Galleria mellonella as a Model System for Studying Listeria Pathogenesis

Essential aspects of the innate immune response to microbial infection are conserved between insects and mammals. This has generated interest in using insects as model organisms to study host-microbe interactions. We used the greater wax moth Galleria mellonella, which can be reared at 37°C, as a model host for examining the virulence potential of Listeria spp. Here we report that Galleria is an excellent surrogate model of listerial septic infection, capable of clearly distinguishing between pathogenic and nonpathogenic Listeria strains and even between virulent and attenuated Listeria monocytogenes strains. Virulence required listerial genes hitherto implicated in the mouse infection model and was linked to strong antimicrobial activities in both hemolymph and hemocytes of infected larvae. Following Listeria infection, the expression of immune defense genes such as those for lysozyme, galiomycin, gallerimycin, and insect metalloproteinase inhibitor (IMPI) was sequentially induced. Preinduction of antimicrobial activity by treatment of larvae with lipopolysaccharide (LPS) significantly improved survival against subsequent L. monocytogenes challenge and strong antilisterial activity was detected in the hemolymph of LPS pretreated larvae. We conclude that the severity of septic infection with L. monocytogenes is modulated primarily by innate immune responses, and we suggest the use of Galleria as a relatively simple, nonmammalian model system that can be used to assess the virulence of strains of Listeria spp. isolated from a wide variety of settings from both the clinic and the environment.Listeriae are rod-shaped, motile, facultative, anaerobic Gram-positive bacteria that are ubiquitously distributed in the environment (28). Of the six species that comprise the genus Listeria, only L. monocytogenes and L. ivanovii are pathogenic and cause disease, while strains of the species L. innocua, L. welshimeri, L. seeligeri, and L. grayi are generally considered to be nonpathogenic (26). L. monocytogenes is a major food-borne pathogen, and listeriosis is an invasive disease that in its severest form can lead to meningitis, meningoencephalitis, septicemia, and abortions (38). Listeriosis occurs primarily in pregnant women, newborn infants, and the elderly as well as in immunocompromised patients, with a mortality rate of about 30% (22, 36). The virulence of L. monocytogenes has been linked to a 9.6-kb pathogenicity island designated vgc (virulence gene cluster) that comprises six genes encoding its major virulence determinants. These are (i) prfA, a master regulator of many known listerial virulence genes; (ii) hly, encoding listeriolysin, a hemolysin required for bacterial escape from the host primary vacuole to the host cytoplasm; (iii) two phospholipase genes denoted plcA and plcB, for facilitating lysis of host cell membranes; (iv) actA, encoding a surface bound protein that directs polymerization of host cell actin and is required for intracellular motility; and (v) mpl, encoding a metalloproteinase which is thought to work together with the plcB product to facilitate cell-to-cell spread (28). Presently, identification and characterization of novel virulence factors rely on assessing mutant bacteria for growth in the organs of infected mice. Nevertheless, the dependence on mouse infection models limits large-scale screening for additional mutants defective in their ability to grow in the host intracellularly or for those required to overcome host innate defenses (33).The possibility of addressing many aspects of mammalian innate immunity in invertebrates has opened a new arena for developing invertebrate models to study human infections. Recently the use of invertebrate models, in particular the fruit fly Drosophila melanogaster, has been introduced for the study of septic listerial infections (37). Listeria mutants attenuated for virulence in a mouse model exhibited lowered virulence in this model. The Drosophila model system has powerful genetic tools available and has thus provided deeper insights into molecular mechanisms of the interactions between Listeria and the insect innate immune system (1, 8-10, 18, 24). However, a recent study has shown that even nonpathogenic L. innocua strains cause lethal infections of Drosophila, limiting it use as a discerning model for the study of virulence potential among pathogenic L. monocytogenes isolates (32).We have a longstanding interest in host-pathogen interactions of the greater wax moth, Galleria mellonella, in particular with entomopathogenic microbes (55). Recently, Galleria has also emerged as a reliable model host to study the pathogenesis of many human pathogens (7, 11, 12, 17, 21, 30, 31, 39-42, 44, 46, 48-51). Among the advantages provided by the Galleria model host (e.g., low rearing costs, convenient injection feasibility, and status as an ethically acceptable animal model), it is of particular importance that Galleria has a growth optimum at 37°C, to which human pathogens are adapted and which is essential for synthesis of many virulence/pathogenicity factors. Significantly, a correlation between the virulence of a pathogen in G. mellonella and that in mammalian models has been established (16, 25).The innate immunity of Galleria is a complex, multicomponent response involving hemolymph coagulation, cellular phagocytosis, and phenol oxidase-based melanization. Importantly, killing of pathogens is achieved similarly to that in mammals, i.e., by enzymes (e.g., lysozymes), reactive oxygen species, and antimicrobial peptides (e.g., defensins). Galleria employs recognition of nonself microbe-associated molecular patterns by germ line-encoded receptors (e.g., Toll and peptidoglycan recognition proteins) (52). Recently, we have found that Galleria also senses pathogens by danger signaling, by detecting either nucleic acids released from damaged cells or peptides resulting from proteolytic cleavage of self proteins by matrix metalloproteinases (3-6).In this work we examined the Galleria model of septic infection for its ability to differentially distinguish between infections caused by strains with different virulence potentials in the mouse infection model, as well as in avirulent strains of Listeria. We found that the Galleria model is highly discriminatory in assessing the pathogenic potential of Listeria spp., and we observed a strong correlation with the virulence previously determined in the mouse model of infection. Here, we present data indicating that the Galleria model also replicates many aspects of innate immune function, such as the constitutive expressions of potential antimicrobial factors following infection. Also, prior induction of immunity in Galleria can protect larvae from septic infection with highly pathogenic L. monocytogenes.