Metamorphosis of Galleria mellonella research

Recently, non-vertebrate hosts, such as amoeba, nematodes, and insects have been explored as alternatives to rodents in infectious disease research.^1,2 In particular, the popularity of the waxmoth Galleria mellonella as a model host has surged. A search of PubMed for articles with “Galleria mellonella” in the title or abstract reveals of list of over 1100 results. Of these, one quarter have been published within the last three years. The advantages of using G. mellonella larvae are numerous. Their use reduces the amount of vertebrate animals used in research, thereby alleviating some of the ethical problems associated with animal welfare. Moreover, they can be obtained easily and inexpensively and no special training, equipment or animal care facilities are required to maintain them.³

Much of the research with G. mellonella focuses on whether known virulence factors play a role in infection of the larvae. For example, clinical isolates from a Legionnaires’ disease outbreak in Edinburgh are able to infect G. mellonella and virulence in the larvae correlates with the type IV secretion system of the bacteria.⁴ In addition, clinical isolates of the yeast Candida albicans known to produce biofilms are associated with increased virulence in G. mellonella.⁵ Other research has studied antibiotic efficacy in the larvae. For example, ciprofloxacin reduces larval killing by extraintestinal pathogenic (ExPEC) Escherichia coli isolates.⁶

These studies demonstrate that G. mellonella is useful for studying virulence factors and antibiotic efficacy, but what about other aspects of pathogenesis? Considering the significant structural and physiological differences between humans and invertebrates, questions remain as to limits of using G. mellonella as a model host. In the accompanying study, “Cryptococcus neoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleria mellonella,” Trevijano-Contador et al. address such questions by testing whether aspects of C. neoformans pathogenesis are replicated during infection of G. mellonella.⁷

Unlike vertebrates, G. mellonella has no adaptive immunity. The first line of defense in insects is the cuticle, a physical barrier rich in chitin that resists invasion. However if the cuticle is breached, other innate defenses are capable of providing protection from infection. Both cellular and humoral immune components can be found in the hemolymph of insects. The cellular immune responses include phagocytosis and encapsulation by hemocytes. Similar to mammalian phagocytes, hemocytes are capable of killing microbes by phagocytosis and oxidative killing. The humoral responses include a variety of secreted molecules with antimicrobial properties. Insects produce antimicrobial peptides capable of directly killing invading pathogens. Additionally, a phenoloxidase system defends against infection using melanin and melanin-intermediates to heal wounds and kill pathogens.^8,9

Cryptococcus neoformans is a fungal pathogen known primarily to infect patients with poor immunity. Usually, infections result from the inhalation of spores or yeast into the lungs. The fungus may disseminate from the lungs to the central nervous system, leading to life-threatening meningoencephalitis. C. neoformans is capable of infecting various organisms besides mammals, such as amoeba, plants and insects, including G. mellonella.^10-12 It is likely that many virulence attributes of C. neoformans are preserved across hosts. In one study, a mutant library was screened for decreased virulence in Caenorhabditis elegans and, for many of the candidates, reduced virulence was seen in both G. mellonella and mice.¹³

C. neoformans has several well-characterized virulence factors that contribute to its pathogenesis. One of the most studied virulence factors is the capsule, a polysaccharide layer surrounding the cell. The capsule is mainly composed of the polysaccharides glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), in addition to a small proportion of mannoproteins. The capsule is dynamic and can change in size and structure under different conditions, including during infection. One of the main functions of the capsule with regard to virulence is the protection of C. neoformans cells from phagocytosis by macrophages. Furthermore, GXM shed from cells during infection interferes with immune cell function.^14,15 Similar to what is seen in mammals, capsule size increases during G. mellonella infection and increased capsule size in correlated with reduced phagocytosis by hemocytes.¹⁶ In the present study, the authors find that capsule and GXM have an effect on lytic activity of the larval hemolymph, a humoral immune response of G. mellonella. Furthermore, they observe that the antigenic properties of the capsule change upon infection.⁷ Together, these findings suggest that capsule dynamics are important in the pathogenesis of C. neoformans in G. mellonella, just as they are in mammalian hosts.

Interaction of the C. neoformans with phagocytes is critical in the pathogenesis of cryptococcal infection of mammals, especially the ability of the fungus to disseminate through the host. Clinical isolates that exhibit a high level of phagocytosis by macrophages and intracellular replication in vitro are associated with death in humans.¹⁷ Upon phagocytosis by macrophages, the phagolysosome forms. After which, there are several possible outcomes. C. neoformans is capable of replication inside this compartment, leading to damage of the phagolysosome membrane, leaking of capsular polysaccharide into the cytoplasm and ultimately lysis of the host cell and release of fungal cells.^18,19 Alternatively, non-lytic release may occur, a unique process of extrusion from macrophages. Within hours of phagocytosis, yeast cells may be expelled from the macrophages. After such events, both the yeast and the host cells are intact and able to reproduce. ^20,21 Lastly, C. neoformans occasionally moves between macrophages by direct transfer of yeast between infected and uninfected cells.^{22, 23} The phenomena of intracellular replication, extrusion and direct transfer suggest a means by which C. neoformans can disseminate through the human body. According to this “Trojan Horse” hypothesis, fungal cells can use phagocytic cells as a place to hide from direct attack by the immune system and be transported through the body and across the blood-brain-barrier.²⁴

Trevijano-Contador et al. recapitulate two important aspects of the interaction of C. neoformans with macrophages in G. mellonella hemocytes. First, C. neoformans cells are observed to replicate inside hemocytes. Second, fungal cells may be released non-lytically from G. mellonella hemocytes.⁷ Interestingly, extrusion of C. neoformans has also been observed in amoeba ²⁵ and Drosophila cell culture.²⁶ The discovery of intracellular replication and extrusion in G. mellonella hemocytes as well suggests evolutionarily conserved mechanisms govern these interactions. It is interesting to consider the purpose of these phenomena in “alternative” hosts like amoeba and insects where dissemination through the bloodstream or across a blood-brain-barrier does not occur. Such questions remain to be addressed in future studies.

The present study shows that G. mellonella research can be expanded beyond identification of virulence factors to more detailed aspects of pathogenesis. This further validates the usefulness of nontraditional models and of G. mellonella in particular. It is fascinating that multiple aspects of C. neoformans pathogenesis are preserved among hosts as different as mammals and insects. Furthermore, the research underscores the idea that virulence is a product of ancient evolutionary interactions that predate the interaction of C. neoformans with mammals.