Legionella pneumophila pathogenesis in the Galleria mellonella infection model

Legionella pneumophila is a facultative intracellular human pathogen and the etiological agent of severe pneumonia known as Legionnaires' disease. Its virulence depends on protein secretion systems, in particular, the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication-permissive vacuole in macrophages. The analysis of the role of these systems and their substrates for pathogenesis requires easy-to-use models which approximate human infection. We examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for L. pneumophila infection. We found that the L. pneumophila strains 130b, Paris, and JR32 caused mortality of the G. mellonella larvae that was strain, infectious dose, growth phase, and T4SS dependent. Wild-type L. pneumophila persisted and replicated within the larvae, whereas T4SS mutants were rapidly cleared. L. pneumophila strain Lp02, which is attenuated in the absence of thymidine but has a functional T4SS, resisted clearance in G. mellonella up to 18 h postinfection without inducing mortality. Immunofluorescence and transmission electron microscopy revealed that L. pneumophila resided within insect hemocytes in a vacuole that ultrastructurally resembled the Legionella-containing vacuole (LCV) observed in macrophages. The vacuole was decorated with the T4SS effector and LCV marker SidC. Infection caused severe damage to the insect organs and triggered immune responses, including activation of the phenoloxidase cascade leading to melanization, nodule formation, and upregulation of antimicrobial peptides. Taken together, these results suggest that G. mellonella provides an effective model to investigate the interaction between L. pneumophila and the host.     

Use of the Galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis

Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using Delta exoU Delta exoY, Delta exoT Delta exoY, and Delta exoT Delta exoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a Delta exoT Delta exoU Delta exoY triple mutant and a Delta pscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.     

Development of Galleria mellonella as an alternative infection model for the Burkholderia cepacia complex

Burkholderia is an important bacterial genus with a complex taxonomy that contains species of both ecological and pathogenic importance, including nine closely related species collectively termed the Burkholderia cepacia complex (BCC). In order to more thoroughly investigate the virulence of this bacterial complex of microorganisms, alternative infection models would be useful. To this end, we have adapted and developed the use of the Galleria mellonella wax moth larvae as a host for examining BCC infections. The experimental conditions affecting the BCC killing of the “wax worm” were optimized. BCC virulence levels were determined using 50% lethal doses, and differences were observed between both species and strains of the BCC. The BCC pathogenicity trends obtained compare favorably with results acquired using other published alternative infection models, as well as mammalian infection models. In addition, BCC killing activity was determined by directly measuring relative bacterial loads in three different BCC strains, thus demonstrating innate differences in BCC strain virulence. Finally, genetically mutated BCC strains were compared to a wild-type BCC strain in order to show concomitant reduction of BCC virulence and increased wax worm survival. For experimentation examining the virulent properties of the BCC, the wax worm has proven to be a useful alternative infection model.     

Colonization and pathogenesis of Cryptococcus neoformans in gnotobiotic mice.

Congenitally immunodeficient nude (nu/nu) mice and their immunocompetent littermates (nu/+) were used to determine whether the absence of thymus-matured T cells would alter the capacity of Cryptococcus neoformans to colonize their mucosal surfaces or enhance their susceptibility to systemic cryptococcosis, or both, following oral challenge. We present data demonstrating that an encapsulated strain of C. neoformans serotype A colonized the alimentary tracts of germfree, conventional, and antibiotic-treated conventional nu/nu mice. Scanning electron microscopy showed that C. neoformans adhered to the epithelial surfaces of the oral cavities, esophagi, and gastrointestinal tracts of monoassociated nu/nu and nu/+ mice, and culture data showed that there were more viable C. neoformans cells in the alimentary tracts of nu/nu mice than of nu/+ mice. Tetracycline-treated conventional nu/nu, but not nu/+, mice were also colonized with C. neoformans following intragastric challenge. C. neoformans-monoassociated and tetracycline-treated conventional nu/nu mice succumbed to disseminated cryptococcosis with cerebral involvement 3 to 4 weeks after oral challenge, whereas no mortality was observed for similarily challenged nu/+ mice. These results demonstrate that an encapsulated strain of C. neoformans can colonize mucosal surfaces and cause systemic cryptococcosis in immunodeficient nu/nu mice, suggesting that the alimentary tract can be a portal of entry for C. neoformans in an immunodeficient host. These data also indicate that functional T cells play an important role in resistance to systemic cryptococcosis of endogenous origin.     

