Academic journal article Genetics

Complex Haploinsufficiency-Based Genetic Analysis of the NDR/Lats Kinase Cbk1 Provides Insight into Its Multiple Functions in Candida Albicans

Academic journal article Genetics

Complex Haploinsufficiency-Based Genetic Analysis of the NDR/Lats Kinase Cbk1 Provides Insight into Its Multiple Functions in Candida Albicans

Article excerpt

WHILE Candida albicans is a component of the normal gastrointestinal flora of humans, it is also one of the most important human fungal pathogens and causes disease in both immunocompetent and immunocompromised individuals (McCarty and Pappas 2015). Superficial mucosal disease predominates in persons with normal immune function and those with altered T-cell function, while invasive, deepseated infections occur in patients with more profound immune deficits affecting innate and adaptive immunity. The ability of C. albicans to cause disease has been linked to its characteristic transition between three distinct morphological forms: yeast, pseudohyphae, and hyphae (Sudbery et al. 2004). C. albicans mutants locked in either yeast or hyphal morphological forms fail to cause disease (Saville et al. 2003): the yeast forms are able to infect mice but cannot cause disease, while those locked as hyphae fail to disseminate and establish infection. Although this dichotomous model for the role of filamentation is consistent with much of the literature, it is almost certainly an oversimplification. Indeed, a large-scale screen of C. albicans mutants comparing the ability to infect mice with morphological phenotypes found that normal filamentation was neither necessary nor sufficient to establish infection (Noble et al. 2010). These observations are not necessarily contradictory to the data from morphologically locked stains, since the ability to cause disease was not studied in the large-scale experiment. One broad but generally accurate interpretation of the vast amount of data regarding C. albicans filamentation is that it is a crucial aspect of this organism's biology. Considered in converse, it also seems broadly accurate that genetic perturbations of many biological processes directly or indirectly result in alterations in C. albicans morphology. As such, phenotypes related to filamentation provide a sensitive assay for the detection of mutations that have some effect on C. albicans biology.

Recently, we have described the use of complex haploinsufficiency as an approach to studying genetic interactions between, and within, pathways in C. albicans (Bharucha et al. 2011). Complex haploinsufficiency involves the generation of heterozygous mutations in two loci and comparing the phenotypes of that strain with the single heterozygotes. If the double heterozygote has a more profound phenotype than either of the single heterozygotes, then this indicates that the two genes interact. This approach is similar in concept to nonallelic, noncomplementation as developed in Saccharomyces cerevisiae (Stearns and Botstein 1988) and has been used to study the genetic interactions of essential genes in S. cerevisiae as well (Haarer et al. 2007). Although the recent discovery and, emerging development, of haploid genetics in C. albicans may allow the simple construction of double haploid mutants at some point (Hickman et al. 2013), the generation and study of complex heterozygotes remains a facile and expedient approach to genetic interaction screening and analysis in C. albicans.

The utility of this approach, however, is dependent on the identification of a sensitive phenotypic read-out to detect the effects of altered gene dosage on C. albicans physiology. Based on the pioneering simple haploinsufficiency screen of Uhl et al. (2003), morphological phenotypes are such a phenotype. Accordingly, we have used filamentation-related phenotypes and complex haploinsufficiency to probe the role of the Regulation of Ace2 and Morphogenesis (RAM) in C. albicans biology (Bharucha et al. 2011). The RAM pathway is centered on the Ndr/Lats family protein kinase Cbk1 (FungiDB: orf19.4909 ) and its transcription factor substrate Ace2 (FungiDB: orf19.6124 ). In addition to these two effector proteins, the RAM network includes Mob2, a Cbk1-binding protein required for kinase activity; Kic1, a kinase of unclear function; and the accessory proteins Tao3, Hym1, and Sog2 (Saputo et al. …

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