Candida glabrata: new tools and technologies-expanding the toolkit

Abstract

In recent years, there has been a noticeable rise in fungal infections related to non-albicans Candida species, including Candida glabrata which has both intrinsic resistance to and commonly acquired resistance to azole antifungals. Phylogenetically, C. glabrata is more closely related to the mostly non-pathogenic model organism Saccharomyces cerevisiae than to other Candida species. Despite C. glabrata's designation as a pathogen by Wickham in 1957, relatively little is known about its mechanism of virulence. Over the past few years, technology to analyse the molecular basis of infection has developed rapidly, and here we briefly review the major advances in tools and technologies available to explore and investigate the virulence of C. glabrata that have occurred over the past decade.

Keywords: Candida; Candida glabrata; S. cerevisiae; technologies; tools; virulence.

Graphical Abstract Figure.

New tools and technologies to investigate the virulence of Candida glabrata.

Figure 1.

18S phylogeny of five Candida species and six hemiascomycetes. 18S sequences and phylogram were aligned and generated using Seaview version 4.5.3 (Gouy, Guindon and Gascuel 2010). Candida glabrata is phylogentically most closely related to S. cerevisiae and is not a member of the CTG clade which other Candida species belong to.

Figure 2.

Overview of the Gateway system. The Gateway system facilitates high-efficiency transfer of genes between different Gateway vectors via site-specific recombination. (A) The gene of interest is cloned in between the attP1/2 sites on the pEntry vector via a BP Clonase reaction to produce a pEntry clone. The pEntry vector harbours a bacterial ‘death’ gene (ccdB) that is exchanged for the gene of interest during the generation of the pEntry clone and transformation of E.coli that are sensitive to the ccdB effects allows for selection of the pEntry clones. (B) The gene of interest can also be cloned directly between the attR1/2 sites of a pDestination vector (containing features of interest such as protein tags, etc.) via a LR Clonase reaction to generate an expression vector thereby omitting the initial step of generating a pEntry clone. The pDestination vectors also contain the bacterial ‘death’ gene, ccdB, to enable selection of the pDestination clones. (C) The generated pEntry clone can be mixed with any available pDestination vector via a LR Clonase reaction to generate pDestination clones. This reaction is reversible using the BP Clonase reaction. pDestination clones with the gene of interest can be used to regenerate/generate the pEntry clones.

Figure 3.

Schematic and applications of the CRISPR-Cas9 system. Archae and bacteria utilize the CRISPR-Cas9 system to degrade foreign DNA by utilizing short RNA strands as guides, and this has been adapted by researchers to (A) edit the genome— gRNA strands are synthesized to complement the target sequence and protospacer adjacent motif (PAM) sequence (the PAM sequence must immediately follow the genomic target sequence for Cas9 to bind). The Cas9 enzyme binds to the gRNA and the target sequence and cleaves both strands to form a DSB. The DSB can be repaired via one of two general repair pathways, the NHEJ DNA repair pathway or the homology directed repair (HDR) pathway. Using the CRISPR system, researchers can either disrupt the gene via NHEJ repair (by not providing a suitable repair template) or edit the gene via HDR repair (by transfecting a suitable repair template into the cell at the same time as the gRNA and Cas9). (B) Activate gene expression—this utilizes a catalytically inactive form of Cas9 (dCas9) fused to a known transcriptional activator such as VP64. The dCas9–transcriptional activator complex binds to a target sequence just upstream from the promoter and causes upregulation of transcription of the target gene. (C) Repress gene expression—by binding dCas9 alone to the target sequence, transcription of the gene is blocked as it prevents the ribosome from binding. Unlike the gene modifications caused by the CRISPR system, both activating and repressing genes using a catalytically inactive form of Cas9 is not permanent as it does not affect the genomic DNA directly.