Galleria mellonella as a model host to study virulence of Candida

Candida spp. are among the most important human fungal pathogens, with infections ranging from relatively benign, superficial manifestation to life-threatening deep-seated candidiasis and disseminated disease. Different mouse models have been developed to recapitulate the different forms of candidiasis and murine models are considered to be the gold standard to study pathogenesis and analyze efficacy of antifungal treatment. However, economical, logistical, and ethical considerations limit the use of mammals in infection experiments, especially when the question at hand requires analysis of a large number of fungal strains. As an alternative approach, different invertebrate infection models have been developed, including Caenorhabditis elegans, Drosophila melanogaster, and larvae of the wax moth Galleria mellonella as hosts. G. mellonella are inexpensive to purchase and do not require specialized facilities for maintenance. The relatively large size of the larvae facilitates easy handling, injection of a defined inoculum, and sampling for downstream analyses. Furthermore, in contrast to other invertebrate hosts, G. mellonella larvae can be maintained at temperatures up to 37 °C, equivalent to the temperature in mammalian hosts. As temperature has been shown to affect expression of Candida virulence traits, this feature is important when assessing virulence of Candida strains. In addition, the larval immune system shows functional and structural similarity to the mammalian innate immune system: Pathogens are recognized by pathogen recognition receptors and can be phagocytosed by the insects’ hemocytes, the functional equivalent to mammalian neutrophils. Similar to neutrophils, hemocytes use reactive oxygen species and lytic enzymes to eliminate microorganisms. Antimicrobial peptides are produced by G. mellonella in response to infection and likely contribute to the host defense, as it has been shown for Candida epithelial infections using mammalian cells. Thus, it is not surprising that G. mellonella larvae are used increasingly as a model for Candida infections, for example to determine the virulence of genetically modified C. albicans strains and to determine the efficacy of antifungal treatment against both C. albicans and non-albicans Candida species. Host mortality after infection with a distinct dose or determination of the LD 50 is commonly used as the primary parameter to assess virulence of microorganisms and to rank the relative virulence of species and strains. Using this approach with systemically infected mice, C. albicans and C. tropicalis were found to be highly virulent while other Candida species such as C. glabrata, C. parapsilosis, and C. krusei induced no mortality, even in immunocompromised mice. The high virulence of C. albicans in murine models correlates with the clinical situation, in which the majority of Candida infections are caused by C. albicans. However, infections with non-albicans Candida species are emerging, including species such as C. glabrata and C. parapsilosis, which rarely cause lethal infections in mice. In the absence of mortality, fungal burden can be used to compare species and strains. However, fungal burden primarily reflects fitness and not necessarily virulence, as illustrated by a C. albicans mutant overexpressing the transcription factor NRG1: This mutant was highly attenuated in a systemic mouse model although fungal burden was comparable to the corresponding wild-type strain. Comparison of the virulence potential of different Candida spp. has also been performed in G. mellonella, confirming C. albicans and C. tropicalis as the most virulent species. In addition, these studies also revealed substantial virulence potential of C. parapsilosis in G. mellonella, leading to significant mortality of infected larvae. Although it is not clear why C. parapsilosis infections are lethal in G. mellonella larvae but not in mice, this observation suggested that G. mellonella could serve as a model organism to study virulence on level of subspecies and strains. Thus, Gago et al. used mortality in the Galleria model as the primary parameter to investigate the virulence potential of the species within in C. parapsilosis complex, C. parapsilosis, C. orthopsilosis, and C. metapsilosis. This study, published recently in Virulence, showed that C. parapsilosis and C. orthopsilosis induced larval mortality at a comparable rate while C. metapsilosis was less virulent. These findings are strongly supported by a recent publication of Németh et al., who obtained comparable results using a different set of strains belonging to the C. parapsilosis complex in a G. mellonella infection model. The results are furthermore consistent with different in vitro approaches, that found C. metapsilosis to be the least virulent species of the parapsilosis complex, and virulence in a vaginal mouse model. Why is C. metapsilosis less virulent than C. parapsilosis and C. orthopsilosis? The ability to secrete proteases and lipases has