1887

Abstract

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of , we recently demonstrated that overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced mRNA stability in AR isolates. Mapping of the 3′ untranslated region (3′ UTR) of revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric 3′ UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that -acting sequences within the 3′ UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the mRNA of AR isolates was ∼35–50 % hyperadenylated as compared with AS isolates. poly(A) polymerase (), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like () locus. Two different alleles, , were recovered from AS (), while a single type of allele () was recovered from AR isolates (). Among the heterozygous deletions of ) and (Δ), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of in the AS isolate. This suggests a dominant negative role of in transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the locus is linked to hyperadenylation and subsequent increased stability of transcripts, thus contributing to enhanced drug resistance.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.035154-0
2010-02-01
2024-12-02
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/2/313.html?itemId=/content/journal/micro/10.1099/mic.0.035154-0&mimeType=html&fmt=ahah

References

  1. Akins R. A. 2005; An update on antifungal targets and mechanisms of resistance in Candida albicans. Med Mycol 43:285–318
    [Google Scholar]
  2. Chen C. G., Yang Y. L., Shih H. I., Su C. L., Lo H. J. 2004; CaNdt80 is involved in drug resistance in Candida albicans by regulating CDR1. Antimicrob Agents Chemother 48:4505–4512
    [Google Scholar]
  3. Coste A. T., Karababa M., Ischer F., Bille J., Sanglard D. 2004; TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2. Eukaryot Cell 3:1639–1652
    [Google Scholar]
  4. Coste A., Turner V., Ischer F., Morschhäuser J., Forche A., Selmecki A., Berman J., Bille J., Sanglard D. 2006; A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 172:2139–2156
    [Google Scholar]
  5. De Micheli M., Bille J., Schuller C., Sanglard D. 2002; A common drug-responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance. Mol Microbiol 43:1197–1214
    [Google Scholar]
  6. Edwalds-Gilbert G., Veraldi K. L., Milcarek C. 1997; Alternative poly(A) site selection in complex transcription units: means to an end?. Nucleic Acids Res 25:2547–2561
    [Google Scholar]
  7. Fonzi W. A., Irwin M. Y. 1993; Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717–728
    [Google Scholar]
  8. Franz R., Kelly S. L., Lamb D. C., Kelly D. E., Ruhnke M., Morschhäuser J. 1998; Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains. Antimicrob Agents Chemother 42:3065–3072
    [Google Scholar]
  9. Franz R., Ruhnke M., Morschhäuser J. 1999; Molecular aspects of fluconazole resistance development in Candida albicans. Mycoses 42:453–458
    [Google Scholar]
  10. Garcia-Vivas J., Lopez-Camarillo C., Zuara-Liceaga E., Orozco E., Marchat L. A. 2005; Entamoeba histolytica: cloning and expression of the poly(A) polymerase EhPAP. Exp Parasitol 110:226–232
    [Google Scholar]
  11. Gaur N. A., Puri N., Karnani N., Mukhopadhyay G., Goswami S. K., Prasad R. 2004; Identification of a negative regulatory element which regulates basal transcription of a multidrug resistance gene CDR1 of Candida albicans. FEMS Yeast Res 4:389–399
    [Google Scholar]
  12. Gerads M., Ernst J. F. 1998; Overlapping coding regions and trancriptional units of two essential chromosomal genes ( CCT8, TRP1) in the fungal pathogen Candida albicans. Nucleic Acids Res 26:5061–5066
    [Google Scholar]
  13. Higgins C. F. 1991; Stability and degradation of mRNA. Curr Opin Cell Biol 3:1013–1018
    [Google Scholar]
  14. Holm L., Sander C. 1995; DNA polymerase β belongs to ancient nucleotidyltransferase superfamily. Trends Biochem Sci 20:345–347
