An Expanding Molecular Toolbox for C albicans

Up until recently the tools available for working with C. albicans were fairly limited, although gene disruption using the URA-blaster cassette has been possible since 1993 (Fonzi & Irwin, 1993). Gene disruptions have become simpler with the development of PCR knockout cassettes (de Hoogt et al., 2000; Wilson et al., 2000; Gola et al., 2003; Taneja et al., 2004) and gene function can be rapidly tested by disruption using the UAU cassette (Enloe et al., 2000).

Unfortunately, the URA-blaster technique has been found to generate mutants that can be affected in their virulence due only to the expression of the URA3 gene at an ectopic locus (Lay et al., 1998; Sundstrom et al., 2002; Staab & Sundstrom, 2003; Brand et al., 2004). This problem can be circumvented by expressing URA3 at a highly expressed locus, such as ENO1 (Sundstrom et al., 2002) or RPS1 (Brand et al., 2004) or by reintroducing URA3 at its native locus (Cheng et al., 2003).

A further advance in gene disruption has been the development of dominant selectable markers and their use in disruption cassettes for C. albicans (Beckerman et al., 2001; Reuss et al., 2004; Morschhauser et al., 2005; Shen et al., 2005). Since URA3 is no longer the selectable marker, the Ura3p effect is no longer a problem and, more importantly, gene disruptions are no longer confined to laboratory strains derived from SC5314. Dominant markers in use for C. albicans include resistance to mycophenolic acid (Beckerman et al., 2001; Morschhauser et al., 2005) and nourseothricin (Reuss et al., 2004; Shen et al., 2005).

The number of regulatable promoters available for use in C. albicans is also increasing, with the tetracycline-regulatable system (Nakayama et al., 2000; Roemer et al., 2003; Park & Morschhauser, 2005b) joining other regulatable promoters (Gerami-Nejad et al., 2004). This system not only allows phenotypes to be examined before and after the gene is switched off in vitro, but can also be used in vivo.

The development of codon-optimised fluorescent reporter genes (GFP (green), YFP (yellow), and CFP (cyan)) (Gerami-Nejad et al., 2001) allows localisation and/or co-localisation of proteins at the level of single cells (Hazan & Liu, 2002; Oberholzer et al., 2002; Gerami-Nejad et al., 2004; Soares-Silva et al., 2004; Karababa et al., 2006). In addition, GFP linked to gene promoters (Barelle et al., 2004) allows visualisation of the induction of those promoters under various conditions, both in vitro and in vivo (Strauss et al., 2001; Gaur et al., 2005; Green et al., 2005a; Barelle et al., 2006). In addition to targeted investigation of C. albicans genes and proteins of interest, the application of transcript profiling experiments or proteomic analyses provide unbiased, global perspectives on gene and protein expression, respectively.

DNA microarrays have been designed and transcript profiling performed to investigate changes in C. albicans gene expression by various groups including Eurogentec (Eurogentec website) in collaboration with the European Galar Fungail Consortium (European Galar Fungail Consortium website) and the NRC Biotechnology Research Institute, Montreal, Canada (Candida albicans@NRC-Biotechnology Research Institute website) as well as many others. Transcript profiling experiments and their contributions to the understanding of C. albicans biology and pathogenesis have recently been reviewed by Garaizar et al. (2006). One of the major uses of transcript profiling for C. albicans has been to examine the global effects on gene expression caused by gene knockouts (Garcera et al., 2005; Garcia-Sanchez et al., 2005; Harcus et al., 2004; Lan et al., 2004; Lane et al., 2001; Lee et al., 2004; Lotz et al., 2004; Murad et al., 2001; Sohn et al., 2003; Tournu et al., 2005; Wang et al., 2006, 2007). These analyses have allowed identification of groups of co-ordinately regulated genes, and have provided insights into pathways involved in certain cellular responses.

Proteomics studies of C. albicans allow greater understanding of changes that occur at the protein level, rather than at the transcription level. Several two-dimensional (2-D) Polyacrylamide gel electrophoresis (PAGE) maps of the C. albicans proteome, both yeast and hyphal forms, have been produced (Hernandez et al., 2004; Yin et al., 2004), with the Cogeme map available at proteomics/ap-Candida-albicans-2d-map.hti. The contributions of proteomics to our understanding of C. albicans and disease have recently been reviewed by Pitarch et al. (2006b, c).

MLST approach for characterisation of C. albicans strains has also been designed (Bougnoux et al., 2002, 2004; Tavanti et al., 2003; Robles et al., 2004), which builds upon previous molecular typing systems for C. albicans (Schmid et al., 1990; Chowdhary et al., 2006). This system allows strains to be typed, their relatedness determined and can also be used to track the development of potential outbreaks (Bougnoux et al., 2006; Chen et al., 2006; Odds et al., 2006; Viviani et al., 2006). This has also demonstrated that the majority of C. albicans isolates causing systemic infection are endogenous to the host (Odds et al., 2006) and that there is some evidence of microevolution of strains in a patient over time (Bougnoux et al., 2002, 2006; Chen et al., 2006; Odds et al., 2006).

The remainder of this chapter describes recent increases in our understanding of C. albicans, its biology and host-fungus interactions, with emphasis on data obtained from transcript profiling and proteomic analyses. Important in the ability of C. albicans to infect and cause disease is its resistance to stresses, especially those experienced during interactions with host immune cells, its ability to adhere to surfaces and host cells, and the ability to form biofilms. The studies focussed upon are those concerned with increasing our understanding of life-threatening systemic infection.

Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

The term vaginitis is one that is applied to any inflammation or infection of the vagina, and there are many different conditions that are categorized together under this ‘broad’ heading, including bacterial vaginosis, trichomoniasis and non-infectious vaginitis.

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