Utilizing the novel invertebrate model Galleria mellonella as a tool to test virulence and culturing

May 2022- March 2023

Pathogen and Microbiome Institute, Barker Laboratory
https://www.bridgetbarker.com/

National Institute of Health - Research Intitative for Scientific Enhancement (NIH-RISE)

Valley fever vaccine

Valley fever collaborative

The environmentally-acquired fungal infection Valley fever is caused by Coccidioides immitis and Coccidioides posadasii, which is endemic to thermic soils of North and South America. Symptoms of Valley fever can range from respiratory illness to a systemic and life-threatening infection. The range of disease severity is hypothesized to be due to virulence, but current research to assess virulence relies on the murine model, which is costly, time-consuming, and has many bioethical restrictions. Alternatively, the novel invertebrate model Galleria mellonella is low in cost, has a similar innate immune system to mammals, and has no bioethical restrictions. We hypothesized that Galleria mellonella might be an alternative in vivo model to assess virulence of Coccidioides spp. Conidia from an attenuated strain of Coccidioides posadasii was quantified by hemocytometer and a series of dilutions were injected into larvae incubated at 37°C. Survivorship was monitored and larvae were sampled to score morphology. The results showed that deaths were higher among larvae groups that received more fungal cells in their inoculum, and spherule formation of Coccidioides posadassii was observed. The results support the hypothesis that Galleria mellonella can be an alternative infection model, and should be further developed as an inexpensive tool to assess virulence of Coccidioides spp. These results could help contribute to our overall goal of research on how virulence of the fungus is associated with clinical presentation and outcome in patients, identifying virulence factors to help design vaccine targets, and overall helping prevent possible deaths associated with Valley fever infection.

Valley fever is an environmentally-acquired fungal infection caused by two species of the genus Coccidioides; Coccidioides immitis and Coccidioides posadasii. Valley fever is typically known to be endemic to the Southwest United States, Central, and South America. Symptoms of Valley fever can range from respiratory illness to a systemic and life-threatening infection. Currently, most strains of Coccidioides spp. for research come from clinical isolates due to the lack of a reliable method to culture Coccidioides spp. from soil . Murine models are currently used for enrichment to culture Coccidioides spp. from soil, but use of mice is costly, time-consuming, and their use in culturing experiments have been largely inconsistent with low fungal recovery. In contrast to mice, the novel invertebrate model Galleria mellonella are low in cost and have the ability for high number of repetitions in experimentation. We hypothesized that Galleria mellonella larvae might be an alternative enrichment method to culture Coccidioides spp. from soil. Here, we took a fungal extraction method used on Arizona soils that were injected with attenuated Coccidioides posadasii, and the injected soil was allowed to incubate for two weeks. The suspension was then injected into the Galleria mellonella larvae as an enrichment step before sacrificing the larvae and plating on selective media. The results showed successful fungal growth confirmed to be Coccidioides posadasii by RT-qPCR. In conclusion, the invertebrate model Galleria mellonella should be further developed as an inexpensive tool to culture Coccidioides spp. from soil. The ability to reliably and cheaply culture from soil will allow researchers, for the first time, considerable access to environmental strains of Coccidioides spp. for investigation in future experiments. The further development of a tool for culturing Coccidioides spp from soil could also potentially allow for the culturing of other difficult-to-culture organisms in soil for use in research.