At Fraunhofer IGB DNA microarray technologies are tested e.g. to identify virulence factors in the pathogenic yeast Candida albicans and to detect point mutations (single nucleotide polymorphisms, SNPs) responsible for the development of resistances against antimycotic drugs in Candida albicans.
To identify potential virulence and resistance factors we applied transcriptional profiling methods that rely on differential mRNA profiles. For the development of genome-wide microarrays we used the genome sequence of Candida albicans known from public databases and converted them into a non-redundant gene set applying automated blast algorithms. Thus we could reduced the number of annotated ORF (open reading frames) from 9,200 to 7,200. The probes were defined in collaboration with the German Cancer Research Center DKFZ in Heidelberg [1, 2].
For specific tasks such as for the analysis of cell wall genes in Candida albicans we tested arrays with a reduced number of gene probes. In the case of cell wall analysis the arrays comprised solely potential cell wall genes. Using this array of about 120 genes we performed investigations on the transcriptional regulation of cell wall genes during the change from yeast to hyphal growth and between virulent and non-virulent strains. The arrays were analyzed by cluster analysis.
For the detection of mutations and polymorphisms (SNPs, pharmacogenomics) in the human genome or for the fast identification of resistance factors in pathogenic microorganisms oligonucleotide arrays can be coupled with enzymatic reactions on specific surfaces.
Using different methods, e.g. differential hybridization, APEX (allele-specific primer extension) or LDR (ligation detection reaction), mutations in an enzyme are identified that lead to resistance against antimycotics in Candida albicans [3, 4].
A coworker of the Molecular Biotechnology Department was awarded the 2003 Max Buchner Prize for her degree thesis work in using a resistance chip to show spot mutations in Candida albicans.
The identification of virulence factors in Candida albicans will be continued using genome-wide transcription profiles. The focus will be laid on the elucidation of host pathogen interactions, which can be modeled with the aid of our in vitro tissue systems. We also will further test SNP arrays and protein arrays.
[1] Sohn, K., Senyürek, I., Fertey, J., Königsdoefer, A., Joffroy, C., Hauser, N., Zelt, G., Brunner, H., Rupp, S. (2006)
An in vitro assay to study the transcriptional response during adherence of Candida albicans to different human epithelia. FEMS Yeast Research 6: 1085-93
[2] Sohn, K., Urban, C., Brunner, H., Rupp, S. (2003)
EFG1 is a major regulator of cell wall dynamics in Candida albicans as revealed by DNA microarrays. Mol. Microbiol. 47: 89-102
[3] Hauser, N. C., Martinez, R., Jacob, A., Rupp, S., Hoheisel, J. D., Matysiak, S. (2006)
Utilising the left-helical conformation of L-DNA for analysing different marker types on a single universal microarray platform. Nucleic Acid Res. 34: 5101-5111
[4] Hauser, N. C., Zeller, Z., Brunner, H., Tovar, G., Rupp, S. (2004)
Array-Techniken zur Diagnostik von human-pathogenen Pilzen. Mycoses 47/8: 362
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB