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-albicansCandida species.-

Host mortality after infection with a distinct dose or determination of the LD50 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 been associated with virulence in C. albicans and C. parapsilosis., Both Gago et al. and Németh et al. analyzed enzymatic activity in the different strains and found C. parapsilosis strains to more frequently express hydrolytic activity. However, as C. orthopsilosis and C. parapsilosis showed comparable virulence in the Galleria model, additional factors must contribute to pathogenicity. Filament formation, mediating penetration of tissue and escape from immune cells upon phagocytosis, is a well described virulence attribute in C. albicans. Most C. parapsilosis and C. orthopsilosis strains were capable of forming pseudohyphae in vitro whereas all C. metapsilosis isolates analyzed produced yeast cells only, suggesting a link between pseudohyphae formation and virulence on the species level., On the strain level, however, individual C. orthopsilosis isolates displayed high virulence in the absence of pseudohyphae formation, suggesting that additional factors are important. Interestingly, partial decoupling of filamentation and virulence has also been observed with defined C. albicans mutants in the G. mellonella model: A C. albicans tec1Δ mutant that still formed filaments exhibited reduced pathogenicity in G. mellonella model. Similarly, restoration of filamentation in C. albicans flo8Δ by overexpression of TEC1 did not restore virulence, both in G. mellonella and in mice. As filament formation enables Candida to escape from immune cells, Gago et al. analyzed the hemocytes in infected G. mellonella larvae. Hemocytes numbers were significantly lower in larvae infected with C. parapsilosis and C. orthopsilosis compared with C. metapsilosis. Correlation of hemocytes numbers with survival after Candida infection has also been observed in other studies,,- and hemocytes function has been clearly linked to the outcome of fungal infections in Galleria., Gago et al. speculated that pseudohyphae production by phagocytosed yeasts may damage hemocytes, thus contributing to the lower number of hemocytes observed. This hypothesis is supported by the results of Németh et al., who demonstrated a greater cytotoxic potential of C. parapsilosis and C. orthopsilosis against mammalian macrophages. However, it yet needs to be demonstrated that Galleria hemocytes are indeed damaged by pseudohyphae and that this process accounts for the lower number of hemocytes in vivo, and in consequence for higher virulence. In this context, the higher rate of phagocytosis of C. metapsilosis by Galleria hemocytes might indicate reduced survival of this fungus in the host, a hypothesis which likewise still needs to be experimentally confirmed.

The study of Gago et al. illustrates the potential of G. mellonella larvae as a model organism to assay Candida virulence and to study pathogenesis. However, many questions remain open, for example which kind of hemocytes respond to Candida infections, which fungal ligands do bind to what hemocyte receptors and whether an unbalanced immune response contributes to pathogenesis, as described in the murine model of disseminated candidiasis and human sepsis. To address these questions, it will be necessary to develop tools that allow investigating different interactions on the cellular and molecular level, as they are available for mice, humans, and other model organisms, such as Drosophila. Useful tools could include immortalized G. mellonella cell lines, antibodies to allow differentiation of hemocytes populations, markers for hemocytes activation, genome data to facilitate development of microarrays and methods for genomic manipulation of G. mellonella. Furthermore, as recently elaborated in other editorials in Virulence, defined G. mellonella lines and standardized protocols for propagation and maintenance are needed to fully develop the potential of G. mellonella larvae as model organisms to study fungal infections and to allow comparison of results obtained in different laboratories. A better understanding of the pathogenesis of fungal infections in Galleria will likely yield important insights into the infection process that can be transferred to mammalian host. There will also be limitations and it is likely that pathogenesis differs in some aspects between different host species. For example, it remains to be elucidated why infection with C. parapsilosis is lethal in G. mellonella larvae but not in mice. These differences, however, if seen in the context of human infections, should not be merely considered a disadvantage of one model over the other. If interpreted with care, understanding both similarities and differences of pathogenesis and host defense in different model hosts will greatly aid in identifying mechanisms that can be transferred to human infections. In addition, it might furthermore help in elucidating specific defects that predispose human patients to infection and possibly allow identifying new approaches for treatment.