Abstract
An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.
Similar content being viewed by others
Abbreviations
- GPI:
-
Glycosylphosphatidylinositol
- NA:
-
Nicotinic acid
- PBS:
-
Phosphate buffered saline
References
Pfaller MA. Nosocomial candidiasis: emerging species, reservoirs, and modes of transmission. Clin Infect Dis. 1996;22:S89–94.
Pfaller MA, Diekema DJ. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida. Diagn Microbiol Infect Dis. 2004;48(2):101–5.
Ramage G, Martínez JP, López-Ribot JL. Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS Yeast Res. 2006;6(7):979–86.
De Groot PWJ, Kraneveld EA, Yin QY, Dekker HL, Groß U, Crielaard W, et al. The cell wall of the human pathogen Candida glabrata: differential incorporation of novel adhesin-like wall proteins. Eukaryot Cell. 2008;7:1951–64.
Klis FM, Sosinska GJ, de Groot PWJ, Brul S. Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulence. FEMS Yeast Res. 2009;9(7):1013–28.
Hoyer LL, Green CB, Oh SH, Zhao X. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family—a sticky pursuit. Med Mycol. 2008;46(1):1–15.
Li F, Palecek SP. EAP1, a Candida albicans gene involved in binding human epithelial cells. Eukaryot Cell. 2003;2(6):1266–73.
Sundstrom P. Adhesion in Candida spp. Cell Microbiol. 2002;4(8):461–9.
Cormack BP, Ghori N, Falkow S. An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Science. 1999;285(5427):578–82.
Iraqui I, Garcia-Sanchez S, Aubert S, Dromer F, Ghigo J-M, d’Enfert C, et al. The Yak1p kinase controls expression of adhesins and biofilm formation in Candida glabrata in a Sir4p-dependent pathway. Mol Microbiol. 2005;55:1259–71.
Jin Y, Samaranayake Y, Yip HK. Biofilm formation of Candida albicans is variably affected by saliva and dietery sugars. Arch Oral Biol. 2004;49:789–98.
Nikawa H, Nishimura H, Hamada T, Kumagai H, Samaranayake LP. Effects of dietary sugars and, saliva, and serum on Candida biofilm formation on acrylic surfaces. Mycopathologia. 1997;139(2):87–91.
Pereira-Cenci T, Deng DM, Kraneveld EA, Manders EM, Del Bel Cury AA, Ten Cate JM, et al. The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces. Arch Oral Biol. 2008;53:755–64.
Schmidt P, Walker J, Selway L, Stead D, Yin Z, Enjalbert B, et al. Proteomic analysis of the pH response in the fungal pathogen Candida glabrata. Proteomics. 2008;8:534–44.
García-Sánchez S, Aubert S, Iraqui I, Janbon G, Ghigo JM, d’Enfert C. Candida albicans biofilms: a developmental state associated with specific and stable gene expression patterns. Eukaryot Cell. 2004;3(2):536–45.
Murillo LA, Newport G, Lan CY, Habelitz S, Dungan J, Agabian NM. Genome-wide transcription profiling of the early phase of biofilm formation by Candia albicans. Eukaryot Cell. 2005;4:1562–73.
Nailis H, Coenye T, Van Nieuwerburgh F, Deforce D, Nelis HJ. Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR. BMC Mol Biol. 2006;7:25. doi:10.1186/1471-2199-7-25.
Nailis H, Kucharíková S, Řičicová M, Van Dijck P, Deforce D, Nelis H, et al. Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression. BMC Microbiol. 2010;10:114. doi:10.1186/1471-2180-10-114.
O’Conner L, Lahiff S, Casey F, Glennon M, Cormican M, Maher M. Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridization probes on the light-cycler™. Mol Cell Probes. 2005;19:153–62.
Yeater KM, Chandra J, Cheng G, Mukherjee PK, Zhao X, Rodriquez-Zas SL. Temporal analysis of Candida albicans gene expression during biofilm development. Microbiology. 2005;151:1051–61.
Domergue R, Castaño I, De las Peñas A, Zupancic M, Lockatell V, Hebel JR, et al. Nicotinic acid limitation regulates silencing of Candida adhesins during UTI. Science. 2005;308(5723):866–70.
Zupancic ML, Cormack BP. Candida cell wall proteins at the host-pathogen interface. In: d’Enfert C, Hube B, editors. Candida comparative and functional genomics. Norfolk: Caister Academic Press; 2007. p. 327–48.
Zupancic ML, Frieman M, Smith D, Alvarez RA, Cummings RD, Cormack BP. Glycan microarray analysis of Candida glabrata adhesin ligand specificity. Mol Microbiol. 2008;68(3):547–59.