Role of alternative oxidase gene in pathogenesis of Cryptococcus neoformans

We identified a homologue of the alternative oxidase gene in a screen to identify genes that are preferentially transcribed in response to a shift to 37 degrees C in the human-pathogenic yeast Cryptococcus neoformans. Alternative oxidases are nucleus-encoded mitochondrial proteins that have two putative roles: they can function in parallel with the classic cytochrome oxidative pathway to produce ATP, and they may counter oxidative stress within the mitochondria. The C. neoformans alternative oxidase gene (AOX1) was found to exist as a single copy in the genome, and it encodes a putative protein of 401 amino acids. An aox1 mutant strain was created using targeted gene disruption, and the mutant strain was reconstituted to wild type using a full-length AOX1. Compared to both the wild-type and reconstituted strains, the aox1 mutant strain was not temperature sensitive but did have significant impairment of both respiration and growth when treated with inhibitors of the classic cytochrome oxidative pathway. The aox1 mutant strain was also found to be more sensitive to the oxidative stressor tert-butyl hydroperoxide. The aox1 mutant strain was significantly less virulent than both the wild type and the reconstituted strain in the murine inhalational model, and it also had significantly impaired growth within a macrophage-like cell line. These data demonstrate that the alternative oxidase of C. neoformans can make a significant contribution to metabolism, has a role in the yeast's defense against exogenous oxidative stress, and contributes to the virulence composite of this organism, possibly by improving survival within phagocytic cells.     

Spores as Infectious Propagules of Cryptococcus neoformans

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (∼95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules.Humans are exposed to infectious agents via inhalation and cutaneous exposure and from the microbiota. Transmission can involve direct human-human transmission, intermediate animal or insect vectors, or exposure to environmental sources. Animals are exposed to pathogenic fungi via direct/fomite transmission of dermatophytes that infect skin and/or nails, animal-animal transmission by the inhalation of the obligate pathogen Pneumocystis, and bloodstream penetration by Candida species from the gastrointestinal microbiota (46).Many pathogenic fungi (dimorphic fungal pathogens, molds such as Aspergillus fumigatus, and the basidiomycete Cryptococcus) are environmental, and exposure occurs via the inhalation of conidia and/or spores, hyphal fragments, or yeast cells. Particles of >5 μm are subject to efficient mucociliary airway clearance, and smaller infectious propagules more readily deposit deep in the lungs and alveoli (18). As such, spores and conidia represent known or suspected infectious propagules for many pathogenic fungi. Moreover, as spores are often stress resistant, abundant, and readily aerially dispersed, animals may encounter spores more often than other infectious forms. Studies of Schizophyllum commune revealed abundant spores present in air above the ocean at distances of up to a mile from shore (24).Cryptococcus neoformans is a common, opportunistic human fungal pathogen that causes meningoencephalitis in immunocompromised individuals and, if untreated, is uniformly fatal (6, 40). Cryptococcosis is caused by three varieties or sibling species that diverged 10 to 40 million years ago and that exhibit different environmental distributions and virulence properties (32, 43). C. neoformans var. grubii (serotype A) is the major cause (95%) of infections worldwide and >99% of infections in AIDS patients (6). C. neoformans var. neoformans (serotype D) strains account for <5% of infections worldwide, but serotype AD hybrids cause up to 20% of clinical infections in Europe (2, 9, 31). The serotype A and D lineages are globally distributed, associated with pigeon guano, and typically infect immunocompromised hosts.The closely related species Cryptococcus gattii (serotypes B and C) is more geographically restricted to tropical and/or subtropical regions, associated with trees, and commonly infects immunocompetent hosts and less frequently AIDS patients and other immunocompromised hosts (6, 29). A C. gattii outbreak has been occurring on Vancouver Island since 1999 and recently expanded to the Canadian mainland and the United States (5, 12, 25, 38, 52).The infectious propagules for many human fungal pathogens are thought to be spores. Humans are exposed to Cryptococcus by inhalation, leading to an initial pulmonary infection that can be asymptomatic or limited or can disseminate. For many individuals, the initial pulmonary infection is cleared; in others, the organism establishes a dormant latent granulomatous form in the hilar lymph nodes (10, 17). Individuals can die harboring granulomas without overt disease (1, 19, 48). In response to immunosuppression, either primary infection or recrudescence of latent infection results in dissemination via the bloodstream. The organism can infect many organs and tissues but exhibits a predilection to infect the central nervous system (45). The suspected infectious propagules for Cryptococcus are spores or small, desiccated, less encapsulated yeast cells that are an ideal size for alveolar deposition. C. neoformans var. neoformans serotype D spores have been reported to be up to 100 times more infectious than yeast cells in an immunocompromised murine inhalation model (50). Studies to purify spores and analyze their pathogenicity in animal models using different infection methods were previously reported, documenting that spores can serve as infectious propagules under certain conditions, including when directly inoculated intracerebrally, intravenously, or by inhalation (7, 50, 59).Cryptococcus neoformans and C. gattii have defined sexual cycles that produce abundant basidiospores, which could represent infectious propagules (13, 23, 27, 28, 39, 42). However, if spores are the infectious propagule, and spores are produced by mating between a and α cells, how and where would this occur in the largely α unisexual population? A potential resolution was the discovery that α isolates can produce hyphae, basidia, and basidiospores via monokaryotic fruiting (11, 54), a modified sexual cycle that requires only one of the two mating types (4, 12, 21, 33-36, 49). This might be a route by which infectious propagules are produced in nature; however, serotype A C. neoformans var. grubii and C. gattii strains do not reproducibly undergo monokaryotic fruiting under laboratory conditions. Population genetic studies provided evidence that they do so in nature (4, 21, 36, 49), but this remains to be established under defined laboratory conditions.As most previous studies of spore infectivity focused on serotype D, we focused our investigations on serotype A spores and virulence properties. Our analysis examined spore size, shape, and surface properties and conditions supporting spore germination. Importantly, we demonstrate that infectious inocula highly enriched for spores serve as efficient infectious propagules in both a murine inhalation model and an invertebrate host. Analyses of isolates recovered from infected animals provide evidence that recombinant spore progeny are infectious. Spores were not more virulent than yeast cells in either virulence assay. Taken together, our studies provide electron microscopic views of the spores and demonstrate that both spores and yeast cells can serve as infectious propagules.     

