIV infected patients while C. parapsilosis is a
commensal and causes candidiasis by transmission through the hands of health workers in
hospital environment.
Azole antifungals are usually the first line drugs of choice for such infections. This class of
drugs act by inhibiting lanosterol 14α-demethylase (CYP51), a rate limiting enzyme essential
for the synthesis of the fungal-specific sterol ergosterol. Azole prophylaxis and/or long-term
use of azole drugs have however led to the acquisition of azole resistance among these
pathogens. Discovery of new drugs to combat the azole resistance is of utmost importance.
The redox partner of CYP51 is NADPH-cytochrome P450 reductase (CPR), which provides
electrons for CYP51 activity. As a part of an approach to structure-directed drug discovery,
this project aims to assess the function, azole susceptibility, biochemistry and structural
features of CYP51s from the fungal pathogens C. neoformans and C. parapsilosis and the
impact of their cognate CPR by using a Saccharomyces cerevisiae host system.
Recombinant full-length, codon optimised and C-terminal hexa-histidine tagged CYP51s of
C. neoformans (CnCYP51), C. parapsilosis (CpCYP51) and the CpCYP51 Y132F mutant
were constitutively overexpressed from the PDR5 locus of a S. cerevisiae host strain deleted
of 7 drug efflux pumps and with the promoter of the native CYP51 gene replaced by a
galactose-regulated promoter (GAL1). A cognate NADPH-cytochrome P450 reductase (CPR)
was constitutively co-expressed from the PDR15 locus of the CYP51 expressing recombinant
strains. Agarose diffusion assays and measurement of minimum inhibitory concentrations
(MIC80) in liquid microdilution assays were used to evaluate the susceptibility of the S.
cerevisiae host and recombinant strains to various short-tailed (fluconazole, voriconazole)
and long-tailed (itraconazole, posaconazole) triazoles, medium-tailed tetrazoles (VT-1161,VT-1129) and an experimental tetrazole MCC-8186, during growth on complex synthetic medium containing glucose. The glucan synthase inhibitor micafungin and the polyene amphotericin B were used as control drugs. Crude membranes of CnCYP51-6 His,
CpCYP51-6 His and CpCYP51-6 His Y132F expressing recombinant strains were used to
analyse the expression levels of these proteins by SDS-PAGE and western blot analysis. The
recombinant proteins were solubilised from the crude membranes using the detergent n-decyl-
β-D-maltoside followed by purification by nickel affinity and size exclusion
chromatography. Purified proteins were used to study the Type I binding of their substrates
(lanosterol and eburicol) and Type II binding of azoles (posaconazole and voriconazole) to
these proteins. Affinity purified CPRs from co-expressing strains were used to assess their
function by the cytochrome c reduction activity. Crystallisation of the proteins with the
triazoles posaconazole and voriconazole was attempted using the hanging-drop vapour diffusion
method.
CnCYP51, CpCYP51 and CpCYP51 Y132F expressed from the PDR5 locus supported
growth in the presence of either galactose or glucose (native ScCYP51 suppressed) and
showed functional activity that was inhibited by azole drugs in tests using agarose diffusion
assays and microdilution assays. In case of CnCYP51 expressing strains, co-overexpression
of the cognate reductase CnCPR showed reduced susceptibility to the short-tailed azoles
fluconazole and voriconazole by 2 to 4-fold, respectively, but not to the long-tailed triazoles
or tetrazoles. A slight reduction in susceptibility to amphotericin B was also induced by
CnCPR co-expression. For the C. parapsilosis CYP51 expressing strains, constitutive
expression in yeast of CpCYP51 Y132F conferred a 10 to 12-fold resistance to fluconazole
and voriconazole, ~6-fold resistance to VT-1161 and VT-1129, and reduced to a 3-fold
resistance to MCC-8186, but did not confer resistance to the long-tailed triazoles. Coexpression
of CpCPR did not cause any change in the susceptibility of recombinant strains
expressing CpCYP51 or its Y132F mutant to the azole drugs.Each of the purified proteins showed spectral characteristics typical of CYP51s. Type I binding assays revealed that the purified CnCYP51 bound preferentially eburicol compared
to lanosterol, while CpCYP51 bound both. Both voriconazole and posaconazole bound to the
purified CnCYP51 producing the characteristic Type II binding spectra. Cytochrome c
reduction activity of the CPRs was detected as a 550 nm peak.Protein crystals were obtained for only the wild type CpCYP51 in complex with posaconazole at pH 8.2 and 8.3 with 44% PEG 400 in 100 mM glycine-NaOH buffer, but
these did not diffract under X-ray exposure.
Structural and functional analysis of recombinant CYP51s from the fungal pathogens C.
neoformans and C. parapsilosis has improved the understanding of their susceptibility to
azole drugs and will help advance structure-directed antifungal discovery.