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

Abstract

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.

Autophagy in Paracoccidioides brasiliensis under normal mycelia to yeast transition and under selective nutrient deprivation

“…cerevisiae . This matches the number identified by Gontijo et al [ 33 ] for Candida albicans and Aspergillus fumigatus ( Table 1 ).…”

“…Although the autophagic process is ubiquitous among eukaryotes, the molecular elements that control them in different organisms have already been quite variable, even within Fungi kingdom. For example, in Cryptococcus neoformans , 21 genes with homology to the autophagy-related (ATG) genes of Saccharomyces cerevisiae [ 33 ], which has 34 ATG genes, are known to date [ 73 – 76 ].…”

“…brasiliensis , apoptosis and autophagy-like mechanisms have recently been implicated in a cyclopalladated 7a-mediated cell death [ 32 ]. Gontijo et al [ 33 ] showed that 21 of 34 autophagy-related genes described in Saccharomyces cerevisiae are present in a basidiomycete, Cryptococcus neoformans , and in Candida albicans and Aspergillus fumigatus , both ascomycetes. Therefore, there is an increasing evidence that autophagy is indeed ubiquitous in the Fungi Kingdom.…”

Conserved Autophagy Pathway Contributes to Stress Tolerance and Virulence and Differentially Controls Autophagic Flux Upon Nutrient Starvation in Cryptococcus neoformans

“…However, there are some differences between our study and that of Ding et al The atg7 and atg8 mutants resulted in slight sensitivity to high temperatures, and these four atg mutants led to smaller capsules (Figure 4 and Supplementary Figure S4). As studies have pointed out that heat stress induces autophagy to insure protein homeostasis at high temperatures (Zhao et al, 2009), deletion of the heat shock transcription factor 1 (HSF1) increases basal autophagy levels (Dokladny et al, 2013), moreover, the transcriptions of ATG genes in C. neoformans are significant increase when transferred from 30 to 37 • C in YPD medium (Gontijo et al, 2017), we speculate that autophagy can also be induced by high temperature in C. neoformans. Atg7 and Atg8 are the core proteins in autophagy process, and they caused slight growth defects at high temperatures which was more significant at 38 • C (data not show).…”

ATGGenes Influence the Virulence of Cryptococcus neoformans through Contributions beyond Core Autophagy Functions

“…Each candidate ATG gene was then tested by reciprocal BLASTp against the S. cerevisiae S288c genome to ensure that the most similar sequence was that of the S. cerevisiae inquiry gene. The ATG1, ATG7, and ATG8 genes from C. neoformans were also previously identified by other groups (17)(18)(19). Single gene deletion mutants were constructed in C. neoformans var.…”