Identification of essential genes of Aspergillus fumigatus and methods of use

• US 20030119013A1
• Filed: 04/23/2002
• Published: 06/26/2003
• Est. Priority Date: 04/23/2001
• Status: Abandoned Application

• Current Assignee: Merck & Co., Inc.
• Original Assignee: Merck & Co., Inc.
• Inventors: Tishkoff, Daniel, Hu, Wenqi, Lemieux, Sebastien, Zamudio, Carlos, Jiang, Bo, Eroshkin, Alexey M.
• Application Number:
  US10/128,714
• Publication Number:
  US 20030119013A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C07K 14/38 from Aspergillus
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 1/145 Fungal isolates
• CPC Class Codes: C12R 2001/68 Aspergillus fumigatus

Abstract

The present invention provides nucleotide sequences, methods and compositions that enable the experimental determination as to whether any gene in the genome of Aspegillus fumigatus is essential, and whether that gene is required for virulence or pathogenicity. The methods involve the construction of genetic mutants in which a target gene is placed under conditional expression. The identification of essential genes and those genes critical to the development of virulent infections, provides a basis for the development of screens for new drugs against Aspergillus fumigatus.

The present invention further provides Aspergillum fumigatus genes that are essential and are potential targets for drug screening. The nucleotide sequence of the target genes can be used for various drug discovery purposes, such as expression of the recombinant protein, hybridization assay and construction of nucleic acid arrays. The uses of proteins encoded by the essential genes, and genetically engineered cells comprising modified alleles of essential genes in various screening methods are also encompassed by the invention.

Claims

1. A purified or isolated nucleic acid molecule comprising a nucleotide sequence encoding a gene product, wherein said gene product consists essentially of an amino acid sequence of one of SEQ ID NO:

3001-3594 or 8001-8603.

2. The nucleic acid molecule of claim 1, wherein said nucleotide sequence is one of SEQ ID NO:

2001-2594 or 7001-7603.

6. A vector comprising a promoter operably linked to the nucleic acid molecule of claim 1, 2, 3, 4, or 5.

7. The vector of claim 6, wherein said promoter is regulatable.

8. A host cell containing the vector of claim 7.

3. A nucleic acid molecule comprising a fragment of one of SEQ ID NO:

1-594, 5001-5603,1001-1594, or 6001-6603, said fragment selected from the group consisting of fragments comprising at least 10, at least 20, at least 25, at least 30, at least 50 and at least 100 consecutive nucleotides of one of SEQ ID NO;

1-594, 5001-5603,1001-1594, or 6001-6603.

4. A nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent condition to a second nucleic acid molecule consisting of (a) a nucleotide sequence selected from the group consisting of one of SEQ ID NO:

1-594, 5001-5603,1001-1594, or 6001-6603, or (b) a nucleotide sequence that encodes a polypeptide consisting of an amino acid sequence selected from the group consisting of one of SEQ ID NO;

3001-3594 or 8001-8603;

wherein said stringent condition comprises hybridization to filter-bound DNA in 6×

sodium chloride/sodium citrate (SSC) at about 45°

C. followed by one or more washes in 0.2×

SSC/0.1% SDS at about 50-65°

C.

5. A purified or isolated nucleic acid molecule obtained from an organism other than Candida albicans or Saccharomyces cerevisiae comprising a nucleotide sequence having at least 30% identity to a sequence selected from the group consisting of SEQ ID NO:

1-594, 5001-5603,1001-1594, or 6001-6603;

fragments comprising at least 25 consecutive nucleotides of SEQ ID NO;

1-594, 5001-5603,1001-1594, or 6001-6603;

the sequences complementary to SEQ ID NO;

1-594, 5001-5603,1001-1594, or 6001-6603; and

the sequences complementary to fragments comprising at least 25 consecutive nucleotides of SEQ ID NO;

1-594, 5001-5603,1001-1594, or 6001-6603, as determined using BLASTN version 2.0 with default parameters.

9. A purified or isolated polypeptide comprising an amino acid sequence selected from the group consisting of one of SEQ ID NO:

3001-3594, or 8001-8603.

