Milestones in Candida albicans gene manipulation

Abstract

In the United States, candidemia is one of the most common hospital-acquired infections and is estimated to cause 10,000 deaths per year. The species Candida albicans is responsible for the majority of these cases. As C. albicans is capable of developing resistance against the currently available drugs, understanding the molecular basis of drug resistance, finding new cellular targets, and further understanding the overall mechanism of C. albicans pathogenesis are important goals. To study this pathogen it is advantageous to manipulate its genome. Numerous strategies of C. albicans gene manipulation have been introduced. This review evaluates a majority of these strategies and should be a helpful guide for researchers to identify gene targeting strategies to suit their requirements.

Molecular genetic techniques for gene manipulation inCandida albicans

“…To ameliorate this problem, it is recommended to place the remaining URA3 copy at a highly expressed locus, including ENO1 , RPS10 , ARG4 , or at the URA3 itself native locus 29 , 34 - 37 . Otherwise, researchers also improve the efficiency of the URA3 cassette and have applied the PCR amplifiable URA3 cassette ( URA3-dpl200 ) and the UAU1 cassette ( ura3 Δ3′- ARG4 - ura3 Δ5′) to disrupt the target gene in C. albicans 19 , 26 , 41 , 42 . The UAU1 cassette on the Tn7 transposon has been used successfully to disrupt several genes in C. albicans 44 …”

“…The parental strains used for these cassettes display no karyotypic changes and have one copy of URA3 expressed at the native locus, which don’t affect the virulence in C. albicans . So far, there is no evidence showing that the ectopic expression of any of the above nutritional markers would affect the virulence in C. albicans 41 , 45 . However, these cassettes cannot be used to disrupt the genes in the wild-type strains.…”

“…The multiple FRT sequences left in the genome might recombine induced by the FLP recombinase, resulting in unexpected deletions or chromosome rearrangements 30 , 40 . The generation of ura3 − clones would exclude the possibility of interchromosomal recombination 38 , 41 . Otherwise, the use of the BSA activates the SAP2 promoter to induce the expression of FLP recombinase in the URA flipper cassette, whereas the BSA also represses the expression of other genes of SAP family except for SAP2 gene 38 , 41 …”

“…The synthetic MET3 - cre fusion of this system to loop out the LoxP -marker- LoxP cassettes in C. albicans is efficient and avoids using positive screen method to select for the desired mutants. The knockout strains generated by the Cre-loxP system are auxotrophic, unlike those by the positive selection markers 41 , 46 . Moreover, the future uses of Cre- loxP system in C. albicans are not limited to gene disruptions; it can be used in the tagging of multiple ORF in situ.…”

Deus ex Candida genetics: overcoming the hurdles for the development of a molecular toolbox in the CTG clade

“…Once identified and isolated, the gene encoding a functional enzyme that complemented the disrupted metabolic pathway was used as dominant marker in the corresponding auxotrophic recipient strain. For example, many C. albicans genetic approaches are based on the BWP17 triple auxotroph (his1 arg4 ura3) in which HIS1, ARG4 and URA3 wild-type genes can be used as selectable markers (Wilson et al, 1999;Samaranayake & Hanes, 2011). These approaches were progressively facilitated by the finding and use of antimetabolites.…”