Gene regulating aureobasidin sensitivity

Details Gene regulating aureobasidin sensitivity Gene regulating aureobasidin sensitivity

1996-09-20

2001-02-27

Wax, Robert A. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S252300, C435S320100, C435S254100, C435S254300, C435S006120, C536S023100, C536S023740, C536S023700

Reexamination Certificate

active

06194166

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a protein regulating a sensitivity to an antimycotic aureobasidin, a gene encoding this protein and to uses of the protein and gene.
DESCRIPTION OF RELATED ART
Systemic mycoses including candidiasis have increased with an increase in immunocompromised patients in recent years due to, for example, the extended use of immunosuppressive drugs and acquired immunodeficiency syndrome (AIDS), and as opportunistic infection due to microbial substitution caused by the frequent use of widespectrum antibacterial antibiotics. Although drugs for treating mycoses such as amphotericin B, flucytosine and azole drugs (for example, fluconazole and miconazole) are now used to cope with this situation, none of them can achieve a satisfactory effect. Also, known diagnostic drugs are insufficient. For candidiasis, in particular, although there have been known several diagnostic drugs (for example, CAND-TEC for detection of candida antigen and LABOFIT for detection of D-arabinitol), none of them gives any satisfactory results in specificity or sensitivity.
The reasons for the delay in the development of remedies and diagnostic drugs for mycoses as described above are that fungi causing the mycoses are eukaryotic organisms similar to the host (i.e., man) and thus are not largely different from man and that knowledges of fungi, in particular, pathogenic fungi are insufficient. Therefore it is difficult to distinguish fungi from man or to selectively kill fungi, which is responsible for the delay in the development of drugs for mycoses.
Recently, the application of genetic engineering techniques such as antisense or PCR to the treatment and diagnosis of mycoses has been expected. However known genes which are applicable thereto and/or proteins coded for by these genes are rare (PCT Pamphlet WO92/03455). Regarding pathogenic fungi, there have been cloned in recent years an acid protease gene, which has been assumed to participate in the pathogenicity of
Candida albicans
(hereinafter referred to simply as
C. albicans
) and
Candida tropicalis
(hereinafter referred to as
C. tropicalis
) causing candidiasis [B. Hube et al.,
J. Med. Vet. Mycol.,
29, 129-132 (1991); Japanese Patent Laid-Open No. 49476/1993; and G. Togni et al.,
FEBS Letters,
286, 181-185 (1991)], a calmodulin gene of
C. albicans
[S. M. Saporito et al.,
Gene,
106, 43-49 (1991)] and a glycolytic pathway enzyme enolase gene of
C. albicans
[P. Sundstrom et al.,
J. Bacteriology,
174, 6789-6799 (1991)]. However, each of these genes and proteins coded for thereby is either indistinguishable from nonpathogenic fungi and eukaryotic organisms other than fungi or, if distinguishable therefrom, cannot serve as a definite action point for exhibiting any selective toxicity.
Aureobasidin [Japanese Patent Laid-Open No. 138296/1990, No. 22995/1991, No. 220199/1991, No. 279384/1993, and No. 65291/1994;
J. Antibiotics,
44 (9), 919-924, ibid., 44 (9), 925-933, ibid., 44 (11), 1187-1198 (1991)] is a cyclic depsipeptide obtained as a fermentation product of a strain
Aureobasidium pullulans
No. R106. It is completely different in structure from other antimycotics. As Tables 1 and 2 show below, aureobasidin A, which is a typical aureobasidin compound, exerts a potent antimycotic activity on various yeasts of the genus Candida including
C. albicans
which is a pathogenic fungus,
Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis
and fungi of the genus Aspergillus and Penicillium (Japanese Patent Laid-Open No. 138296/1990) but has an extremely low toxicity in mammal. Thus this compound is expected to be useful as an antimycotic being excellent in selective toxicity.
Hereinafter, Candida, Cryptococcus and Aspergillus will be abbreviated respectively as C, Cr. and A.
TABLE 1
Test Strain
TIMM No.
MIC(&mgr;g/ml)
