Caenorhabditis elegans: A Nematode Infection Model for Pathogenic Fungi
Recent work suggests that fungal virulence factors important in human disease have evolved through interactions with environmental predators such as amoebae, nematodes, and insects. This has allowed the use of simple model hosts for the study of fungal pa
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1. Introduction Caenorhabditis elegans, a free-living soil nematode, is a widely used invertebrate in vivo model for infection and has been utilized in the study of host-pathogen interactions and antimicrobial compound discovery. C. elegans has many advantages favorable for use as a model host, such as its short reproductive cycle producing genetically identical progeny, an evolutionary conserved immune response pathway, and availability of molecular tools (such as an annotated genome and RNA interference libraries). The nematode also provides a cost-efficient and ethically acceptable alternative to mammalian models. Furthermore, in drug discovery, applications of this model system can give clues about compound toxicity and an indication if the compound has any interaction with the host immune system.
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_31, © Springer Science+Business Media, LLC 2012
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In the laboratory, the nematode can be easily maintained and propagated on nematode growth medium (NGM), and fed on a lawn of Escherichia coli (auxotrophic strain OP50). There are two sexual forms of C. elegans : the predominant hermaphrodite and males. The hermaphrodite produces both eggs and sperm and subsequently can self-fertilize producing hundreds of progeny. The time to complete the C. elegans life cycle is dependent on temperature. For example, when the nematode is maintained at 15°C, it takes approximately 5 days for progeny to develop and lay eggs; however, at 25°C the process is completed in two and a half days. C. elegans has been utilized as a model host for several clinically relevant fungal pathogens, including Candida albicans, Candida glabrata, Cryptococcus neoformans, and Histoplasma capsulatum (1–4). Two assay types utilizing C. elegans to study host-pathogen interactions have been used: the liquid media assay and the solid media assay. Choosing the appropriate assay depends on the purpose of the experiment. For example, C. albicans readily forms hyphae in the liquid environment, and the liquid assay would be the choice if the goal is to study the role of filamentation in virulence. Use of the liquid media assay is also preferred in drug discovery since it facilitates the study of large libraries of chemical compounds. In this chapter, we will summarize representative assays that have been developed to allow C. elegans to be used in the study of fungal pathogenesis and antifungal compound discovery.
2. Materials 2.1. Caenorhabditis elegans Growth
1. Caenorhabditis elegans strains (glp4; sek1 strain, N2 Bristol strain) (see Note 1). 2. 5 mg/mL cholesterol: Make up solution in ethanol. 3. 1 M KPO4 buffer: Add 108.3 g KH2PO4 and 35.6 g K2HPO4 to 1,000 mL distilled water and adjust to pH 6. Autoclave and keep at room temperature. 4. Nematode growth medium (NGM) agar: Add 3 g NaCl, 2.5 g peptone, and 17 g agar to 975 mL distilled water and autoclave. After
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