XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:109-1

Poster (Painel)

109-1	Low levels of reactive oxygen and nitrogen species (ROS/RNS) induce proliferation of Paracoccidioides brasiliensis
Authors:	Juliana Terzi Maricato (UNIFESP - Universidade Federal de São Paulo) ; Ana Eliza Coronel Janú Haniu (UNIFESP - Universidade Federal de São Paulo) ; Patricia Xander Batista (UNIFESP-DIADEMA - Universidade Federal de São Paulo) ; Wagner Luiz Batista (UNIFESP - Universidade Federal de São PauloUNIFESP-DIADEMA - Universidade Federal de São Paulo)

Abstract

Paracoccidioides brasiliensis acts as a facultative intracellular pathogen able to survive and replicate inside macrophages not activated. However, macrophages efficiently activated by cytokines are able to kill the fungus, probably due to intense oxidative stress and hostile environment inside activated macrophages. Among the molecules that exert fungicidal action in the phagosome are hydrogen peroxide (H2O2), nitric oxide (NO) and its derivatives. Nevertheless, proteins with antioxidant functions have been identified in pathogenic fungi which, in some cases, may protect fungal cells against oxidative stress generated by phagocytic cells. In this work we aimed to determine the concentrations of nitrosative and oxidative agents that induce death or survival of P. brasiliensis. Briefly, P. brasiliensis yeast cells were expanded for 7 days at 36°C in liquid modified YPD under agitation. Cells were pelleted by centrifugation and rinsed three times with an excess volume of phosphate-buffered saline (PBS). Washed cells were suspended in F12 medium and incubated for 24 h under agitation. Cells were exposed to different concentration of H2O2 or Nitrite (NO release in cultures mildly acidified) for 5 h. The number of viable fungi after stimulation was determined by CFU counts. We observed that high concentrations of H2O2 or Nitrite (10 mM and 1μM, respectively) induced fungal death (30±1.8 and 47±11 CFU) when compared with control without treatment (131.6±8.6 and 135±7.8 CFU). Intermediate dilutions of the nitrosative and oxidative agents led to P. brasiliensis survive (127±16.6 and 109±13 CFU). Surprisingly, low concentrations of H2O2 or Nitrite (0.1 mM H2O2 or 100-250 nM nitrite) stimulated the proliferation of P. brasiliensis (228.5±9.6 and 178±16.5 CFU) when compared with no treated yeast cells. Western blot analyses showed an increase in the profile of phosphorylation and nitration proteins, after stimulation with different concentrations of H2O2 and nitrite. Phosphoproteomics studies will be performed to understand the signaling pathways committed to survival, proliferation or death of the fungus against oxidative and nitrosative stimuli. Financial support: CNPq

Keyword: nitric oxide, phosphorylation, reactive oxygen species

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