An analysis of the interaction of environmental and clinical isolates of Aspergillus fumigatus with vertebrate and invertebrate immune responses

The ubiquitous saprophytic filamentous fungus Aspergillus fumigatus is a significant pathogen among individuals undergoing chemotherapy, allogenic stem cell transplantation and in patients with underlying pulmonary conditions or chronic granulomatous disease patients. Central to the virulence of A. fumigatus is the production of various secondary metabolites, proteolytic enzymes and iron sequestering molecules which coupled to the rapidly growing nature of the fungus aid its persistence in host tissue. Assessment of the virulence of A. fumigatus isolates has previously centred on the use of murine models of aspergillosis however in recent years there has been an increasing body of evidence suggesting the use of invertebrates as a viable alternative. The work presented here sought to further develop the use of the Greater wax moth Galleria mellonella as a novel in vivo tool to assess the pathogenicity of A. fumigatus isolates as an alternative to murine models of infection. The data presented here characterises the pathogenicity of clinical and environmental isolates of A. fumigatus in G. mellonella. Results here suggest that mycotoxins are produced at varying levels in the A. fumigatus isolates with culture filtrates demonstrating immunosuppressive properties. In vivo mycotoxin production by A. fumigatus ATCC 26933 in G. mellonella was characterised and demonstrated that high quantities of fumagillin were produced in the first 24 hours of invasive infection. A study was performed to assess the effect of fumagillin on human neutrophils and G. mellonella haemocytes. Fumagillin inhibited phagocytosis and pathogen directed killing in both cell types and disrupted oxygen consumption through reduced translocation of p47phox and its insect homologue. Exposure of neutrophils and haemocytes to fumagillin also disrupted degranulation. Experimental evidence presented here suggests that the inhibition to cellular processes is mediated by reduced F-actin assembly. A proteomic analysis of G. mellonella haemolymph revealed evidence to suggest that fumagillin mediates an oxidative stress in vivo and disrupted the humoral immune response. Recent literature has suggested that the immune system of G. mellonella could be primed to subsequent infection. Larvae primed with specific sub-lethal inocula of conidia demonstrated improved resistance to subsequent infection through increased haemocyte density, protein activity, and synthesis of proteins involved in oxygen transport, pathogen recognition and iron sequesterisation and increased expression of antimicrobial peptides. This project has further developed the G. mellonella model as a model system to investigate the pathogenicity of A. fumigatus isolates and the immunomodulatory effects of fungal secondary metabolites due to an immune system which shows a high level of conservation with the human innate immune system