Analysis of Subtelomeric Silencing in Candida glabrata

Abstract

Analysis of gene function often involves detailed studies of when a given gene is expressed or silenced. Transposon mutagenesis is a powerful tool to generate insertional mutations that provide with a selectable marker and a reporter gene that can be used to analyze the transcriptional activity of a specific locus in a variety of microorganisms to study gene regulation. Then the reporter gene expression can be easily measured under different conditions to gain insight into the regulation of the particular locus of interest. We have used transposon mutagenesis as a tool to generate insertional mutations with a modified Tn7 transposon containing the reporter gene URA3 (Tn7-URA3) to study subtelomeric silencing in the opportunistic fungal pathogen Candida glabrata. This method consists of two major steps: an in vitro Tn7-URA3 mutagenesis of a plasmid containing the desired subtelomeric region to be analyzed, followed by homologous recombination into the target region of the C. glabrata genome. As an alternative, a fusion PCR protocol can also be used in which the URA3 reporter gene can be "fused" together with the 5' and 3' regions of the desired insertion point by a two step PCR protocol. This fusion product can be introduced into the C. glabrata genome by homologous recombination after transformation in the same way as the Tn7-URA3 mutagenesis products. Once the URA3 reporter gene has been introduced in the desired locus in the C. glabrata genome, a simple plate growth assay is performed to assess the expression of the reporter gene.

Adhesins in Candida glabrata

“…Because these subtelomeric regions contain sequence repeats, they are prone to rearrangements or non-allelic homologous recombination, which could explain the expansion of the adhesin-encoding gene families in C. glabrata . For some of these adhesins, it has been already shown that their expression is under control of subtelomeric silencing mediated by Rap1, the Sir-complex, and Ku70/80 [ 30 , 31 , 32 , 33 , 34 ]. An update on the effect of chromatin structure and pathways controlling this subtelomeric silencing on adherence in C. glabrata can be found in a separate review in this issue by López-Fuentes et al, 2018.…”

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

“…In conclusion, although many studies employing transposon mutagenesis are performed in S. cerevisiae and C. albicans, there is a growing body of work using transposons in other pathogenic fungi including Cryptococcus, C. glabrata and Histoplasma [111,[135][136][137][138][139]. Since transposon mutagenesis allows genome-scale DNA modification, this strategy serves a powerful tool to generate insertional mutations.…”

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

“…The technology has been adapted for diploid organisms using the parasexual cycle, haploid insufficiency, and homologous recombination (Carr et al, 2010;Davis et al, 2002;Firon et al, 2003;Juarez-Reyes et al, 2011;Spreghini et al, 2003;Uhl et al, 2003). The use of transposons has superseded auxotrophic and STM approaches.…”

“…Resulting constructs were integrated into subtelomeric regions of C. glabrata by homologous recombination. It was possible to select for 'silenced' URA3 on 5-FOA media (Juarez-Reyes et al, 2011).…”