10. A purified or isolated polypeptide obtained from an organism other than Candida albicans or Saccharomyces cerevisiae comprising an amino acid sequence having at least 30% similarity to an amino acid sequence selected from the group consisting of one of SEQ ID NO:

3001-3594 or 8001-8603, as determined using FASTA version 3.0t78 with the default parameters.

35. An antibody preparation which binds the polypeptide of claim 9 or 10.

36. The antibody preparation of claim 35 which comprises a monoclonal antibody.

11. A fusion protein comprising a fragment of a first polypeptide fused to a second polypeptide, said fragment consisting of at least 6 consecutive residues of an amino acid sequence selected from one of SEQ ID NO:

3001-3594 or 8001-8603.

12. A method of producing a polypeptide, said method comprises introducing into a cell, a vector comprising a promoter operably linked to a nucleotide sequence encoding a polypeptide consisting of an amino acid sequence selected from the group consisting of one of SEQ ID NO:

3001-3594 or 8001-8603; and

culturing the cell such that the nucleotide sequence is expressed.

13. A method of producing a polypeptide, said method comprising providing a cell which comprises a heterologous promoter operably linked to a nucleotide sequence encoding a polypeptide consisting of an amino acid sequence selected from the group consisting of one of SEQ ID NO:

3001-3594 or 8001-8603; and

culturing the cell such that the nucleotide sequence is expressed.

14. A method for identifying a compound which modulates the activity of a gene product encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of one of SEQ ID NO:

2001-2594 or 7001-7603, said method comprising;

(a) contacting said gene product with a compound; and

(b) determining whether said compound modulates the activity of said gene product.

15. The method of claim 14, wherein the activity of the gene product is inhibited.

16. The method of claim 14, wherein said gene product is a polypeptide and said activity is selected from the group consisting of an enzymatic activity, carbon compound catabolism activity, a biosynthetic activity, a transporter activity, a transcriptional activity, a translational activity, a signal transduction activity, a DNA replication activity, and a cell division activity.

17. A method of eliciting an immune response in an animal, comprising introducing into the animal a composition comprising an isolated polypeptide, the amino acid sequence of which comprises at least 6 consecutive residues of one of SEQ ID NO:

3001-3594 or 8001-8603.

18. An isolated strain of Aspergillus fumigatus wherein the gene comprising a nucleotide sequence selected from the group consisting of one of SEQ ID NO:

1-594, 5001-5603,1001-1594, 6001-6603, 2001-2594, and 7001-7603, is inactive or placed under the control of a heterologous promoter.

20. The strain of claim 18 or 19, wherein said heterologous promoter is regulatable.

37. A method for evaluating a compound against a target gene product encoded by a nucleotide sequence comprising one of SEQ ID NO:

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603, said method comprising the steps of;

(a) contacting wild type fungal cells with the compound and generating a first protein expression profile;

(b) determining the protein expression profile of the fungal cells of claim 18 or 19, which have been cultured under conditions wherein the target gene is substantially underexpressed, not expressed or overexpressed and generating a second protein expression profile for the cultured cells; and

(c) comparing the first protein expression profile with the second protein expression profile to identify similarities in the profiles.

38. A collection of Aspergillus fumigatus strains of claim 18 or 19, wherein the cells of each strain further comprises one or more molecular tags each of about 20 nucleotides, wherein the sequences of each tag in a cell is unique to the strain of cells.

39. The collection of claim 38, wherein the molecular tag(s) is disposed within the gene disruption cassette.

40. An isolated strain of Aspergillus fumigatus of claim 18 or 19, wherein the cells of said strain further comprises one or more molecular tags each of about 20 nucleotides, wherein the sequence of each tag in said cells is unique to said strain of cells.

19. An isolated strain of Aspergillus fumigatus comprising a nucleic acid molecule comprising a nucleotide sequence selected from one of SEQ ID NO:

1-594, 5001-5603,1001-1594, 6001-6603, 2001-2594, and 7001-7603, under the control of a heterologous promoter.

21. A method of identifying a compound or binding partner that binds to a polypeptide comprising an amino acid sequence selected from the group consisting of one of SEQ ID NO:

3001-3594 or 8001-8603, or a fragment thereof said method comprising;

(a) contacting the polypeptide or fragment thereof with a plurality of compounds or a preparation comprising one or more binding partners; and

(b) identifying a compound or binding partner that binds to the polypeptide or fragment thereof.

22. A method for identifying a compound having the ability to inhibit growth or proliferation of Aspergillus fumigatus, said method comprising the steps of:

(a) reducing the level or activity of a gene product encoded by a nucleic acid selected from the group consisting of SEQ ID NO;

2001-2594 or 7001-7603, in a Aspergillus fumigatus cell relative to a wild type cell, wherein said reduced level is not lethal to said cell;

(b) contacting said cell with a compound; and

(c) determining whether said compound inhibits the growth or proliferation of said cell.

23. The method of claim 22, wherein said step of reducing the level or activity of said gene product comprises transcribing a nucleotide sequence encoding said gene product from a regulatable promoter under conditions in which said gene product is expressed at said reduced level.

24. The method of claim 23, wherein said gene product is a polypeptide comprising a sequence selected from the group consisting of polypeptides encoded by SEQ ID NO:

3001-3594 or 8001-8603.

25. A method for inhibiting growth or proliferation of Aspergillus fumigatus cells comprising contacting the cells with a compound that (i) reduce the level of or inhibit the activity of a nucleotide sequence selected from the group consisting of SEQ ID NO:

1-594, 5001-5603,1001-1594, 6001-6603, 2001-2594, and 7001-7603, or (ii) reduce the level of or inhibit the activity of a gene product encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO;

1-594, 5001-5603,1001-1594, 6001-6603, 2001-2594, and 7001-7603,.

26. The method of claim 25, wherein said gene product is a polypeptide comprising an amino acid sequence selected from the group consisting of polypeptides encoded by SEQ ID NO:

3001-3594 or 8001-8603.

27. The method of claim 25, wherein the compound is an antibody, a fragment of an antibody, an antisense nucleic acid molecule, or a ribozyme.

28. A method for manufacturing an antimycotic compound comprising the steps of:

(a) screening a plurality of candidate compounds to identify a compound that reduces the activity or level of a gene product encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO;

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603; and

(b) manufacturing the compound so identified.

29. The method of claim 28, wherein said gene product is a polypeptide comprising an amino acid sequence selected from the group consisting of polypeptides encoded by SEQ ID NO:

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603.

30. A method for treating an infection of a subject by Aspergillus fumigatus comprising administering a pharmaceutical composition comprising a therapeutically effective amount of a compound that reduces the activity or level of a gene product encoded by a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603, and a pharmaceutically acceptable carrier, to said subject.

31. The method of claim 30, wherein the compound is an antibody, a fragment of an antibody, an antisense nucleic acid molecule, or a ribozyme.

34. The method of claim 30, wherein said subject is selected from the group consisting of a plant, a vertebrate, a mammal, an avian, and a human.

32. A method for preventing or containing contamination of an object by Aspergillus fumigatus comprising contacting the object with a composition comprising an effective amount of a compound that reduces the activity or level of a gene product encoded by a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603.

33. A pharmaceutical composition comprising a therapeutically effective amount of an agent which reduces the activity or level of a gene product encoded by a nucleic acid selected from the group consisting of SEQ ID NO:

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603, in a pharmaceutically acceptable carrier.

41. A nucleic acid molecule microarray comprising a plurality of nucleic acid molecules, wherein each nucleic acid molecule comprises a nucleotide sequence that is hybridizable to a target nucleotide sequence selected from the group consisting of SEQ ID NO:

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603.

42. A computer or a computer readable medium that comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NO.:

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603, or at least one amino acid sequence selected from the group consisting of SEQ ID NO.;

3001-3594 and 8001-8603.

43. A method assisted by a computer for identifying a putatively essential gene of a fungus, comprising detecting sequence homology between a fungal nucleotide sequence or fungal amino acid sequence with at least one nucleotide sequence selected from the group consisting of SEQ ID NO.:

1-594, 5001-5603, 1001-1594, 6001-6603, 2001-2594, and 7001-7603, or at least one amino acid sequence selected from the group consisting of SEQ ID NO.;

3001-3594 and 8001-8603.