    [Google Scholar]
  15. Hsu S. I., Cohen D., Kirschner L. S., Lothstein L., Hartstein M., Horwitz S. B. 1990; Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells. Mol Cell Biol 10:3596–3606
    [Google Scholar]
  16. Hull C. M., Johnson A. D. 1999; Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans. Science 285:1271–1275
    [Google Scholar]
  17. Jiang B., Xu D., Allocco J., Parish C., Davison J., Veillette K., Sillaots S., Hu W., Rodriguez-Suarez R. other authors 2008; PAP inhibitor with in vivo efficacy identified by Candida albicans genetic profiling of natural products. Chem Biol 15:363–374
    [Google Scholar]
  18. Karnani N., Gaur N. A., Jha S., Puri N., Krishnamurthy S., Goswami S. K., Mukhopadhyay G., Prasad R. 2004; SRE1 and SRE2 are two specific steroid-responsive modules of Candida drug resistance gene 1 ( CDR1) promoter. Yeast 21:219–239
    [Google Scholar]
  19. Kusov Y. Y., Shatirishvili G., Dzagurov G., Gauss-Muller V. 2001; A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA. Nucleic Acids Res 29:E57
    [Google Scholar]
  20. Lopez-Camarillo C., Luna-Arias J. P., Marchat L. A., Orozco E. 2003; EhPgp5 mRNA stability is a regulatory event in the Entamoeba histolytica multidrug resistance phenotype. J Biol Chem 278:11273–11280
    [Google Scholar]
  21. Lopez-Ribot J. L., McAtee R. K., Lee L. N., Kirkpatrick W. R., White T. C., Sanglard D., Patterson T. F. 1998; Distinct patterns of gene expression associated with development of fluconazole resistance in serial Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob Agents Chemother 42:2932–2937
    [Google Scholar]
  22. Magee B. B., Magee P. T. 2000; Induction of mating in Candida albicans by construction of MTLa and MTLα strains. Science 289:310–313
    [Google Scholar]
  23. Manoharlal R., Gaur N. A., Panwar S. L., Morschhäuser J., Prasad R. 2008; Transcriptional activation and increased mRNA stability contribute to overexpression of CDR1 in azole-resistant Candida albicans. Antimicrob Agents Chemother 52:1481–1492
    [Google Scholar]
  24. McCarthy J. E. 1998; Post-transcriptional control of gene expression in yeast. Microbiol Mol Biol Rev 62:1492–1553
    [Google Scholar]
  25. Morschhäuser J., Barker K. S., Liu T. T., Blaß-Warmuth J., Homayouni R., Rogers P. D. 2007; The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog 3:e164
    [Google Scholar]
  26. Murad A. M., Lee P. R., Broadbent I. D., Barelle C. J., Brown A. J. 2000; CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast 16:325–327
    [Google Scholar]
  27. Pesole G., Liuni S. 1999; Internet resources for the functional analysis of 5′ and 3′ untranslated regions of eukaryotic mRNAs. Trends Genet 15:378
    [Google Scholar]
  28. Pesole G., Liuni S., Grillo G., Licciulli F., Mignone F., Gissi C., Saccone C. 2002; UTRdb and UTRsite: specialized databases of sequences and functional elements of 5′ and 3′ untranslated regions of eukaryotic mRNAs. Update. Nucleic Acids Res 30:335–340
    [Google Scholar]
  29. Prasad R., De W. P., Goffeau A., Balzi E. 1995; Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals. Curr Genet 27:320–329
    [Google Scholar]
  30. Prokipcak R. D., Raouf A., Lee C. 1999; The AU-rich 3′ untranslated region of human MDR1 mRNA is an inefficient mRNA destabilizer. Biochem Biophys Res Commun 261:627–634
    [Google Scholar]
  31. Puri N., Krishnamurthy S., Habib S., Hasnain S. E., Goswami S. K., Prasad R. 1999; CDR1, a multidrug resistance gene from Candida albicans, contains multiple regulatory domains in its promoter and the distal AP-1 element mediates its induction by miconazole. FEMS Microbiol Lett 180:213–219
    [Google Scholar]
  32. Reuss O., Vik A., Kolter R., Morschhäuser J. 2004; The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341:119–127
    [Google Scholar]