The role of mating type and morphology in Cryptococcus neoformans pathogenesis

Cryptococcus neoformans is a major fungal pathogen of both humans and animals. The fungus can be divided into two varieties, with each variety being composed of two serotypes. A sexual phase has been identified, which classifies C. neoformans as a bipolar heterothallic fungus with two mating types, MATa and MATalpha. The analysis of mating and mating type in this organism is important for a number of reasons. Both clinical and environmental isolates display a severe bias of the MATalpha mating type over MATa. MATalpha cells are also more virulent than MATalpha cells. Molecular and genetic analyses of the genes that make up the mating pathway have revealed that some of these genes are required for virulence. Finally, although it is well known that infection begins in the lungs after inhalation of infectious particles, it still remains unclear what constitutes the infectious particle. This review will discuss current information about what is known about the role that mating type and morphology play in virulence.     

Immediate hypersensitivity to Cryptococcus neoformans.

The role of the capsular polysaccharide in anaphylactic reactions to Cryptococcus neoformans was investigated. Groups of mice were sensitized with viable cells of either a moderately encapsulated strain of C. neoformans or a non-encapsulated variant. Anaphylactic reactions were observed in both groups of mice to a similar extent when challenged with whole cells. Mice sensitized with the encapsulated strain and challenged with homologous polysaccharide showed only mild hypersensitivity symptoms. Mice sensitized with either the encapsulated or the nonencapsulated strain showed cross-reactivity when challenged with killed cells of the heterologous strain. These data indicate that the capsular polysaccharide plays a minor role in anaphylactic reactions to C. neoformans and that the sensitizing antigen is probably located in the cell wall of the yeast.     

