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The role of autophagy in Cryptococcus neorformas high temperature (37°C) growth and virulence

Grant number: 15/04400-9
Support type:Regular Research Grants
Duration: December 01, 2015 - November 30, 2018
Field of knowledge:Biological Sciences - Genetics
Principal Investigator:Marcelo Afonso Vallim
Grantee:Marcelo Afonso Vallim
Home Institution: Instituto de Ciências Ambientais, Químicas e Farmacêuticas (ICAQF). Universidade Federal de São Paulo (UNIFESP). Campus Diadema. Diadema , SP, Brazil
Associated grant(s):16/50185-5 - The role of autophagy on the pathogenicity of Cryptococcus neoformans and as a potential drug target, AP.R SPRINT

Abstract

Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37°C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system (AMT) to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. (Gontijo et al., 2014). Among several mutants unable to grow at 37°C, we explored one bearing an interruption in the APE4 gene. This gene encodes an aspartyl aminopeptidase that in Saccharomyces cerevisiae is involved in Cvt (cytoplasm to vacuole targeting) and autophagy. The C. neoformans ape4 mutant is not able to grow at 37°C and it is not virulent in animal model (mouse). In C. neroformans the Cvt and autophagy processes are poorly understood. Among five proteins involved in the S. cerevisiae Cvt and autophagy processess (APE1, APE4, AMS1. ATG19 and ATG8), only three are apparently encoded by C. neoformans (APE4, AMS1 and ATG8). In order to get insights into the autophagic mechanism in C. neoformans and its role on pathogenesis the aims of this project are to delete each of these genes from this yeast genome and check the mutants phenotypes associated to nutritional status, their role on high temperature growth and virulence. Also, in this project we will study the sub cellular localization and transit of the respective proteins by GFP translational fusions (green fluorescent protein). Moreover, a proteomics and Yeast two hybrid system will be used to search for proteins that interact with APE4 and AMS1 in order to place them correctly with the ATG8-membrane bound during the vesicle formation. The broad goal of this project is to elucidate part of the autophagic mechanism in C. neoformans, which seems to be diverse from S. cerevisiae and associate this knowledge to virulence and pathogenesis, in order to propose novel therapies to this clinically important pathogen. (AU)

Scientific publications (5)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MARTHO, KEVIN FELIPE; BRUSTOLINI, OTAVIO J. B.; VASCONCELOS, ANA TEREZA; VALLIM, MARCELO A.; PASCON, RENATA C. The Glycerol Phosphatase Gpp2: A Link to Osmotic Stress, Sulfur Assimilation and Virulence in Cryptococcus neoformans. FRONTIERS IN MICROBIOLOGY, v. 10, NOV 26 2019. Web of Science Citations: 0.
DE MELO, AMANDA TEIXEIRA; MARTHO, KEVIN FELIPE; ROBERTO, THIAGO NUNES; NISHIDUKA, ERIKA S.; MACHADO JR, JOEL; BRUSTOLINI, OTAVIO J. B.; TASHIMA, ALEXANDRE K.; TEREZAVASCONCELOS, ANA; VALLIM, MARCELO A.; PASCON, RENATA C. The regulation of the sulfur amino acid biosynthetic pathway in Cryptococcus neoformans: the relationship of Cys3, Calcineurin, and Gpp2 phosphatases. SCIENTIFIC REPORTS, v. 9, AUG 15 2019. Web of Science Citations: 2.
CALVETE, CRISLAINE LAMBIASE; MARTHO, KEVIN FELIPE; FELIZARDO, GABRIELLE; PAES, ALEXANDRE; NUNES, JOAO MIGUEL; FERREIRA, CAMILA OLIVEIRA; VALLIM, MARCELO A.; PASCON, RENATA C. Amino acid permeases in Cryptococcus neoformans are required for high temperature growth and virulence; and are regulated by Ras signaling. PLoS One, v. 14, n. 1 JAN 25 2019. Web of Science Citations: 4.
GONTIJO, FABIANO DE ASSIS; DE MELO, AMANDA TEIXEIRA; PASCON, RENATA C.; FERNANDES, LARISSA; PAES, HUGO COSTA; ALSPAUGH, J. ANDREW; VALLIM, MARCELO A. The role of Aspartyl aminopeptidase (Ape4) in Cryptococcus neoformans virulence and authophagy. PLoS One, v. 12, n. 5 MAY 25 2017. Web of Science Citations: 4.
CRUZ MARTHO, KEVIN FELIPE; DE MELO, AMANDA TEIXEIRA; FERNANDES TAKAHASHI, JULIANA POSSATO; GUERRA, JULIANA MARIOTTI; DA SILVA SANTOS, DAYANE CRISTINA; PURISCO, SONIA UEDA; CARVALHO MELHEM, MARCIA DE SOUZA; FAZIOLI, RAQUEL DOS ANJOS; PHANORD, CLERLUNE; SARTORELLI, PATRICIA; VALLIM, MARCELO A.; PASCON, RENATA C. Amino Acid Permeases and Virulence in Cryptococcus neoformans. PLoS One, v. 11, n. 10 OCT 3 2016. Web of Science Citations: 6.

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