Galleria mellonella Model of Coccidioidomycosis for Drug Susceptibility Tests and Virulence Factor Identification

Abstract

Coccidioidomycosis (CM) can manifest as respiratory and disseminated diseases that are caused by dimorphic fungal pathogens, such as Coccidioides species. The inhaled arthroconidia generated during the saprobic growth phase convert into multinucleated spherules in the lungs to complete the parasitic lifecycle. Research on coccidioidal virulence and pathogenesis primarily employs murine models typically associated with low lethal doses (LD₁₀₀ < 100 spores). However, the Galleria model has recently garnered attention due to its immune system bearing both structural and functional similarities to the innate system of mammals. Our findings indicate that Coccidioides posadasii can convert and complete the parasitic cycle within the hemocoel of the Galleria larva. In Galleria, the LD₁₀₀ is between 0.5 and 1.0 × 10⁶ viable spores for the clinical isolate Coccidioides posadasii C735. Furthermore, we demonstrated the suitability of this model for in vivo antifungal susceptibility tests to validate the bioreactivity of newly discovered antifungals against Coccidioides. Additionally, we utilized this larva model to screen a Coccidioides posadasii mutant library showing attenuated virulence. Similarly, the identified attenuated coccidioidal mutants displayed a loss of virulence in a commonly used murine model of coccidioidomycosis. In this study, we demonstrated that Galleria larvae can be applied as a model for studying Coccidioides infection.

Keywords: Coccidioides; Galleria mellonella; Valley Fever; drug susceptibility; fungi; mini-host; virulence factors.

Figure 1

Galleria mellonella model of coccidioidomycosis. An illustration of the hemocoel injection method (A). Survival plots of groups of Galleria larvae (n = 20) that were challenged with an indicated dose of viable arthroconidia prepared from the virulent C. posadasii C735 isolate (B,C). Larvae incubated at 37 °C with 10% CO₂ and monitored daily for survival (**** p < 0.0001, *** p < 0.001), via the log-rank test. (D) Representative larvae showing the degree of melanization ranging from scores of 0 to 5: 0, no melanization present; 1, one to three pigmented segments of larvae; 2, four or more melanization segments; 3, partial light-gray pigmentation along the back and tail; 4, visible intensified black spots across the whole light-gray body; and 5, intensified melanization covering the whole body. (E) Plots of melanization scores for each challenged group and controls for a period of 7 days (** p < 0.05 via Mann–Whitney test).

Figure 2

Parasitic growth and propagation of Coccidioides posadasii in G. mellonella larvae. Galleria larvae (n = 10 per time point) were challenged with 5 × 10⁵ C.posadasii C735 spores. (A) Fungal burdens measured on day 2, 5, and 7 post-challenge. * p < 0.05 and **** p < 0.0001 via the Kruskal–Wallis test. (B) Micrographs of G. mellonella larvae infected with Coccidioides at 5 dpc. Galleria larvae sections were stained with Gomori methenamine silver stain (GMS). The left panel shows nodule formation in the fat tissue under the outer cuticle layer of the larvae. The enlarged insert on the right panel shows various developmental morphologies and stages of Coccidioides; the red arrows indicate the release of endospores from a ruptured spherule, the yellow arrows indicate hypha-like cells, the white arrows indicate a segmenting spherule, and the blue arrows indicate small spherules.

Figure 3

Application of Galleria larva model of coccidioidomycosis for antifungal discovery. (A) Timeline and treatment: groups of larvae (n = 10) challenged with 5 × 10⁵ spores of C735, rested for 2 h, and then treated with Amphotericin B (AmB) (B), Sanguinarine (SANG) (C), or Closantel (CLO) (D) at indicated doses. Treatment with PBS served as a control. The larvae received two additional doses of each drug at 2- and 4-days post-challenge. Unchallenged larvae that received each drug alone were used to assess drug toxicity. **** p < 0.0001, ** p < 0.01 and * p < 0.05 via the log-rank test.

Figure 4

Screening attenuated mutants of Coccidioides posadasii using the larva model. Groups of Galleria larvae (n = 10) were challenged with 5 × 10⁵ viable spores prepared from parental C. posadasii C735 and indicated Ti-DNA insertion mutant strains (CpTs). The larvae were incubated at 37 °C. and monitored daily for (A) survival rate of mutants were compared to C735 by log-rank test Mantel-Cox *** p < 0.001, ** p < 0.01, * p < 0.05). (B) mean melanization scores for a period of 7 days. (C) Fungal burden at day 7 post-challenge were compared to C735 via one-way ANOVA with Kruskal–Wallis test (**** p < 0.0001).

Figure 5

Growth of the CpT13 and CpT30 attenuated mutants compared to the C735 parental isolate on GYE and Converse media. (A) Colony size was measured (in mm) for each strain grown on a GYE plate at 30 °C for a 7-day period. Representative photographs of colonies were taken at 3 and 6 days post-inoculation. Their growth curves were comparable. (B) Micrographs of hyphae isolated from the 7-day culture of the two mutants and the parental strains in GYE medium showing similar morphologies. They also formed visually identical arthroconidia with similar sizes and morphologies. (C) Debris and small fungal cells (gray) and large gated spherules (red) were gated from 4-day cultures of the mutants and parental strains in the Converse medium using image flow cytometry as described in the Materials and Methods section. The insert image in each panel is of a representative spherule of each strain. (D) Spherule images labeled with Calcofluor white selected from gated spherule populations of each strain. The images are representative of spherules of an average size in each population.

Figure 6

Evaluation of virulence of Coccidioides posadasii Ti-DNA insertion mutants using a mouse model of coccidioidomycosis. Groups of BALB/c mice (n = 7–8) were challenged with 450 viable spores prepared from parental Cp C735 and indicated Ti-DNA insertion mutant strains (CpTs). (A) Mice monitored for daily survival for a period of 30 days. (B) Fungal burdens in the lungs and the spleen determined at day 14 post-challenge in C57BL/6 mice. The blue dashed line represents the challenge dose. The survival rate, **** p < 0.0001 via the log-rank (Mantel–Cox) test compared to that of C735. The fungal burden, **** p < 0.0001, *** p < 0.001 and ** p < 0.01, was evaluated via one-way ANOVA with the Kruskal–Wallis test.