Yin QY, De Groot PWJ, Dekker HL, De Jong L, Klis FM, De Koster CG. Comprehensive proteomic analysis of Saccharomyces cerevisiae cell walls: identification of proteins covalently attached via glycosylphosphatidylinositol remnants or mild alkali-sensitive linkages. J Biol Chem. 2005;280(21):20894–901.
Sherman DJ, Martin T, Nikolski M, Cayla C, Souciet JL, Durrens P, et al. Génolevures: protein families and synteny among complete hemiascomycetous yeast proteomes and genomes. Nucleic Acids Res. 2009;37(Database issue):D550–4.
Guiver M, Levi K, Oppenheim BA. Rapid identification of candida species by TaqMan PCR. J Clin Pathol. 2001;54(5):362–6.
Sanguinetti M, Posteraro B, Fiori B, Ranno S, Torelli R, Fadda G. Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance. Antimicrob Agents Chemother. 2005;49(2):668–79.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034.
Weig M, Jänsch L, Groß U, De Koster CG, Klis FM, De Groot PWJ. Systematic identification in silico of covalently bound cell wall proteins and analysis of protein-polysaccharide linkages of the human pathogen Candida glabrata. Microbiology. 2004;150(Pt 10):3129–44.
Coutinho PM, Henrissat B. Carbohydrate-active enzymes: an integrated database approach. In: Gilbert HJ, Davies G, Henrissat B, Svensson B, editors. Recent advances in carbohydrate bioengineering. Cambridge: The Royal Society of Chemistry; 1999. p. 3–12.
Martinez-Lopez R, Monteoliva L, Diez-Orejas R, Nombela C, Gil C. The GPI-anchored protein CaEcm33p is required for cell wall integrity, morphogenesis and virulence in Candida albicans. Microbiology. 2004;150:3341–54.
Pardo M, Monteoliva L, Vazquez P, Martinez R, Molero G, Nombela C, et al. PST1 and ECM33 encode two yeast cell surface GPI proteins important for cell wall integrity. Microbiology. 2004;150(Pt 12):4157–70.
Ecker M, Deutzmann R, Lehle L, Mrša V, Tanner W. PIR-proteins of Saccharomyces cerevisiae are attached to b-1, 3-glucan by a new protein-carbohydrate linkage. J Biol Chem. 2006;281(17):11523–9.
De Groot PWJ, De Boer AD, Cunningham J, Dekker HL, De Jong L, Hellingwerf KJ, et al. Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell. 2004;3(4):955–65.
Sosinska GJ, de Koning LJ, de Groot PWJ, Manders EM, Dekker HL, Hellingwerf KJ, et al. Mass spectrometric quantitation of the adaptations in the wall proteome of Candida albicans in response to ambient pH. Microbiology. 2011;157(1):136–46.
Kaur R, Ma B, Cormack BP. A family of glycosylphosphatidylinositol-linked aspartyl proteases is required for virulence of Candida glabrata. Proc Natl Acad Sci USA. 2007;104(18):7628–33.
Castaño I, Pan SJ, Zupancic M, Hennequin C, Dujon B, Cormack BP. Telomere length control and transcriptional regulation of subtelomeric adhesins in Candida glabrata. Mol Microbiol. 2005;55(4):1246–58.
De Las Peñas A, Pan SJ, Castano I, Alder J, Cregg R, Cormack BP. Virulence-related surface glycoproteins in the yeast pathogen Candida glabrata are encoded in subtelomeric clusters and subject to RAP1- and SIR-dependent transcriptional silencing. Genes Dev. 2003;17(18):2245–58.
Li M, Petteys BJ, McClure JM, Valsakumar V, Bekiranov S, Frank EL, et al. Thiamine biosynthesis in Saccharomyces cerevisiae is regulated by the NAD+ -dependent histone deacetylase Hst1. Mol Cell Biol. 2010;30(13):3329–41. doi:10.1128/MCB.01590-09.
Sandmeier JJ, Celic I, Boeke JD, Smith JS. Telomeric and rDNA silencing in Saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway. Genetics. 2002;160(3):877–89.
Desai C, Mavrianos J, Chauhan N (2011) Candida glabrata Pwp7p and Aed1p are required for adherence to human endothelial cells. FEMS Yeast Res. doi:10.1111/j.1567-1364.2011.00743.x
Acknowledgments
PG was supported by an INCRECyT fellowship from Parque Científico y Tecnológico de Albacete.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kraneveld, E.A., de Soet, J.J., Deng, D.M. et al. Identification and Differential Gene Expression of Adhesin-Like Wall Proteins in Candida glabrata Biofilms. Mycopathologia 172, 415–427 (2011). https://doi.org/10.1007/s11046-011-9446-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11046-011-9446-2