The role of Aspartyl aminopeptidase (Ape4) in Cryptococcus neoformans virulence and authophagy

PLOS ONE, Dec 2019

In order to survive and cause disease, microbial pathogens must be able to proliferate at the temperature of their infected host. We identified novel microbial features associated with thermotolerance in the opportunistic fungal pathogen Cryptococcus neoformans using a random insertional mutagenesis strategy, screening for mutants with defective growth at 37°C. Among several thermosensitive mutants, we identified one bearing a disruption in a gene predicted to encode the Ape4 aspartyl aminopeptidase protein. Ape4 metalloproteases in other fungi, including Saccharomyces cerevisiae, are activated by nitrogen starvation, and they are required for autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway. However, none have been previously associated with altered growth at elevated temperatures. We demonstrated that the C. neoformans ape4 mutant does not grow at 37°C, and it also has defects in the expression of important virulence factors such as phospholipase production and capsule formation. C. neoformans Ape4 activity was required for this facultative intracellular pathogen to survive within macrophages, as well as for virulence in an animal model of cryptococcal infection. Similar to S. cerevisiae Ape4, the C. neoformans GFP-Ape4 fusion protein co-localized with intracytoplasmic vesicles during nitrogen depletion. APE4 expression was also induced by the combination of nutrient and thermal stress. Together these results suggest that autophagy is an important cellular process for this microbial pathogen to survive within the environment of the infected host.

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The role of Aspartyl aminopeptidase (Ape4) in Cryptococcus neoformans virulence and authophagy

May The role of Aspartyl aminopeptidase (Ape4) in Cryptococcus neoformans virulence and authophagy Fabiano de Assis Gontijo 1 2 Amanda Teixeira de Melo 1 2 Renata C. Pascon 1 2 Larissa Fernandes 0 2 Hugo Costa Paes 2 J. Andrew Alspaugh 2 Marcelo A. Vallim 1 2 0 Universidade de BrasÂõlia, Faculdade de Ceilaà ndia , Ceil aÃndia, DF , Brazil , 3 Universidade de BrasÂõlia, Faculdade de Medicina , BrasÂõlia, DF , Brazil , 4 Duke University School of Medicine, Department of Medicine , Durham, NC , United States of America 1 Universidade Federal de São Paulo, Departamento de Ciências Biolo gicas , Diadema, SP , Brazil 2 Editor: Kirsten Nielsen, University of Minnesota , UNITED STATES In order to survive and cause disease, microbial pathogens must be able to proliferate at the temperature of their infected host. We identified novel microbial features associated with thermotolerance in the opportunistic fungal pathogen Cryptococcus neoformans using a random insertional mutagenesis strategy, screening for mutants with defective growth at 37ÊC. Among several thermosensitive mutants, we identified one bearing a disruption in a gene predicted to encode the Ape4 aspartyl aminopeptidase protein. Ape4 metalloproteases in other fungi, including Saccharomyces cerevisiae, are activated by nitrogen starvation, and they are required for autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway. However, none have been previously associated with altered growth at elevated temperatures. We demonstrated that the C. neoformans ape4 mutant does not grow at 37ÊC, and it also has defects in the expression of important virulence factors such as phospholipase production and capsule formation. C. neoformans Ape4 activity was required for this facultative intracellular pathogen to survive within macrophages, as well as for virulence in an animal model of cryptococcal infection. Similar to S. cerevisiae Ape4, the C. neoformans GFP-Ape4 fusion protein co-localized with intracytoplasmic vesicles during nitrogen depletion. APE4 expression was also induced by the combination of nutrient and thermal stress. Together these results suggest that autophagy is an important cellular process for this microbial pathogen to survive within the environment of the infected host. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funded by FAPESP grants (2007/505363 and 2015/04400-9) to MAV, (2011/51298-4 and 2016/14542-8) to RCP and NIH grants (AI050128 and AI074677) to JAA. Competing interests: The authors have declared that no competing interests exist. Introduction C. neoformans is a fungal pathogen with worldwide distribution; it is found in the environment on rotting wood and is often associated with bird excreta. This yeast can cause fatal respiratory and neurological infections, especially in immunocompromised populations. Recent surveys estimate more than 500,000 deaths from C. neoformans every year, especially in patients with AIDS and other diseases that compromise the immune system [ 1 ]. The immunocompromised population has increased worldwide due to many reasons and among them the AIDS pandemic and a growing number of transplant patients. Together, these factors have transformed this yeast into an important pathogen [ 2, 3 ]. The options for antifungal therapies for cryptococcosis are limited. The most commonly used drugs for treatment are polyenes (amphotericin B-based drugs), antimetabolites (flucytosine) and azoles [ 4 ]. However, drug toxicity and acquired resistance are still important issues in treating this type of infection [ 5, 6 ]. In order to contribute to the development of new antifungal therapies, our laboratory and others have been searching for genetic and physiological traits that can affect the virulence factors which allow C. neoformans to survive and multiply in the host. Among these virulence factors, the most well studied are the production of polysaccharide capsule, melanin, phospholipase and growth at 37ÊC [7±10]. The ability to grow at human physiological temperature is very important for virulence, and it is a trait controlled by a number of genes [11±15]. To identify additional genetic elements involved in high temperature growth, we screened a random insertion mutant library, induced by the Agrobacterium tumefaciens gene delivery system, in order to identify mutants unable to growth at 37ÊC. Among several thermo-sensitive mutants, we explored one bearing an insertion in the aspartyl aminopeptidase (APE4) gene. In S. cerevisiae the protein aspartyl aminopeptidase (Ape4) plays a role during autophagy [16]. In eukaryotes, autophagy is defined as a group of processes that occurs inside the vacuoles leading to degradation of cytoplasmic components such as parts of the cytosol, macromolecular complexes and organelles. This process is important to maintain the balance between catabolism and anabolism, allowing the cell to (...truncated)


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Fabiano de Assis Gontijo, Amanda Teixeira de Melo, Renata C. Pascon, Larissa Fernandes, Hugo Costa Paes, J. Andrew Alspaugh, Marcelo A. Vallim. The role of Aspartyl aminopeptidase (Ape4) in Cryptococcus neoformans virulence and authophagy, PLOS ONE, 2017, Volume 12, Issue 5, DOI: 10.1371/journal.pone.0177461