Background

Cryptococcus neoformans is a basidiomycetous polysaccharide-encapsulated fungus, pathogenic for multiple species (Kwon-Chung et al., 2014). In humans, C. neoformans can cause serious infections, primarily in immunocompromised patients with clinical manifestations of pneumonia and meningitis (Lui et al., 2006; Kwon-Chung et al., 2014). Due to high mortality, lack of available vaccines, limited treatment options, and increasing resistance to the fluconazole—a drug commonly used for treatment of cryptococcosis—it is estimated that the total number of cryptococcosis-related deaths exceeds 180,000 annually worldwide (Cogliati, 2013; Perfect and Bicanic, 2015). Cryptococcus spp. have several characteristic virulence factors that are essential for establishing infection and providing defense mechanisms against the host immune system. Important cryptococcal virulence factors include the polysaccharide capsule, melanin, and formation of titan cells (Zaragoza, 2019). Researchers have developed several methods to stimulate the production of those virulence factors in culture conditions; while this allows for precise functional analysis, it is not sufficient for a more holistic understanding of virulence (Casadevall et al., 2000; Liu et al., 2008; Crabtree et al., 2012; Almeida et al., 2015).

The study of virulence usually requires the use of an animal model organism. Animal testing plays an essential role in the medical and biomedical field of research. Unfortunately, research performed on mice, rats, and other mammals comes with serious limitations, including medium to high costs, requirement for specialized animal testing facilities, and concerns about the use of vertebrate animals in research. Additionally, mouse and mammalian studies can take several months depending on the virulence of the microbe and disease progression. To avoid those problems, many researchers performed studies, including virulence assays, on invertebrate models like the fruit fly Drosophila melanogaster or the larvae of the Greater wax moth Galleria mellonella. G. mellonella has been shown to be a useful model organism to test pathogenicity of multiple fungal species, including C. neoformans, which was first used in a G. mellonella model by the Mylonakis group in 2005 (Reeves et al., 2004; Mylonakis et al., 2005; Pereira et al., 2018; Maurer et al., 2019). The advantage of G. mellonella as a model host is its flexibility for changes in the infection protocol that allow the study of variables not possible in vertebrate and invertebrate models, such as host temperature. In contrast to other invertebrate model systems, infected larvae of G. mellonella can be incubated at room temperature, 30 °C, and 37 °C (Mylonakis et al., 2005). This temperature range is important because it allows experiments at mammalian temperatures. G. mellonella larvae are between 2–3 cm in length, an easy size to handle and manipulate. In addition, since G. mellonella are relatively easy to infect and store in high numbers, this model enables researchers to perform virulence or survival screens of many different conditions, strains, or mutants, before narrowing those down to use in a resource and time-intensive mouse model (Mylonakis et al., 2005; Firacative et al., 2020).

G. mellonella are used as models for C. neoformans infections, as several of the virulence factors important for C. neoformans infections in mammalian hosts also play comparable roles in G. mellonella. These include laccase, capsule, and the production of titan cells, which allow the fungus to evade immune clearance (Mylonakis et al., 2005; García-Rodas et al., 2011; Eisenman et al., 2014). Additionally, C. neoformans undergoes similar interactions with the immune systems of G. mellonella and mammals (Browne et al., 2013; Trevijano-Contador and Zaragoza, 2018; Stączek et al., 2020; Smith and Casadevall, 2021). C. neoformans is phagocytosed by both insect hemocytes (immune cells) and mammalian macrophages (Browne et al., 2013; Pereira et al., 2018). The fungus is also encapsulated within immune nodules of G. mellonella, which are aggregates of insect immune cells that neutralize the infection, comparable to the granulomas that form around C. neoformans within the mammalian lung. Here we present a standardized protocol for the virulence survival assay of C. neoformans performed in G. mellonella larvae.