Investigation of bcr1 Gene Expression in Candida albicans Isolates by RT-PCR Technique and its Impact on Biofilm Formation

Authors
F. Nikoomanesh 1
F. Nikoomanesh 1
S. Roudbarmohammadi 1
M. Roudbary 2
M. Bayat 3
G. Heidari 4
1 Department of medical mycology, Faculty of medical science, Tarbiat Modares University, Tehran, Iran
2 Department of Medical Mycology and Parasitology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
3 Science and Research Branch, Islamic Azad University, Tehran, Iran
4 Food and Drug Organization, Tehran, Iran
Abstract
Background: Adhesion and biofilm formation are two important steps in Candida pathogenesis. The aim of the current study was to investigate the presence of bcr1 gene in Candida albicans (C. albicans) isolates from women with vaginal candidiasis and its impact on biofilm formation. Methods: We used 50 clinical isolates which confirmed C. albicans by PCR-RFLP. Then total RNA was extracted from C. albicans isolates by glass bead and lysis buffer, and cDNA was synthesized using reverse transcriptase enzyme. RT-PCR (Reverse Transcriptase PCR) was used to evaluate the expression of bcr1 gene. Biofilm formation was evaluated in 96-well microplate and then tetrazolium reduction was assayed. All data were analyzed using t-test by SPSS software. Results: Fifty clinical isolates out of 150 were confirmed as C. albicans by using PCR-RFLP method. All the isolates were resistant to fluconazole, 47/50(94%) isolates had bcr1 gene by using PCR, and 45(95.7%) out of 47 isolates, showed BCR1 expression by the RT-PCR. Isolates which harbored bcr1 gene was succeed to form a dense biofilm on microplate. Comparison of the results of the tetrazolium reduction assay on the two isolates that had BCR1expression and two isolates that had no BCR1 expression showed significant differences (p=0.014). Conclusion: According to our result, all of the isolates that had bcr1 gene expression according to RT-PCR, were also resistant to fluconazole in disk diffusion test and additionally, their adherence was higher compared to the control group. These results indicate that there is a positive relation between expression of bcr1 gene and biofilm formation.

Keywords

  1. Harriott MM, Lilly EA, Rodriguez TE, Fidel PLJr, Noverr MC. Candida albicans forms biofilm on the vaginal mucosa. Microbiology. 2010; 156(Pt 12): 3635-44.

  2. Finkel JS, Matichell AP. Genetic control of Candida albicans biofilm development. Nat Rev Microbiol. 2011; 9(2):109-18.

  3. Fux CA, Costerton JW, Stewart PS, Stoodley P. Survival steratgies of Infectious biofilms. Trends Microbiol. 2005; 13(1): 34-40.

  4. Nobile CJ, Mitchel AP. Genetics and genomics of Candida albicans biofilm formation. Cell Microbiol. 2006; 8(9): 1382-91.

  5. Haswer SP, Douglas LJ. Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect Immun. 1994; 62(3):915-21.

  6. Yang YL. Virulence factors of Candida species. J Microbiol Immunol Infect. 2003; 36(4):223-8.

  7. Douglas LJ. Candida biofilms and their role in infection. Trends Microbiol. 2003; 11(1): 30-6.

  8. Nobile CJ, Mitchell AP. Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor bcr1p. Curr Biol. 2005; 15(12):1150-5.

  9. Nobile CJ, Andes DR, Nett JE, Smith FJ, Yue F, Phan QT, et al. Critical role of Bcr1-dependent adhesions in C. albicans biofilm formation in vitro and in vivo . PLoS Pathog. 2006; 2(7): e63.

  10. Ding C, Vidanes GM, Maguire SL, Guida A, Synnott JM, Andes DR, et al. Conserved and divergent roles of Bcr1 and CFEM proteins in Candida parapsilosis and Candida albicans. PLoS One. 2011; 6(12):e28151.

  11. Fanning S, Xu W, Solis N, Woolford CA, Filler SG, Mitchell AP. Divergent target of Candida albicans biofilm regulator Bcr1 in vitro and in vivo. Eukaryotic Cell .2012; 11(7): 896-904.

  12. Dwivedi P, Thompson A, Xie Z, Kashleva H, Ganguly S, Mitchell AP, et al. Role of Bcr1-activated genes hwp1 and hyr1 in Candida albicans oral mucosal biofilm and neutrophil evasion. PLoS One. 2011; 6(1): e16218.

  13. Yamada Y, Makimura K, Merhendi H, Ueda K, Nishiyama Y, Yamaguchi H, et al. Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts. Jpn J Infect Dis. 2002; 55(4):122-5.

  14. Mårdh PA, Rodrigues AG, Genç M, Novikova N, Martinez-de-Oliveira J, Guaschino S. Facts and myths on recurrent Vulvovaginal Candidosis: a review on epidemiology, clinical manifestations, pathogenesis and therapy. Int J STD AIDS. 2002; 13(8):522-39.

  15. Stanley NR, Lazazzera BA. Environmental signals and regulatory pathways that influence biofilm formation. Mol Microbiol. 2004; 52(4): 917-24.

  16. Sundstrom P. Adhesion in Candida spp. cell. Cell Microbiol. 2002; 4(8):461-9.

  17. Hoyer LL. The als gene family of Candida albicans. Trends Microbiol. 2001; 9(4): 176-80.

  18. Finkel JS, Xu W, Huang D, Hill EM, Desai JV, Woolford C, et al. Portrait of Candida albicans adherence regulators. PLoS Pathog. 2012; 8(2):e1002525.

Infection Epidemiology and Microbiology
Volume 2, Issue 1 - Serial Number 2
January 2016
Pages 22-24