US2004121468A1PendingUtilityA1

Methods for regulating bud-hypha transitions and cAMP levels by the adenylate cyclase-associated protein gene, CAP1

Priority: Mar 9, 2001Filed: Jan 13, 2004Published: Jun 24, 2004

Est. expiryMar 9, 2021(expired)· nominal 20-yr term from priority

Inventors:Paula Sundstrom

A61K 38/37

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Claims

Abstract

The infection of a mammalian host by a microorganism can be prevented or treated through the disruption of the C. albicans homologue of adenylate cyclase-associated protein gene. These methods may be used in the identification, prevention or treatment of microbial infection of mammalian hosts such as immunocompromised or immunosuppressed humans, for example, those having AIDS or undergoing transplantation or anti-cancer therapy.

Claims

exact text as granted — not AI-modified

I claim:

1 . A method for interfering with morphogenic transitions of a fungus comprising the step of:
interfering with biochemical signaling pathways upon which a fungus relies for morphogenic transition.

2 . The method of claim 1 , wherein said method further comprises the step of reducing the virulent properties of said fungus.

3 . The method of claim 2 , wherein said virulent properties comprise adhesive properties.

4 . The method of claim 3 , wherein said adhesive properties comprise abilities of fungi to adhere to one or more human tissues.

5 . The method of claim 4 , wherein said human tissues are one or more human tissues selected from the group consisting of vaginal, penile, oral, esophageal, gastrointestinal, and umbilical tissues.

6 . The method of claim 2 , wherein said virulent properties comprise invasive properties.

7 . The method of claim 6 , wherein said invasive properties comprise abilities of fungi to degrade extracellular matrix proteins.

8 . The method of claim 6 , wherein said invasive properties comprise abilities of fungi to block neutrophil oxygen radical production and degranulation.

9 . The method of claim 2 , wherein said virulent properties comprise proliferative properties.

10 . The method of claim 9 , wherein said proliferative properties comprise abilities of fungus cells to induce their exit from other cells that have engulfed said fungus cells.

11 . The method of claim 10 , wherein said exit from other cells comprises the physical interaction by fungus cells of said other cells by hyphae.

12 . The method of claim 1 , wherein said fungus comprises a pathogenic yeast strain.

13 . The method of claim 12 , wherein said pathogenic yeast strain comprises C. albicans.

14 . The method of claim 1 , wherein said morphogenic transitions comprise transitions from the budding form to the hyphal growth form.

15 . The method of claim 1 , wherein said biochemical signaling pathways comprise cAMP-PKA signaling pathways of said fungus.

16 . The method of claim 15 , wherein said interfering with said multifunctional cAMP-PKA signaling or other Cap1 function pathways of a fungus comprises the disruption of a gene associated with said pathway.

17 . The method of claim 16 , wherein said gene comprises the C. albicans homolog of adenylate cyclase-associated protein (CAP1) gene.

18 . The method of claim 17 , wherein said disruption comprises interfering with the coding region of said C. albicans homolog of adenylate cyclase-associated protein (CAP1) gene.

19 . The method of claim 17 , wherein said homolog comprises the following isolated DNA sequence SEQ. ID. NO. 2:

ATGTCAACCGAGGAGAGTCAATTCAATGTTCAAGGTTACAATATTATCAC

AATCTTGAAAAGATTAGAGGCAGCAACGTCTCGTCTTGAGGACATTACCA

TTTTCCAAGAGGAAGCAAACAAAAACCACTATGGAGTTGATTCTCTCACT

GAAAAGGGAACCCCCAAAAGCAGAACTGTTGAATCGTCAGAAGCAACTTC

CGATGGTAAATCACTCGAATCTACATCATTTGCCACTTTTTCTGAAGCTC

CTGTAGAAAAATCCAAATTGATTGTGGAATTTGAGAACTTTGTTGAAAGC

TACGTTCATCCACTTGTTGAAACATCCAAAAAGATCGATTCCTTGGTGGG

GGAGTCCGCCCAATATTTTTATGAGGCATTTGTCGAACAAGGGAAATTTT

TGGAGCTTGTATTGCAATCCCAACAACCAGATATGACTGATCCAGCTTTG

GCAAAGGCATTAGAACCAATGAATGCTAAATGCACCAAAATTAACGAATT

AAAAGATTCCAATCGTAAATCTCCATTCTTCAATCATTTAAGCACTTTCA

GTGAAAGTAATGCCGTTTTTTATTGGATTGGGATCCCTACACCAGTCTCG

TACATTACTGATACTAAAGATACAGTCAAATTTTGGTCTGACAGAGTTTT

AAAAGAATACAAGACCAAAGACCAAGTGCATGTTGAATGGGTAAAACAAA

CATTATCTGTTTTTGACGAATTGAAGAATTATGTTAAAGAATATCACACA

ACTGGTGTTGCTTGGAACCCCAAAGGAAAGCCTTTTGCAGAAGTTGTATC

TCAGCAAACAGAGAGTGCTGCTAAGAATTCTTCGTCTGCTTCTGGTTCTG

CAGGAGGAGCAGCTCCACCACCACCTCCACCTCCACCTCCAGCAACGTTT

TTTGATGACACTGAAAAAGACAGTGAAAATCCATCTCCAGCTTCAGGTGG

TATTAATGCGGTTTTTGCTGAATTGAATCAAGGTGCCAACATCACATCTG

GTTTAAAAAAAGTCGACAAATCTGAGATGACGCATAAGAACCCTGAATTA

AGAAAACAGCCACCAGTTGCACCAAAAAAACCAGCACCCCCAAAGAAGCC

ATCTAGTTTATCCGGTGGTGTGAGTTCAGCTCCAGTAAAGAAGCCTGCTA

AGAAGGAGTTGATTGACGGTACAAAATGGATAATTCAAAATTTTACAAAA

GCTGATATTTCCGATTTGAGTCCAATTACCATTGAAGTTGAGATGCATCA

ATCTGTTTTCATTGGTAATTGTAGTGATGTCACCATTCAGTTGAAAGGTA

AAGCAAATGCAGTGTCGGTATCGGAAACCAAGAATGTGGCTCTTGTCATT

GATTCGTTGATTTCCGGAGTCGATGTTATTAAATCCTACAAGTTTGGTAT

ACAAGTTTTAGGTTTGGTACCAATGTTGAGTATTGATAAATCAGATGAAG

GGACTATCTATTTGTCGCAAGAAAGCATTGACAATGATAGTCAGGTTTTT

ACTAGTAGCACTACAGCACTCAACATCAATGCACCAAAGGAAAATGATGA

TTATGAAGAATTGGCTGTTCCTGAACAATTTGTTAGTAAGGTTGTGAATG

GCAAATTAGTCACTCAAATTGTTGAACATGCTGGATAA;

including any insertions, deletions, mutations, or modifications.

20 . The method of claim 17 , wherein said homolog encodes a polypeptide having the following sequence (SEQ. ID. NO. 1):

MSTEESQFNVQGYNIITILKRLEAATSRLEDITIFQEEANKNHYGVDSLT

EKGTPKSRTVESSEATSDGKSLESTSFATFSEAPVEKSKLIVEFENFVES

YVHPLVETSKKIDSLVGESAQYFYEAFVEQGKFLELVLQSQQPDMTDPAL

AKALEPMNAKCTKINELKDSNRKSPFFNHLSTFSESNAVFYWIGIPTPVS

YITDTKDTVKFWSDRVLKEYKTKDQVHVEWVKQTLSVFDELKNYVKEYHT

TGVAWNPKGKPFAEVVSQQTESAAKNSSSASGSAGGAAPPPPPPPPPATF

FDDTEKDSENPSPASGGINAVFAELNQGANITSGLKKVDKSEMTHKNPEL

RKQPPVAPKKPAPPKKPSSLSGGVSSAPVKKPAKKELIDGTKWIIQNFTK

ADISDLSPITIEVEMHQSVFIGNCSDVTIQLKGKANAVSVSETKNVALVI

DSLISGVDVIKSYKFGIQVLGLVPMLSIDKSDEGTIYLSQESIDNDSQVF

TSSTTALNINAPKENDDYEELAVPEQFVSKVVNGKLVTQIVEHAG.

