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

• Grant number: 15/04400-9
• Support Opportunities: Regular Research Grants
• Start date: December 01, 2015
• End date: November 30, 2018
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Marcelo Afonso Vallim
• Grantee: Marcelo Afonso Vallim
• Host Institution: Instituto de Ciências Ambientais, Químicas e Farmacêuticas (ICAQF). Universidade Federal de São Paulo (UNIFESP). Campus Diadema. Diadema , SP, Brazil
• Associated research 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

(11)

(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)

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. (16/14542-8, 16/23890-0, 15/04400-9)

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; et al. Amino Acid Permeases and Virulence in Cryptococcus neoformans. PLoS One, v. 11, n. 10, OCT 3 2016. (15/04400-9)

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. (15/04400-9, 16/14542-8)

OLIVEIRA, LIDIANE; FERRARINI, MARCIO; DOS SANTOS, ALEXANDRE PAES; VARELA, MARINA THEMOTEO; SANTOS CORREA, ISABELA TERESA; TEMPONE, ANDRE GUSTAVO; MELHEM, MARCIA S. C.; VALLIM, MARCELO A.; FERNANDES, JOAO PAULO S.; PASCON, RENATA C.. Coumaric acid analogues inhibit growth and melanin biosynthesis in Cryptococcus neoformans and potentialize amphotericin B antifungal activity. European Journal of Pharmaceutical Sciences, v. 153, OCT 1 2020. (18/10279-6, 18/03918-2, 15/04400-9, 16/25028-3, 16/14542-8)

ROBERTO, THIAGO NUNES; LIMA, RICARDO FERREIRA; PASCON, RENATA CASTIGLIONI; IDNURM, ALEXANDER; VALLIM, MARCELO AFONSO. Biological functions of the autophagy-related proteins Atg4 and Atg8 in Cryptococcus neoformans. PLoS One, v. 15, n. 4, APR 6 2020. (16/50185-5, 16/14542-8, 15/04400-9)

COURROL, LILIA CORONATO; VALLIM, MARCELO AFONSO. Spectroscopic Analysis of Chicken Meat Contaminated with E. coli, Salmonella, and Campylobacter. FOOD ANALYTICAL METHODS, v. 14, n. 3, p. 512-524, MAR 2021. (17/23686-6, 15/04400-9)

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. (16/14542-8, 15/04400-9)

COURROL, LILIA CORONATO; VALLIM, MARCELO AFONSO. Characterization of chicken meat contaminated with Salmonella by fluorescence spectroscopy. SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY, v. 261, NOV 15 2021. (15/04400-9, 17/23686-6)

VALLIM, MARCELO A.; FELIZARDO, GABRIELLE; PASCON, RENATA C.. Role of Pep4 protein in Cryptococcus neoformans cell survival and virulence factors. Medical Mycology, v. 56, p. 1-pg., 2018-06-01. (15/04400-9, 16/14542-8)

DA SILVA, JEYSON PEREIRA; MENEGHINI, MARIANA REIS; SANTOS, RONALDO SILVA; ALVES, VERONICA LIRA; MARTHO, KEVIN FELIPE DA CRUZ; VALLIM, MARCELO AFONSO; PASCON, RENATA CASTIGLIONI. ATP sulfurylase atypical leucine zipper interacts with Cys3 and calcineurin A in the regulation of sulfur amino acid biosynthesis in Cryptococcus neoformans. SCIENTIFIC REPORTS, v. 13, n. 1, p. 16-pg., 2023-07-20. (18/20390-1, 20/01000-8, 21/06290-7, 19/12051-5, 21/00725-1, 15/04400-9)

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. (16/14542-8, 18/20390-1, 17/20106-9, 15/04400-9)