Role of the Apt1 Protein in Polysaccharide Secretion by Cryptococcus neoformans

Abstract

e Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1⌬ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1⌬ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection.

The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans

“…Our previous studies also demonstrated that aptl Δ cells had defective GXM secretion during in vitro regular growth, in the presence of host cells and in vivo, which is in agreement with the observation of aberrant Golgi morphology [26]. Since GXM synthesis is Golgi-associated [19], we asked whether APT1 deletion would impact intracellular GXM detection.…”

“…Previous studies demonstrated that deletion of APT1 in C. neoformans did not affect PS exposition and lipid translocation across the plasma membrane [26]. Instead, aptlΔ cells showed an abnormal distribution of Golgi membranes and a defect in membrane trafficking during endocytosis [26,37], suggesting intracellular localization of the protein encoded by APT1 .…”

“…Instead, aptlΔ cells showed an abnormal distribution of Golgi membranes and a defect in membrane trafficking during endocytosis [26,37], suggesting intracellular localization of the protein encoded by APT1 . To determine whether organelle membranes were affected in aptlΔ cells, ultrastructural aspects were analyzed by transmission electron microscopy (TEM) after cultivation of C. neoformans for 48 or 72 h. Compared to WT and complemented cells, the aptlΔ mutant showed abnormal vacuolar morphology with irregular structural patterns and aberrant endomembrane folding after 48 h of growth (Fig.…”

“…Most of the extracellular molecules in fungi are secreted through non-canonical mechanisms that are regulated at multiple levels. Regulators of non-canonical secretion in fungi include Golgi reassembly and stacking protein (Grasp) [24], sucrose non-fermenting protein 7 (Snf7) [25], and the putative aminophospholipid translocase (flippase) Aptl [26]. …”

“…Since this asymmetry is fundamental for both intracellular and plasma membrane architecture [28], flippases directly impact endocytic and secretory pathways [29–36]. In C. neoformans, deletion of APT1 , the gene coding for a putative flippase, resulted in disturbed GXM secretion in vitro and in vivo, increased cryptococcal susceptibility to antifungal agents, reduced fungal survival inside macrophages and attenuated fungal virulence in a murine model of infection [26,37]. Similarly, deletion of cryptococcal CDC50 (the gene encoding a non-catalytic subunit of flippases) affected iron acquisition, GXM secretion, susceptibility to macrophages and virulence in a mice model of cryptococcosis [38,39].…”