  33. Rognon B., Kozovska Z., Coste A. T., Pardini G., Sanglard D. 2006; Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance. Microbiology 152:3701–3722
    [Google Scholar]
  34. Ross J. 1996; Control of messenger RNA stability in higher eukaryotes. Trends Genet 12:171–175
    [Google Scholar]
  35. Russell J. E., Morales J., Makeyev A. V., Liebhaber S. A. 1998; Sequence divergence in the 3′ untranslated regions of human ζ- and α-globin mRNAs mediates a difference in their stabilities and contributes to efficient α- to - ζ gene development switching. Mol Cell Biol 18:2173–2183
    [Google Scholar]
  36. Sanglard D., Odds F. C. 2002; Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2:73–85
    [Google Scholar]
  37. Sanglard D., Kuchler K., Ischer F., Pagani J. L., Monod M., Bille J. 1995; Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39:2378–2386
    [Google Scholar]
  38. Sanglard D., Ischer F., Monod M., Bille J. 1996; Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob Agents Chemother 40:2300–2305
    [Google Scholar]
  39. Sanglard D., Ischer F., Monod M., Bille J. 1997; Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterisation of CDR2, a new multidrug ABC transporter gene. Microbiology 143:405–416
    [Google Scholar]
  40. Sparks K. A., Dieckmann C. L. 1998; Regulation of poly(A) site choice of several yeast mRNAs. Nucleic Acids Res 26:4676–4687
    [Google Scholar]
  41. Talibi D., Raymond M. 1999; Isolation of a putative Candida albicans transcriptional regulator involved in pleiotropic drug resistance by functional complementation of a pdr1 pdr3 mutation in Saccharomyces cerevisiae. J Bacteriol 181:231–240
    [Google Scholar]
  42. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
    [Google Scholar]
  43. Trzaska D., Dastych J. 2005; Role of AURE sequences in the regulation of mRNA stability. Postepy Biochem 51:28–35
    [Google Scholar]
  44. Uhl M. A., Johnson A. D. 2001; Development of Streptococcus thermophilus lacZ as a reporter gene for Candida albicans. Microbiology 147:1189–1195
    [Google Scholar]
  45. White T. C. 1997; Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41:1482–1487
    [Google Scholar]
  46. White T. C., Pfaller M. A., Rinaldi M. G., Smith J., Redding S. W. 1997; Stable azole drug resistance associated with a substrain of Candida albicans from an HIV-infected patient. Oral Dis 3 (suppl. 1):S102–S109
    [Google Scholar]
  47. White T. C., Marr K. A., Bowden R. A. 1998; Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11:382–402
    [Google Scholar]
  48. Wirsching S., Michel S., Kohler G., Morschhäuser J. 2000; Activation of the multiple drug resistance gene MDR1 in fluconazole-resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor. J Bacteriol 182:400–404
    [Google Scholar]
  49. Yang Y. L., Lin Y. H., Tsao M. Y., Chen C. G., Shih H. I., Fan J. C., Wang J. S., Lo H. J. 2006; Serum repressing efflux pump CDR1 in Candida albicans. BMC Mol Biol 7:22
    [Google Scholar]
  50. Zhao J., Hyman L., Moore C. 1999; Formation of mRNA 3′ ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol Mol Biol Rev 63:405–445
    [Google Scholar]
  51. Znaidi S., Weber S., Zin Al-Abdin O., Bomme P., Saidane S., Drouin S., Lemieux S., De D. X., Robert F., Raymond M. 2008; Genome-wide location analysis of Candida albicans Upc2p, a regulator of sterol metabolism and azole drug resistance. Eukaryot Cell 7:836–847
    [Google Scholar]
  52. Zuker M., Mathews D. H., Turner D. H. 1999; Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In RNA Biochemistry and Biotechnology pp 11–43 Edited by Barciszewski J., Clark B. F. C. Boston, MA: Kluwer Academic Publishers;
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.035154-0
Loading
/content/journal/micro/10.1099/mic.0.035154-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

PDF

Supplementary material 3

PDF

Supplementary material 4

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error