Leadership In Controlled Gene Expression

Candida albicans is a commensal organism of the human gastrointestinal tract that is responsible for various nonlife-threatening infections and for most Candida infections. Under certain circumstances it can become pathogenic and is a major cause of mortality in bloodstream infections (sepsis) particularly in patients which are immune-compromised due to HIV infection, organ transplantation or chemo- and radiation therapy. There is an increasing need for new drugs to fight fungal infections but antifungal drug discovery is limited by the availability of novel therapeutic targets. 'Elitra' scientists established a method that permits large scale target identification and target validation in diploid pathogenic microorganisms like C. albicans (Roemer et al., 2003).

Their elegant two step procedure termed GRACE (Gene Replacement and Conditional Expression) allows the conditional knockout of genes essential for viability and growth. Based on the available genomic sequence of Candida albicans, essentially any gene can be analyzed. While one allele is disrupted by PCR-based gene replacement, the promoter region of the second allele is replaced by a Tet-responsive promoter. Expression of the second allele will occur only in the absence of Dox in this Tet-Off-dependent system. Thus addition of Dox will inactivate the Tet-responsive gene allowing identification of essential genes of the pathogen by the conditional growth phenotype of the corresponding strain.

Systematic application of this approach revealed almost 600 essential genes in C. albicans of which many may represent new drug target candidates. Further analysis of this set of genes was achieved by a search for orthologues in the human and yeast genome. Consequently while genes and their respective proteins missing in humans but conserved in e.g. S. cerevisiae may represent broad spectrum targets, C. albicans specific genes with no human or yeast counterpart may be suitable as narrow spectrum targets.

In addition to comparative analyses, strains identified by GRACE where directly used in an in vivo mouse model for definitive target validation. In these experiments which resemble a human infection more closely, mice were infected with strains conditionally expressing three different genes that had previously been shown to be essential for survival of the pathogen. All mice receiving Doxycycline prior to infection to abolish expression of the essential genes survived and remained healthy while all control mice that had not received any Dox succumbed to infection within 10 days.  These experiments proved that all three GRACE strains are virulent in the absence of Dox and all three targets are necessary to establish a fungal infection. Although the GRACE strain collection is not freely available it was subsequently used in gene annotation studies (Oh et al., 2010) and in efforts to identify therapeutic agents (Xu et al., 2009).