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Novel lysine biosynthetic gene sequences (LYS1 and LYS5) used as PCR targets for the detection of the pathogenic Candida yeast

Abstract

We report here a sensitive and specific polymerase chain reaction (PCR) detection assay for the pathogenic Candida yeast based on the novel LYS1 [encoding saccharopine dehydrogenase (SDH)] and LYS5 [encoding phosphopantetheinyl transferase (PPTase)] gene sequences of the fungal unique lysine biosynthetic pathway. Both LYS1 and LYS5 DNA-specific PCR primers SG1, SG2 and SG3, SG4, respectively, amplified predicted 483 and 648-bp fragments from Candida albicans genomic DNA but not from other selected fungal, bacterial, or human DNA. The 18S rDNA control primers exhibited positive amplifications in all PCR assays. The LYS1-and LYS5-specific primers strongly amplified C. albicans and Candida tropicalis target sequences; however, the LYS1 primers also weakly amplified fragments from Candida kefyr and Candida lusitaniae DNA. Both sets of primers amplified target sequences from less than 10 pg of serially diluted C. albicans DNA, and the LYS1 specific primers also detected DNA isolated from serially diluted 50 C. albicans cells. The PCR primers reported here are sufficiently sensitive and specific for the potential early detection of Candida infections with no possibility of false positive results from cross-contamination with bacterial or human DNA.

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References

  1. Ahmad S, Mustafa AS, Khan Z, Al-Rifaiy AI, Khan ZU (2004) PCR-enzyme immunoassay of rDNA in the diagnosis of candidemia and comparison with amplicon detection by agarose gel electrophoresis. Int J Med Microbiol 294:45–51

  2. Arancia S, Sandini S, Cassone A, De Bernardis F, La Valle R (2004) Construction and use of PCR primers from a 65 kDa mannoprotein gene for identification of C. albicans. Mol Cell Probes 18:171–175

  3. Ausubel FM (1998) Current protocols in molecular biology. Wiley, New York, New York, p 13.11.1

  4. Bhattacharjee JK (1992) Evolution of α-aminoadipate pathway for the synthesis of lysine in fungi. In: Mortlock RP (ed) Evolution of metabolic function. CRC, Boca Raton, Florida, pp 47–80

  5. Bhattacharjee JK, Suvarna K, Bhattacherjee V (2002) Reagents and kits for detecting fungal pathogens in biological sample. U.S. Patent No. 6455248 B1

  6. Broquist HP (1971) Lysine biosynthesis in yeast. Methods Enzymol 17B:112–129

  7. Burgener-Kairuz P, Zuber JP, Jaunin P, Buchman TG, Bille J, Rossier M (1994) Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-alpha-demethylase (L1A1) gene fragment. J Clin Microbiol 32:1902–1907

  8. Crampin AC, Matthews RC (1993) Application of the polymerase chain reaction to the diagnosis of candidosis by amplification of an HSP 90 gene fragment. J Med Microbiol 39:233–238

  9. Ehmann DE, Gehring AM, Walsh CT (1999) Lysine biosynthesis in Saccharomyces cerevisia: mechanism of α-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5. Biochem 38:6171–6177

  10. Garrad RC, Bhattacharjee JK (1992) Lysine biosynthesis in selected pathogenic fungi: characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans. J Bacteriol 174:7379–7384

  11. Garrad RC, Schmidt TM, Bhattacharjee JK (1994) Molecular and functional analysis of the LYS1 gene of Candida albicans. Infect Immun 62:5027–5031

  12. Guo S, Bhattacharjee JK (2003) Molecular characterization of the Candida albicansLYS5 gene and site-directed mutational analysis of the PPTase (Lys5p) domains for lysine biosynthesis. FEMS Microbiol Lett 224:261–267

  13. Guo S, Bhattacharjee JK (2004) Posttranslational activation, site-directed mutation and phylogenetic analyses of the lysine biosynthesis enzymes alpha-aminoadipate reductase Lys1p (AAR) and the phosphopantetheinyl transferase Lys7p (PPTase) from Schizosaccharomyces pombe. Yeast 21:1279–1288

  14. Guo S, Evans SA, Wilkes MB, Bhattacharjee JK (2001) Novel posttranslational activation of the LYS2-encoded alpha-aminoadipate reductase for biosynthesis of lysine and site-directed mutational analysis of conserved amino acid residues in the activation domain of Candida albicans. J Bacteriol 183:7120–7125

  15. Guo S, Garrad RC, Bhattacharjee JK (2006) Functional analysis through site-directed mutations and phylogeny of the Candida albicansLYS1-encoded saccharopine dehydrogenase. Mol Genet Genomics (MGG) 275:74–80

  16. Hijarrubia MJ, Aparicio JF, Casqueiro J, Martin JF (2001) Characterization of the lys2 gene of Acremonium chrysogenum encoding a functional α-aminoadipate activating and reducing enzyme. Mol Gen Genet (MGG) 264:755–762

  17. Kan VL (1993) Polymerase chain reaction for the diagnosis of candidemia. J Infect Dis 168:779–783

  18. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acid Res 30:3059–3066

  19. Miyakawa Y, Mabuchi T (1994) Characterization of a species-specific DNA fragment originating from the Candida albicans mitochondrial genome. J Med Vet Mycol 32:71–75

  20. Pfaller MA, Diekema DJ (2002) Role of sentinel surveillance of candidemia: trends in species distribution and antifungal susceptibility. J Clin Microbiol 40:3551–3557

  21. Reiss E, Obayashi T, Orle K, Yoshida M, Zancope-Oliveira RM (2000) Non-culture based diagnostic tests for mycotic infections. Med Mycol 38(Suppl 1):147–159

  22. van Burik JA, Magee PT (2001) Aspects of fungal pathogenesis in humans. Annu Rev Microbiol 55:743–772

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Acknowledgements

We thank G.R. Janssen for his valuable comments on the manuscript and Barbara J. Stahl for the preparation of the manuscript. We also thank Dr. D.J. Diekema for sending us several Candida strains. This research was supported by the National Institute of General Medical Science grant 1R15GM065153, grant from Eli Lilly and Company and Miami University.

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Correspondence to J. K. Bhattacharjee.

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Guo, S., Bhattacharjee, J.K. Novel lysine biosynthetic gene sequences (LYS1 and LYS5) used as PCR targets for the detection of the pathogenic Candida yeast. Appl Microbiol Biotechnol 72, 416–420 (2006). https://doi.org/10.1007/s00253-006-0470-y

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Keywords

  • Polymerase Chain Reaction Primer
  • Systemic Candidiasis
  • LYS5 Gene
  • Candida Lusitaniae
  • Polymerase Chain Reaction Target