| Congresso Brasileiro de Microbiologia 2023 | Resumo: 20-1 | ||||
Resumo:Free-living amoebas play important roles in various ecosystems, including the regulation of microbial populations in soil. Hence, several evidence suggest that amoebas are natural predators of pathogenic fungi to humans. The fungi belonging to the genus Cryptococcus, such as C. neoformans and C. gattii, are encapsulated basidiomycetes and important human pathogens. Host cells employ a strategy known as nutritional immunity, wherein they sequester various nutrients, such as zinc, within vacuoles and other organelles, in order to reduce their accessibility to invading pathogens, such as C. gattii. The proper metabolism of zinc plays a crucial role in the virulence of C. gattii, which is regulated by the transcription factor Zap1. This factor is directly associated with the activation of proteins expression containing the ZIP domain, such as ZIP1, which mobilizes zinc for the cytoplasm. Considering that the ACA1_271600 gene, which encodes a ZNT Family A metal carrier from the SLC30 family was identified in Acanthamoeba castellanii, which is potentially involved in the internalization of nutrients into vacuoles. The objective of this study was to assess the influence of zinc homeostasis on the A. castellanii antifungal activity against C. gattii. We performed gene silencing of ACA1_271600 and evaluated the phagocytosis and intracellular proliferation rates of wild-type (WT) cells, as well as null mutants for the ZIP1 gene of C. gattii, which is known to be important for proper development of cryptococcal cells under zinc deprivation conditions, thus serving as a biomarker for zinc avalibility. To confirm the gene silencing, real-time PCR was performed, revealing a near 80% decrease in the relative levels of transcripts of the ACA1_271600 gene in the lineage treated with siRNA compared to non-related siRNA treatment. To assess whether the reduction in the expression of ACA1_271600 alters C. gattii survival inside amoebas, we evaluated the rate of phagocytosis and intra-amoeba replication. Both the wild lineage and the zip1 mutant showed higher levels of proliferation when ACA1_271600 was silenced compared to scrambled siRNA treatment. To understand why zinc deprivation changes the replicative capacity of C. gattii, we performed a systems biology analysis to recover direct interactors of the ZIP1 and ZAP1 genes. We detected various enriched processes associated with key components of cell viability, such as transcription and DNA repair. The results suggest that the silencing of the ACA1_271600 gene influences A. castellanii predatory capacity, influencing nutrient availability, including zinc, in the intracellular environment. Palavras-chave: Acanthamoeba castellanii, Cryptococcus gattii, Homeostasis, Zinc Agência de fomento:CNPq; CAPES; FAPERGS | |||||