Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms.

Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.

Evaluation of Caenorhabditis elegans as a host model for Paracoccidioides brasiliensis and Paracoccidioides lutzii.

Paracoccidioidomycosis is a systemic fungal infection affecting mainly Latin American countries that is caused by Paracoccidioides brasiliensis and Paracoccidioides lutzii. During the study of fungal pathogenesis, in vivo studies are crucial to understand the overall mechanisms involving the infection as well as to search for new therapeutic treatments and diagnosis. Caenorhabditis elegans is described as an infection model for different fungi species and a well-characterized organism to study the innate immune response. This study evaluates C. elegans as an infection model for Paracoccidioides spp. It was observed that both species do not cause infection in C. elegans, as occurs with Candida albicans, and one possible explanation is that the irregular size and shape of Paracoccidioides spp. difficult the ingestion of these fungi by the nematode. Besides this difficulty in the infection, we could observe that the simple exposition of C. elegans to Paracoccidioides species was able to trigger a distinct pattern of expression of antimicrobial peptide genes. The expression of cnc-4, nlpl-27 and nlp-31 was superior after the exposure to P. brasiliensis in comparison to P. lutzii (P < 0.05), and these findings demonstrate important differences regarding innate immune response activation caused by the two species of the Paracoccidioides genus.