Cryptococcus neoformans gene involved in mammalian pathogenesis identified by a Caenorhabditis elegans progeny-based approach

Caenorhabditis elegans can serve as a substitute host for the study of microbial pathogenesis. We found that mutations in genes of the fungal pathogen Cryptococcus neoformans involved in mammalian virulence allow C. elegans to produce greater numbers of progeny than when exposed to wild-type fungus. We used this property to screen a library of C. neoformans mutants for strains that permit larger C. elegans brood sizes. In this screen, we identified a gene homologous to Saccharomyces cerevisiae ROM2. C. neoformans rom2 mutation resulted in a defect in mating and growth defects at elevated temperature or in the presence of cell wall or hyperosmolar stresses. An effect of the C. neoformans rom2 mutation in virulence was confirmed in a murine inhalation infection model. We propose that a screen for progeny-permissive mutants of microorganisms can serve as a high-throughput method for identifying novel loci related to mammalian pathogenesis.     

Cane sugar factor as an inducing agent of immunity in Galleria mellonella

The injection of cane sugar factor (CSF) into Galleria mellonella larvae results in an immune response similar to that produced by a formalized Pseudomonas aeruginosa vaccine. Vaccination with CSF is followed by: an increase in the LD₅₀ of Pseudomonas aeruginosa; an in vivo protective response to P. aeruginosa the development of which can be inhibited by cobra venom factor (CVF); an antibacterial activity in hemolymph 24 h after the injection of CSF; the development of a transferrable immune response in hemolymph of donor larvae capable of protecting recipient larvae against a lethal challenge of Pseudomonas aeruginosa; an increase in extracellular lysozyme equal to that induced by Pseudomonas vaccine; a reduction in total hemocyte count during the period of protective immunity; and the presence in hemolymph of new basic proteins, with electrophoretic mobilities and appearance times after the CSF injection, identical to those induced by the formalized vaccine.

CSF was shown to be composed primarily of glucose.     

Binding of cryptococcal polysaccharide to Cryptococcus neoformans

Radioiodinated cryptococcal polysaccharide was used to study binding of the soluble polysaccharide to encapsulated and non-encapsulated cryptoccoci. Binding of polysaccharide to non-encapsulated cryptococci occurred rapidly over a 30-min period and was largely complete after 2 h. Bound, labeled polysaccharide was slowly eluted from Cryptococcus neoformans after the addition of unlabeled polysaccharide, indicating reversibility of binding. Non-encapsulated cryptococci bound polysaccharide in two ways. Specific binding to the yeast was saturable by ca. 82 ng of polysaccharide per 10(6) yeast cells. Nonspecific binding also occurred which was not saturable under the conditions used in our experiments. Phagocytosis of the non-encapsulated yeast strain was inhibited when the specific binding was ca. 50% saturated. Binding of polysaccharide to an encapsulated strain showed nonspecific, nonsaturable binding, but little specific binding occurred. Presumably the specific binding sites were saturated in the encapsulated strain. Polysaccharides obtained from a hypocapsular mutant (A61) and a normally encapsulated strain competed effectively with labeled serotype D polysaccharide for binding sites on non-encapsulated cryptococci and had identical phagocytosis-inhibiting properties. Similarly, polysaccharides from all four cryptococcal serotypes competed effectively with labeled serotype D polysaccharide for binding sites on the non-encapsulated strain, and all four polysaccharides inhibited phagocytosis of non-encapsulated Cryptococcus neoformans. Unmodified, de-O-acetylated, carboxyl-reduced, periodate-oxidized and reduced (polyalcohol), and Smith-degraded polysaccharides competed with labeled polysaccharide for binding sites on the cell. The unmodified, de-O-acetylated and carboxyl-reduced polysaccharides inhibited phagocytosis of non-encapsulated cells, but the polyalcohol and Smith product were unable to inhibit phagocytosis.     

Hypersensitivity pneumonitis with antibodies to Cryptococcus neoformans

Clinical and immunological studies were made in forty-two patients diagnosed as suffering from hypersensitivity pneumonitis at Osaka Prefectural Habikino Hospital between 1973 and 1977. All the sera from forty-one patients tested had high litres of antibody against Cryptococcus neoformans in indirect fluorescent antibody tests, and twelve also had precipitins against Cryptococcus neoformans polysaccharide. Only about 10% of control sera from patients with otherlung diseases had low titresof antibody against Cryptococcus neoformans. Antibody against Cryptococcus neoformans was also found frequently in the sera of asymptomatic members of the families of the patients. A possible relationship of Cryptococcus neoformans to hypersensitivity pneumonitis is suggested.     

