Regulation of fungal gene expression

Details Regulation of fungal gene expression Regulation of fungal gene expression

1998-11-10

2001-10-16

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

Reexamination Certificate

active

06303302

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to methods of identifying and isolating genes which are involved in the regulation of fungal gene expression. The invention also relates to methods useful for identifying fungal virulence factors. It further relates to a method of identifying agents which increase or decrease the expression or activity of a gene that regulates or is required for fungal pathogenesis. The invention also relates to the use of such agents as fungicides or fungistats.
Fungi are a large and diverse group of organisms with enormous importance to humans. Pathogenic fungi are a significant cause of human disease, particularly in the rapidly increasing proportion of the population whose immune system has been compromised by disease, chemotherapy, or immunosuppressive drugs. A wide variety of plant-pathogenic fungi (e.g., blights, rusts, molds, smuts, mildews) cause huge food crop loss and damage to ornamental plants. Plant diseases are caused by a myriad of invasive fungal pathogens falling into many genera, for example: soft rot (e.g., Rhizopus), leaf curl (e.g., Taphrina), powdery mildew (e.g., Sphaerotheca), leaf spots (e.g., Fulvia), blight (e.g., Alternaria), blast (e.g., Magnaporthe), black rot (e.g., Guignardia), scab (e.g., Venturia), wilts (e.g., Fusarium), rusts (e.g., Puccinia), smuts (e.g., Ustilago), and cankers (e.g., Rhizoctonia). In addition, fungal species are the commercial source of a great many medicinally useful products, such as antibiotics (e.g., beta-lactam antibiotics such as penicillin, cephalosporin, and their derivatives), anti-hypercholesterolemic agents (e.g., lovastatin and compactin), immunosuppressives (e.g., cyclosporin), and antifungal drugs (e.g., pneumocandin and echinocandin). All of these drugs are fungal secondary metabolites, small secreted molecules that fungi utilize against competitors in their microbial environment. Finally, fungi also produce commercially important enzymes (e.g., cellulases, proteases, and lipases) as well as other products (e.g., citric acid, gibberellic acid, natural pigments, and flavorings).
The specifics by which fungi invade their growth substrate are not understood in detail. However, two important themes regarding the fungal invasion process have emerged in recent years. First, important human fungal pathogens, such as Candida sp., Aspergillus sp., Mucor sp., Rhizopus sp., Fusarium sp, Penicillium marneffei, Microsporum sp. and Trichophyton sp. invade through host tissues as filamentous hyphae. The virulence of
Candida
(C.)
albicans
has been shown to be dependent upon invasion of host tissues; mutations in any of several genes required for invasive growth substantially reduce virulence in a mouse model of systemic infection. Pathogenesis of the plant fungal pathogen
Ustilago
(U.)
maydis
also requires invasion. Second, there is a correlation between genes that regulate agar invasion in
Saccharomyces
(S.)
cerevisiae
and genes that control invasion in pathogenic yeast. As
S. cerevisiae
is amenable to genetic studies, it can be utilized to molecularly dissect the genetics of fungal invasion.
Homologs of certain
S. cerevisiae
genes required for invasion also regulate the production of secondary metabolites and secreted catabolic enzymes in other fungi. For example, activating mutations in Aspergillus homologs of the
S. cerevisiae
INV genes cause increased production of the secondary metabolite penicillin and a secreted alkaline phosphatase (Orejas et al., Genes Dev. 1995, 9:1622).
SUMMARY OF THE INVENTION
In one aspect, the invention features a method for determining whether a candidate compound decreases the expression of a gene operably linked to a fungal invasin gene promoter. The method generally includes the steps of (a) providing a fungus expressing the gene operably linked to a fungal invasin gene promoter; (b) contacting the fungus with the candidate compound; and (c) detecting or measuring expression of the gene following contact of the fungus with the candidate compound. In preferred embodiments, the fungus is a wild-type strain (e.g.,
Saccharomyces cerevisiae, Candida albicans
, or
Aspergillus nidulans
); a mutant strain; or a transgenic fungus. In other preferred embodiments, the gene used in the method of the invention is a fungal invasin gene. Exemplary fungal invasin genes include, without limitation, AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, FLO11, IRA1, MCM1, MGA1, MUC1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB1, SSD1, STE21, STP22, SWI4, TPK2, TPK3, RIM1, or YPR1.
In still other preferred embodiments, the gene used in the method of the invention is a reporter gene. Exemplary reporter genes useful in the methods of the invention include, without limitation, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), &bgr;-galactosidase (lacZ), luciferase, URA3, or HIS3.
In preferred embodiments, the fungal invasin gene promoter utilized in the methods of the invention is derived from the FLO11, MUC1, STA1, STA2, or STA3 gene promoter. In other preferred embodiments, the fungal invasin gene promoter includes a promoter sequence derived from an AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, RIM1, INV14, INV15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2, TPK3, or YPR1 gene promoter. Preferably, the fungal invasin gene promoter is a fragment or a deletion of the fungal invasin gene promoter (e.g., a fragment of the FLO11 gene promoter); and, if desired, the fragment is fused to a basal promoter (e.g., a basal promoter from a PGK1, ADH1, GAL1-10, tet-R, MET25, CYC1 or CUP1 gene).
Typically, the expression of the gene (e.g., the endogenous FLO11 or a recombinant reporter gene expressed under the control of the FLO11 gene promoter or fragment thereof) is measured by assaying the RNA or protein levels or both of the expressed gene. For example, the polypeptide expressed by the fungal invasin gene or by the reporter gene produces a detectable signal under conditions such that the compound causes a measurable signal to be produced. Quantitatively determining the amount of signal produced requires comparing the amount of signal produced to the amount of signal detected in the absence of any compound being tested or upon contacting the cell with any other compound as is described herein. The comparison permits the identification of the compound as one which causes a change in the detectable signal produced by the expressed gene (e.g., at the RNA or protein level) and thus identifies a compound that is capable of inhibiting fungal invasion. A decrease in the expression of the fungal invasin gene is generally accompanied by an inhibition of fungal invasion or an inhibition of the developmental switch from yeast form to pseudohyphal growth or both.
In related aspects, the invention also features a method for determining whether a candidate compound increases the expression of a gene operably linked to a fungal invasin gene promoter. The method generally includes the steps of (a) providing a fungus expressing the gene operably linked to a fungal invasin gene promoter; (b) contacting the fungus with the candidate compound; and (c) detecting or measuring expression of the gene following contact of the fungus with the candidate compound. In preferred embodiments, the method further includes determining whether the candidate compound increases the production of a secondary metabolite in the fungus.
In another aspect, the invention features a method for determining whether a candidate compound inhibits fungal invasion. The method generally includes the steps of (a) contacting a fungus with a candidate compound under conditions suitable for invasion and (b) measuring or detecting invasion by the fungus following contact with the candidate compound. In preferred embodiments, the fungus is
Candida albicans
or
Saccharomyces cerevisiae.
In another aspect, the invention features a method for determ

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