Non-mammalian models have been used to investigate fungal virulence. In this work we have explored the use of Galleria mellonella as an infection model for the pathogenic dimorphic fungi Histoplasma capsulatum and Paracoccidioides lutzii. In mammalian models these fungi cause similar infections, and disease outcomes are influenced by the quantity of the infective inocula. We describe a similar aspect in a G. mellonella model and characterize the pathogenesis features in this system. Infection with P. lutzii or H. capsulatum, in all inoculum used, killed larvae at 25 and 37°C. However, there was a lack of correlation between the number of yeast cells used for infection and the time to larvae death, which may indicate that the fungi induce protective responses in a dynamic manner as the lowest concentrations of fungi induced the most rapid death. For both fungi, the degree of larvae melanization was directly proportional to the inocula size, and this effect was visibly more apparent at 37°C. Histological evaluation of the larvae showed a correlation between the inoculum and granuloma-like formation. Our results suggest that G. mellonella is a potentially useful model to study virulence of dimorphic fungi.

Paracoccidioidomycosis is a systemic granulomatous disease caused by Paracoccidioides spp. A peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4(+) helper-1 immune response in mice and protects against intratracheal challenge with virulent P. brasiliensis. Previously, we evaluated the efficacy of the P10 peptide alone or combined with antifungal drugs in mice immunosuppressed and infected with virulent isolate of P. brasiliensis. In the present work, our data suggest that P10 immunization leads to an effective cellular immune response associated with an enhanced T cell proliferative response. P10-stimulated splenocytes increased nitric oxide (NO) production and induced high levels of IFN-γ, IL-1β and IL-12. Furthermore, significantly increased concentrations of pro-inflammatory cytokines were also observed in lung homogenates of immunized mice. P10 immunization was followed by minimal fibrosis in response to infection. Combined with antifungal drugs, P10 immunization most significantly improved survival of anergic infected mice. Administration of either itraconazole or sulfamethoxazole/trimethoprim together with P10 immunization resulted in 100 % survival up to 200 days post-infection, whereas untreated mice died within 80 days. Hence, our data show that P10 immunization promotes a strong specific immune response even in immunocompromised hosts and thus P10 treatment represents a powerful adjuvant therapy to chemotherapy.

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