A Streptococcus pneumoniae infection model in larvae of the wax moth Galleria mellonella

The bacterium Streptococcus pneumoniae is a leading human opportunistic pathogen. The limitations of the current vaccine have led to increased recognition of the need to understand bacterial behaviour and competitive dynamics using in vivo models of infection. Here, we investigate the potential application of the larvae of the wax moth Galleria mellonella as an informative infection model. Larvae were challenged with a range of doses of S. pneumoniae isolates differing in known virulence factors to determine the LD(50) values. Infection dynamics were determined by obtaining bacterial counts from larvae over a time course. Differences in virulence between serotypes could be distinguished in this host. Infection with strains differing in known virulence factors demonstrated predicted differences in virulence. Acapsulate and pneumolysin-negative strains were less virulent than their respective wild types. A large reduction in virulence was seen in strains lacking cell wall D-alanylation. The mortality of G. mellonella larvae is attributable to bacterial growth within larvae, while surviving larvae are able to clear infections by reducing bacterial numbers. These data demonstrate that G. mellonella larvae represent an in vivo infection model with applications for investigating aspects of bacterial-host interactions such as the role of antimicrobial peptide activity and resistance.     

Karyotyping of Cryptococcus neoformans as an epidemiological tool.

Karyotyping of Cryptococcus neoformans var. neoformans can be used as an epidemiological tool for C. neoformans infections. In this study of over 40 isolates from both clinical and environmental sources, 90% had a unique chromosome banding by pulsed-field electrophoresis. There was no conserved pattern associated with body site of infection, geographical location of the isolate, or human immunodeficiency virus status. Karyotypes of individual isolates remained stable during both in vitro passage and in vivo infections. Karyotype was used to exclude the possibility of nosocomial spread of C. neoformans in one clinical situation and supported relapse in two other cases. Because of its variable sizes between isolates, karyotyping of C. neoformans is a convenient method for molecular identification of different strains.     

Respiratory and skin allergy to Galleria mellonella (bee moth)

This case report describes a patient with bee moth-induced rhinoconjunctivitis, asthma and contact urticaria. Immunoblot analysis showed IgE reactivity to two distinct bee moth proteins at 23 and 70 kDa, respectively. ELISA inhibition studies excluded cross-reactivity to the other popular live bait, fly larva.     

Calreticulin enriched as an early-stage encapsulation protein in wax moth Galleria mellonella larvae

To investigate the molecular mechanism of the early-stage encapsulation reaction in insects, we purified a 47kDa protein from injected beads into Galleria mellonella larvae. When a cDNA clone was isolated, the 47kDa protein showed high homology with Drosophila and human calreticulin. Western blotting analysis showed that the 47kDa protein was present in the hemocytes, but not in the plasma. When the early-stage encapsulated beads were coated with 47kDa protein antibody and reinjected into G. mellonella larvae, any further encapsulation reaction was inhibited. These results suggest that calreticulin is involved in non-self recognition in invertebrate cellular defense reactions.     

Cyclosporin A inhibits the growth of Cryptococcus neoformans in a murine model.

Cryptococcus neoformans is a frequent opportunistic infectious agent in patients with decreased T-lymphocyte-mediated immune function, including those with acquired immune deficiency syndrome. Cyclosporin A (CsA), a potent inhibitor of T-lymphocyte function, was administered subcutaneously to mice to study the pathogenesis of C. neoformans infections in the setting of impaired T-cell function. Surprisingly, survival was prolonged indefinitely in animals that received immunosuppressive doses of CsA following either intratracheal or intravenous inoculations of C. neoformans. Furthermore, following intratracheal inoculation, mice treated with CsA cleared C. neoformans from their lungs more rapidly than did control mice. CsA directly inhibited the growth of C. neoformans when it was added to cultures in vitro at concentrations comparable to the blood levels achieved in experimental mice. Thus, CsA inhibited both in vitro and in vivo growth of C. neoformans. While these results must be extended to studies in humans, these data suggest that patients who now receive CsA-immunosuppressive therapy may be fortuitously protected against infections with C. neoformans. Furthermore, research into cyclosporin derivatives may yield compounds with less immunosuppressive properties and enhanced antifungal activity.