Processes of screening for an antimycotic agent

Details Processes of screening for an antimycotic agent Processes of screening for an antimycotic agent

1999-11-23

2002-08-13

Duffy, Patricia A. (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S004000, C435S007200, C435S007310, C435S007320, C435S029000, C435S069100, C435S070100, C435S822000, C435S911000

Reexamination Certificate

active

06432664

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a protein regulating the sensitivity to an antimycotic aureobasidin and a gene coding for this protein, namely, a gene coding for a protein regulating aureobasidin sensitivity. The present invention further relates to a series of the uses of the protein and the gene. Furthermore, it relates to an antibody against this protein and the use of the same.
2. 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 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 W092/03455). 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 and No. 279384/1993, Japanese Patent Application No. 303177/1992, 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, 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 (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 conventional antimycotics with a weak toxicity shows only a fungistatic effect, which has been regarded as a clinical problem. In contrast, aureobasidin has a fungicidal effect. From this point of view, it has been urgently required to clarify the mechanism of the selective toxicity of fungi of aureobasidin. However this mechanism still remains unknown.
Under these circumstances, the present invention aims at finding a novel protein regulating aureobasidin sensitivity through the clarification of the mechanism of the selective toxicity to fungi of aureobasidin. Accordingly, the present invention aims at finding a gene coding for a protein regulating aureobasidin sensitivity, providing a process for cloning this gene and the protein regulating aureobasidin sensitivity which is encoded by this gene, further providing an antisense DNA and an antisense RNA of this gene, providing a nucleic acid probe being hybridizable with this gene, providing a process for detecting this gene with the use of the nucleic acid probe, providing a process for producing the protein regulating aureobasidin sensitivity by using this gene and providing an antibody against the protein regulating aureobasidin sensitivity, and a process for detecting the protein regulating aureobasidin sensitivity by using this antibody.
SUMMARY OF THE INVENTION
The present invention may be summarized as follows. Namely, the first invention of the present invention relates to an isolated gene coding for a protein regulating aureobasidin sensitivity, that is, a gene regulating aureobasidin sensitivity. The second invention relates to a process for cloning a gene regulating aureobasidin sensitivity which is characterized by using the gene regulating aureobasidin sensitivity of the first invention or a part thereof as a probe. The third invention relates to a nucleic acid probe which is hybridizable with a gene regulating aureobasidin sensitivity and comprises a sequence consisting of 15 or more bases. The fourth invention relates to an antisense DNA of a gene regulating aureobasidin sensitivity. The fifth invention relates to an antisense RNA of a gene regulating aureobasidin sensitivity. The sixth invention relates to a recombinant plasmid having a gene regulating aureobasidin sensitivity contained therein. The seventh invention relates to a transformant having the above-mentioned plasmid introduced thereinto. The eighth invention relates to a process for producing a protein regulating aureobasidin sensitivity by using the above-mentioned transformant. The ninth invention relates to an isolated protein regulating aureobasidin sensitivity. The tenth invention relates to an antibody against a protein regulating aureobasidin sensitivity. The eleventh invention relates to a process for detecting a protein regulating aureobasidin sensitivity by using the above-mentioned antibody. The twelfth invention relates to a process for detecting a gene regulating aureobasidin sensitivity by the hybridization which is characterized by using the nucleic acid probe of the third invention of the present invention. The thirteenth invention relates to a process for screening an antimycotic by using the abov

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