21 . The method of claim 15 , wherein said cAMP-PKA signaling pathways operate to increase levels of cAMP.

22 . The method of claim 21 , wherein said increased levels of cAMP operate to stimulate morphogenic transitions of said fungus.

23 . The method of claim 1 , wherein said biochemical signaling pathway is within a fungus that has infected a human.

24 . The method of claim 23 , wherein said human suffers from a disease.

25 . The method of claim 24 , wherein said disease is human immunodeficiency virus.

26 . The method of claim 24 , wherein said disease comprises complications associated with acquired immune deficiency syndrome.

27 . The method of claim 24 , wherein said disease comprises complications associated with an acquired immune deficiency syndrome related complex.

28 . The method of claim 24 , wherein said disease comprises one or more diseases selected from the group consisting of HIV, mucosal candidiasis, oral candidiasis, esophageal candidiasis, thrush, hemoatogenously disseminated candidiasis, and candida vaginitis.

29 . The method of claim 23 , wherein said human is immunocompromised.

30 . The method of claim 23 , wherein said human is an organ transplant recipient.

31 . The method of claim 23 , wherein said human is undergoing a treatment regimen.

32 . The method of claim 31 , wherein said treatment regimen is chemotherapy.

33 . The method of claim 31 , wherein said treatment regimen is a drug regimen.

34 . The method of claim 33 , wherein said drug regimen suppresses the immune system.

35 . The method of claim 33 , wherein said drug regimen incorporates the use of one or more drugs that are selected from the group consisting of azathioprine, steroids, cyclosporine, antilymphocyte globulins, monoclonal anti-T cell antibodies, prednisone, methylprednisone, and cyclophosphamide.

36 . A microarray comprising at least one nucleotide sequence or fragment thereof, of the CAP1 gene.

37 . A method for detecting a gene expression product capable of stimulating increases in cAMP levels in a microorganism, comprising the steps of:
hybridizing labeled cDNA created from isolated mRNA from a fungal strain onto a microarray comprising at least one nucleotide sequence or fragment thereof, of the CAP1 gene; assessing expression level or lack thereof of a gene expression product of said fungal strain.

38 . The method of claim 37 , wherein said fungal strain contain the C. albicans homolog of the adenylate cyclase-associated protein (CAP1) gene.

39 . The method of claim 37 , wherein said increase in cAMP levels promote bud/hypha transitions of said microorganism.

40 . The method of claim 37 , wherein said microorganism is selected from the group consisting of bacteria, yeast and fungus.

41 . The method of claim 37 , wherein said microorganism is a yeast.

42 . The method of claim 37 , wherein said microorganism is C. albicans.

43 . A method for identifying potential virulence genes in fungus comprising the step of comparing the gene expression products in at least two different fungal strains.

44 . The method of claim 43 , wherein said comparing step comprises:
co-hybridizing a labeled first cDNA from isolated mRNA from a first fungal strain and a labeled second cDNA from isolated mRNA from a second fungal strain to a microarray, wherein said microarray comprises at least one fungal nucleotide sequence or fragment thereof of C. albicans; comparing hybridization signals from said cDNA of said first fungal strain and the said cDNA of said second fungal strain.

45 . The method of claim 44 , further comprising the step of isolating cDNA of interest.

46 . The method of claim 44 , wherein said first fungal strain is a mutant fungal strain.

47 . The method of claim 46 , wherein said fungal strain contain the C. albicans homolog of the adenylate cyclase-associated protein (CAP1) gene.

48 . The method of claim 44 , wherein said second fungal strain is a parental fungal strain.

49 . The method of claim 48 , wherein said fungal strain contain the C. albicans homolog of the adenylate cyclase-associated protein (CAP1) gene.

50 . The method of claim 44 , wherein said second fungal strain is a revertant fungal strain.

51 . The method of claim 50 , wherein said fungal strain contain the C. albicans homolog of the adenylate cyclase-associated protein (CAP1) gene.

52 . The method of claim 43 , wherein said fungus is a pathogenic or nonpathogenic yeast strain.

53 . The method of claim 52 , wherein said pathogenic yeast strain is Candida albicans.

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