DNA encoding sterol methyltransferase

Details DNA encoding sterol methyltransferase DNA encoding sterol methyltransferase

1998-03-13

2001-05-01

Stole, Einar (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S254100, C435S254110, C435S254220, C536S023200

Reexamination Certificate

active

06225075

ABSTRACT:

BACKGROUND OF THE INVENTION
The frequency of occurrence of human fungal infections has been increasing over the past decade in response to a combination of factors (Georgopapadakou et al., 1994). These factors include advances in invasive surgical techniques which allow for opportunistic pathogen access, the administration of immunosuppressive agents employed in transplantation, and an increase in the number of immunosuppressed patients resulting from chemotherapy and disease states such as AIDS. The threat to human health is further compounded by the increased frequency with which resistance to the commonly employed antifungal agents is occurring.
Currently, the most common antifungals include the polyenes and the azoles. The polyenes bind to ergosterol, the fungal membrane sterol, and induce lethal cell leakage (Brajtburg et al., 1990). However, polyenes often have negative side effects and resistance to polyenes has been reported (Hebeka et al., 1965; Powderley et al., 1988). The azoles are fungistatic agents that inhibit the cytochrome P450-mediated removal of the C-14 methyl group from the ergosterol precursor, lanosterol (Vanden Bossche et al., 1987). Resistance to azoles has been reported in
Candida albicans
(Clark et al., 1996; Sanglard et al., 1996; Sanglard et al., 1995; White, 1997a; White, 1997b) as well as in other species of Candida (Moran et al., 1997; Parkinson et al., 1995), and in other fungal pathogens, including species of Histoplasma (Wheat et al., 1997), Cryptococcus (Lamb et al., 1997; Venkateswarlu al., 1997), and Aspergillus (Denning et al., 1997).
The pathway for fungal sterol biosynthesis is one target for antifungal development. In particular, fungal genes that catalyze a step in sterol biosynthesis that is not found in cholesterol biosynthesis (Pinto et al., 1983) are of interest in this regard. One such fungal gene is the sterol methyltransferase gene (ERG6). Non-recombinant
Saccharomyces cerevisiae
erg6 mutants have been available for some time (Molzhan et al., 1972). The
S. cerevisiae
ERG6 gene has been isolated, and recombinant strains prepared (i.e., via genetic engineering) in which the gene has been disrupted (Gaber et al., 1989). Although the absence of the ERG6 gene product in
S. cerevisiae
was not lethal, it did result in several severely compromised phenotypes (Bard et al., 1978; Kleinhans et al., 1979; Lees et al., 1979; Lees et al., 1980).
S. cerevisiae
erg6 mutants have been shown to have diminished growth rates as well as limitations on utilizable energy sources (Lees et al., 1980), reduced mating frequency (Gaber et al., 1989), altered membrane structural features (Kleinhans et al., 1979; Lees et al., 1979), and low transformation rates (Gaber et al., 1989). In addition, several lines of evidence have indicated that
S. cerevisiae
erg6 mutants have severely altered permeability characteristics. This has been demonstrated using dyes (Bard et al., 1978), cations (Bard et al., 1978), and spin labels used in electron paramagnetic resonance studies (Kleinhans et al., 1979). These early observations have been corroborated recently by the cloning of the
S. cerevisiae
LIS1 gene (Welihinda et al., 1994), mutants of which were selected on the basis of hypersensitivity to sodium and lithium. Sequencing of LIS1 has indicated identity to ERG6. In addition, studies using the Golgi inhibitor, brefeldin A, have routinely employed erg6 mutants because of their remarkably increased permeability to the compound (Vogel et al., 1993). However, as
S. cerevisiae
and
Candida albicans
differ in their ability to survive and grow on various sterol intermediates, and as
S. cerevisiae
is rarely the cause of a human disease, it was unknown whether the ERG6 gene in the common fungal pathogen,
C. albicans
, effected similar properties.
Thus, a continuing need exists for fungal genes and strains that can aid in the identification of agents that increase the susceptibility of pathogenic fungi to conventional anti-fungal or anti-metabolic agents.
SUMMARY OF THE INVENTION
The present invention provides an isolated nucleic acid segment comprising a nucleic acid sequence encoding a
Candida albicans
sterol methyltransferase (ERG6), a biologically active variant or subunit thereof. As described hereinbelow, the
Candida albicans
ERG6 gene was isolated by complementation of a
Saccharomyces cerevisiae
erg6 mutant using a
Candida albicans
genomic library. Preferably, the sterol methyltransferase of the invention comprises SEQ ID NO:2, which is encoded by a DNA having SEQ ID NO:1. Thus, the invention further provides isolated, purified recombinant
Candida albicans
sterol methyltransferase, a biologically active variant or subunit thereof, e.g., a polypeptide having SEQ ID NO:2. Methods to isolate and purify sterol methyltransferase are known to the art (see, for example, Ator et al., 1989).
ERG6 can be used in a method to identify antifungals targeted specifically to sterol methyltransferase. Therefore, the invention also provides a method to identify inhibitors of fungal sterol methyltransferase. The method comprises contacting an amount of isolated, purified recombinant
Candida albicans
sterol methyltransferase, or a biologically active variant or subunit thereof, with an amount of an agent. The activity of the sterol methyltransferase in the presence of the agent is then determined or detected relative to an amount of sterol methyltransferase not contacted with the agent.
The isolation and characterization of a
Candida albicans
ERG6 gene also permits the preparation of recombinant
Candida albicans
isolates that lack a functional sterol methyltransferase, e.g., isolates which have decreased or reduced amounts of sterol methyltransferase, or lack sterol methyltransferase activity. Inhibiting the functional ERG6 gene product may make the cell hypersensitive to exogenous compounds, and thus could increase the effectiveness of new or existing antifungals. Thus, as described hereinbelow, the first copy of the Candida ERG6 gene was disrupted by transforming a wild type isolate with the ura blaster system. The second copy of the Candida ERG6 gene was disrupted by ura blaster transformation or mitotic recombination. The resulting erg6 strains were shown to be more susceptible to a number of sterol synthesis and metabolic inhibitors including terbinafine, tridemorph, fenpropiomorph, fluphenazine, cycloheximide, cerulenin and brefeldin A, relative to the corresponding isolate of
Candida albicans
which encodes a functional sterol methyltransferase. No increase in susceptibility to azoles was noted.
Therefore, recombinant
Candida albicans
isolates lacking, or having reduced, sterol methyltransferase activity are useful in a method to identify antifungal agents that would otherwise have low or no ability to permeate the fungal cell membrane and thus may be overlooked as therapeutic agents. Moreover, the administration of inhibitors of the ERG6 gene product to a host organism having a fungal infection may make the fungal cell increasingly susceptible to antifungals or other agents which normally would be excluded, e.g., due to their lack of ability to permeate the cell, and may permit clinical treatment at lower dosages. Hence, the invention provides a method to enhance the efficacy of an agent such as an antifungal agent, comprising: administering to a mammal having, or at risk of having, a fungal infection, an amount of agent that inhibits
Candida albicans
sterol methyltransferase and an amount of an anti-fungal agent effective to inhibit or treat the infection. Preferably, the sterol methyltransferase inhibitor is administered in an amount that reduces or decreases the effective amount of the anti-fungal agent administered relative to the effective amount of the anti-fungal agent administered in the absence of the inhibitor.
Also provided is a method to identify inhibitors of fungal sterol methyltransferase. The method comprises contacting an isolate of a fungus, e.g.,
Candida albicans
, with an amount of an agent, wherein the genome of the is

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