A Pathogenesis Assay Using Saccharomyces cerevisiae and C aenorhabditis elegans Reveals Novel Roles for Yeast AP-1, Yap1, and Host Dual Oxidase BLI-3 in Fungal Pathogenesis

Abstract

Treatment of systemic fungal infections is difficult because of the limited number of antimycotic drugs available. Thus, there is an immediate need for simple and innovative systems to assay the contribution of individual genes to fungal pathogenesis. We have developed a pathogenesis assay using Caenorhabditis elegans, an established model host, with Saccharomyces cerevisiae as the invading fungus. We have found that yeast infects nematodes, causing disease and death. Our data indicate that the host produces reactive oxygen species (ROS) in response to fungal infection. Yeast mutants sod1⌬ and yap1⌬, which cannot withstand ROS, fail to cause disease, except in bli-3 worms, which carry a mutation in a dual oxidase gene. Chemical inhibition of the NADPH oxidase activity abolishes ROS production in worms exposed to yeast. This pathogenesis assay is useful for conducting systematic, whole-genome screens to identify fungal virulence factors as alternative targets for drug development and exploration of host responses to fungal infections.Nosocomial microbial infections are a growing health problem. Among these, fungal infections are especially threatening, with an estimated mortality rate of 40% (47). The key reason for this alarming mortality rate is the limited range of antifungal agents. Identification of new drug targets requires highthroughput infection assays that are complicated by the very fact that they involve two organisms: a host and a pathogen.We have taken a reductionistic approach to studying hostpathogen interactions and have developed a Saccharomyces cerevisiae-based assay to understand the genetic and molecular mechanisms of fungal pathogenesis. Using Caenorhabditis elegans as a model host, we have found that S. cerevisiae infects the worm, producing visible disease phenotypes. The two organisms used in our study are specifically suited for host-pathogen infection studies because both genomic sequences have been completely determined and mutants are readily available. A complete genome knockout collection is available for S. cerevisiae, a resource that does not exist for any fungal pathogen. Likewise an RNA interference (RNAi)-mediated knockdown genomic library is available for C. elegans. These unique tools are key in the context of a genetic screen and allow us to systematically scan the entire genomes to identify fungal virulence factors and modulators of host immunity that combat a fungal pathogen.The budding yeast S. cerevisiae has recently been described as an emerging pathogen and has been isolated from human patients (34,35). It is routinely used as a model for pathogenic fungi because a large proportion of its genes are conserved in pathogenic fungi (for a review, see reference 32). Homologs of genes and pathways identified in S. cerevisiae have been shown to be important in bona fide pathogens. It has also been used for the identification of gene products important for fungal survival in the mammalian host environment (21, 46). For example, the SSD1 allele type affects pathogenicity of yea...

Zinc Cluster Transcription Factors Alter Virulence in Candida albicans

“…A rich body of literature demonstrates that molecular mechanisms of infectious disease progression in C. elegans are mechanistically similar to humans ( Pukkila-Worley et al 2009, 2011; also reviewed in Engelmann and Pujol 2010;Marsh and May 2012). We identified seven mutants, CMP1, IFF11, SAP8, DOT4, ZCF15, orf19.1219, and orf19.6713, representing 10% of the mutants screened, that were unable to illicit the Dar response, previously described as a robust disease phenotypes in C. elegans ( Figure S4A), in particular, a deformity in the post anal region (Dar) (Jain et al 2009). CMP1, IFF11, and SAP8 have previously been implicated in the virulence of C. albicans, thus validating our screening methods (Schaller et al 1999a,b;Bader et al 2003;Bates et al 2007).…”

“…In order to identify novel C. albicans virulence determinants, we screened a collection of 724 C. albicans mutants (12% of the genome) for their abilities to induce the Dar phenotype (Jain et al 2009) in Caenorhabditis elegans. Live nematodes were infected with C. albicans to identify mutants that exhibited altered phenotype in the worms.…”

“…The assays have previously been described (Jain et al 2013). Briefly, 30 healthy C. elegans were exposed to C. albicans, which was mixed into their diet (Jain et al 2009). The percentage of worms showing signs of infection (Jain et al 2013) were scored manually 4 days post infection.…”

“…We employed experimentally tractable in vivo and ex vivo models to elucidate the role of these ZCF TFs in the innate immune system (Jain et al 2009(Jain et al , 2013. Among the 35 ZCFs, these mutants exhibit similar redox sensitivity profiles in phagocytes ex vivo, and nematodes in vivo.…”

Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis

“…We used the previously described deformed anal region (Dar) tail-swelling phenotype (16,32) as a biomarker to monitor fungal infections in nematode hosts. A small quantity of C. albicans was introduced with the nematode's standard diet of Escherichia coli OP50 cells, and E. coli growth was attenuated with the antibiotic streptomycin to avoid interactions with C. albicans (33). Here, we used a dose of C. albicans that caused all untreated worms to display Dar disease (Fig.…”