C. albicans
0136
≦0.04
C. albicans
var.
stellatoidea
1308
≦0.04
C. tropicalis
0312
0.08
C. kefyr
0298
0.16
C. parapsilosis
0287
0.16
C. krusei
0270
≦0.04
C. guilliermondii
0257
0.08
C. glabrata
1062
<0.04
Cr. neoformans
0354
0.63
Cr. terreus
0424
0.31
Rhodotorula rubra
0923
0.63
A. fumigatus
0063
20
A. clavatus
0056
0.16
TABLE 2
Test Strain
TIMM No.
MIC(&mgr;g/ml)
A. nidulans
0112
0.16
A. terreus
0120
5
Penicillium commune
1331
1.25
Trichophyton mentagrophytes
1189
10
Epidermophyton floccosum
0431
2.5
Fonsecaea pedrosoi
0482
0.31
Exophiala werneckii
1334
1.25
Cladosporium bantianum
0343
0.63
Histoplasma capsulatum
0713
0.16
Paracoccidioides brasiliensis
0880
0.31
Geotrichum candidum
0694
0.63
Blastomyces dermatitidis
0126
0.31
Each of the existing antimycotics with a low toxicity shows only a fungistatic action, which causes a clinical problem. In contrast, aureobasidin exerts a germicidal action. Although it has been required to clarify the mechanism of the selective toxicity of aureobasidin from these viewpoints, this mechanism still remains completely unknown.
As described in Canadian Patent Laid-Open No. 2124034, the present inventors have previously found out that
Saccharomyces cerevisiae
(hereinafter referred to simply as
S. cerevisiae
) and
Schizosaccharomyces pombe
(hereinafter referred to simply as
Schizo. pombe
) are sensitive to aureobasidin. We have further mutated sensitive cells of
S. cerevisiae
or
Schizo. pombe
into resistant cells and successfully isolated a gene capable of imparting a resistance to aureobasidin (a resistant gene) therefrom. We have furthermore successfully isolated a gene capable of imparting aureobasidin sensitivity (a sensitive gene) from the corresponding sensitive cells.
We have also isolated a gene regulating aureobasidin sensitivity from
C. albicans
with the use of the gene regulating aureobasidin sensitivity or a part thereof as a probe. However no gene regulating aureobasidin sensitivity has been found in molds including those belonging to the genus Aspergillus.
There have been known techniques for introducing useful genes into monoploid fungal cells to be used in a laboratory, for example,
Saccharomyces cerevisiae
(hereinafter referred to simply as
S. cerevisiae
),
Schizosaccharomyces pombe
(hereinafter referred to simply as
Schizo. pombe
) and
Aspergillus nidulans
(hereinafter referred to simply as
A. nidulans
). Since the incorporation and fixation of plasmid DNAs into fungal cells are relatively scarcely successful, it is required to use selective markers in the identification of transformants. In the most common case, selection can be achieved by introducing an auxotrophic mutation into host cells. Examples of the mutation generally employed in, for example,
S. cerevisiae
include ura3, leu2, trp1 and his3. A plasmid carries a wild type copy of one of these genes. Since the wild type copy on the plasmid is dominant over the chromosomal allele of the host, cells having the plasmid introduced thereinto can be screened in a minimal medium which contains no nutrient required by the auxotrophic host cells. Also there have been published some reports, though in a small number, relating to the use of drug resistance in the screening of transformants. Namely, there have been reported replication vectors and chromosome integration vectors containing genes which are resistant against antibiotics such as a neomycin homologue G418, hygromycin and cerulenin. A replication vector has a DNA replication origin acting in a cell. This plasmid is held outside the chromosome as a cyclic episome and continuously reduced at a ratio of several percent with the proliferation of the cells. An integration vector is inserted into the chromosome of a host cell and thus held in a stable state. In this case, therefore, it is unnecessary to further add a drug to the medium in order to exert the selection function for maintaining the sequence of the vector.
In the case of industrial fungi, it is required to sustain the useful character, which has been imparted thereto, in a stable state. A chromosome integration vector is useful for this purpose.
Fungi have been widely applied t

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