Gontijo FA et al. (2017), The role of Aspartyl aminopeptidase (Ape4) in C...

ECB-ART-45487

PLoS One 2017 May 08;125:e0177461. doi: 10.1371/journal.pone.0177461.

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

Gontijo FA , de Melo AT , Pascon RC , Fernandes L , Paes HC , Alspaugh JA , Vallim MA .

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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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Genes referenced: LOC100888042 LOC105444942 LOC115919910 LOC590902 LOC591473 pelp1 stk36

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References [+] :

Alspaugh, RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans. 2000, Pubmed


	Fig 2. Ape4 is required for C. neoformans capsule production.The wild type (KN99α), ape4, and ape4+APE4 strains where inoculated in CO2-independent medium and capsule production was evaluated after 72 hours of incubation at 30°C and 37°C. Scale bar represents 10 μm.
	Fig 3. Reduced phospholipase activity in the ape4 mutant is demonstrated by reduced precipitation zone on egg yolk agar medium compared to control strains.(The scale represents 1 cm).
	Fig 4. Cell wall integrity and osmotic stress were altered in the ape4 mutant compared to wild type (KN99α) and reconstituted (ape4+APE4) strains.The cell wall integrity was tested on YPD plates at 30°C, supplemented with 0.5% Congo Red (A) and the effect of osmotic stress was assessed on YPD plates supplemented with 0.75M and 1.5 M of NaCl (B) and KCl (C).


	Fig 7. Ape4 is required for full C. neoformans virulence in a murine model.The wild type (KN99α), mutant (ape4) and the reconstituted (ape4+APE4) strains were inoculated by nasal inhalation in C57BL/6 mice. Survival was followed during the course of the infection up to 40 days. p value was <0.001 for the comparisons between ape4, and wild type and ape4+APE4.

	Fig 9. Influence of different nitrogen sources on C. neoformans growth.The wild type (KN99α), mutant (ape4) and the reconstituted (ape4+APE4) strains were cultured in rich medium (YPD) (A), synthetic dextrose (SD) supplemented with ammonium sulfate and amino acids (B), L-proline (C) or uric acid (D). All assays were performed at 30°C in triplicates up to 72 hours. Values were statistically validated by ANOVA (Bonferroni post-test, p<0.05 GraphPad Prism program 5).

	Fig 11. GFP-Ape4 sub-cellular localization.The C. neoformans Ape4 protein was fused to GFP (GFP-Ape4) to show its cellular localization during the yeast growth (KN990α) in rich (YPD) and defined medium without nitrogen source (SD—NS) at 30°C and 37°C. (A) Yeast cells images were captured by Differential Interference Contrast (DIC) microscopy, (B) FM4-64 demonstrates endocytic vesicles, and (C) epifluorescent microscopy demonstrates cell localization of GFP-Ape4. (D) Merged images of FM4-64 and GFP-Ape4. Arrows indicate FM4-64 stained vesicles, GFP labeled Ape4 and co-localization of both. All images were processed using the Zen 2011 software (Zeiss). Scale bar represent 5 μm.