Banca de DEFESA: Clara Luna Freitas Marina

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : Clara Luna Freitas Marina
DATE: 27/02/2023
TIME: 14:00
LOCAL: Meio virtual
TITLE:

Analysis of Latent Cryptococcal Infection and Cryptococcus neoformans Dormant Cell Reactivation Associated with Macrophage Cellular Metabolism

KEY WORDS:

Cryptococcus neoformans, dormancy, metabolism, macrophage, reactivation

PAGES: 104
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

The ability to remain dormant or in a latent state is an adaptation observed in several cells and organisms, in which cells reduce their metabolism, transcription and translation, to remain alive in conditions not ideal for their growth. Thus, they resume active growth when the environment returns to favorable conditions. Such adaptation is also observed in Cryptococcus neoformans yeasts, the fungus that causes cryptococcal meningitis in immunocompromised patients. In this work we analyze the mechanisms involved in the reactivation of C. neoformans during macrophage infection and whether yeasts induce changes in the cellular metabolism of this phagocytes. Furthermore, we infected immunosuppressed and immunocompetent WT and KO mice to analyze whether the presence of IFN-γ or NO prevented C. neoformans reactivation also in an in vivo context, as this pattern was previously demonstrated in vitro by our research group. We observed that it is not necessary for the fungus to have phagocytosis for it to reactivate, with only contact with extracellular compounds released by macrophages being sufficient, although yeasts reactivate at more expressive rates when there is phagocytosis. We also observed that the extracellular vesicles released by macrophages are important in this process of fungal reactivation. Furthermore, we observed that our model of dormant C. neoformans (DCn) was only able to reactivate in mice immunosuppressed with dexamethasone (C57Bl/6) deficient in IFN-γproduction or in immunodeficient mice (Balb/c nude), in which the fungus maintained its virulence and migrated to the CNS, where it resumed its proliferation. For analysis of cellular metabolism, we infected macrophages with DCn, Stat and heat-killed (HK) + 1% Stat for 24h. Next, we analyzed mitochondrial mass, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, glucose and fatty acid uptake by flow cytometry. Furthermore, we analyzed the respiratory profile of infected cells by oximetry and performed RT-qPCR to quantify important genes in cell metabolism. We observed that DCn doesn’t significantly alter most of the aspects analyzed regarding mitochondrial metabolism, characterizing a virulence strategy to prevent activation of the immune system, favoring its survival in the phagolysosome. However, all fungi induced extracellular acidification, indicating an increase in the performance of glycolysis by macrophages, while Stat prevents the increase in glucose uptake induced by treatment with LPS and IFN-𝛾, possibly representing an active strategy to avoid pro-inflammatory activation. Interestingly, DCn induces a greater uptake of fatty acids by macrophages pre-treated with LPS and IFN-𝛾, despite reducing the ability and dependence of macrophages to use this energy source. This demonstrates the preference of DCn for the use of lipids as an energy source and the ability to modulate the metabolism of the host macrophage in its favor. Finally, DCn modulated the expression of genes related to glucose and fatty acid metabolism, although to a lesser extent than Stat, showing that for phenotypic expression, a longer infection time should be necessary.

BANKING MEMBERS:
Externo à Instituição - Milton Adriano Pelli de Oliveira - UFG
Presidente - 2223020 - ANAMELIA LORENZETTI BOCCA
Interna - 1207115 - ILDINETE SILVA PEREIRA
Externa ao Programa - 3088161 - JULIANA LOTT DE CARVALHO
Externo à Instituição - PEDRO MANOEL MENDES DE MORAES VIEIRA - UNICAMP