The endosomal sorting complex required for transport machinery influences haem uptake and capsule elaboration in Cryptococcus neoformans

Abstract

Summary Iron availability is a key determinant of virulence in the pathogenic fungus Cryptococcus neoformans. Previous work revealed that the ESCRT (endosomal sorting complex required for transport) protein Vps23 functions in iron acquisition, capsule formation and virulence. Here, we further characterized the ESCRT machinery to demonstrate that defects in the ESCRT-II and III complexes caused reduced capsule attachment, impaired growth on haem and resistance to non-iron metalloprotoporphyrins. The ESCRT mutants shared several phenotypes with a mutant lacking the pH-response regulator Rim101 and, in other fungi, the ESCRT machinery is known to activate Rim101 via proteolytic cleavage. We therefore expressed a truncated and activated version of Rim101 in the ESCRT mutants and found that this allele restored capsule formation but not growth on haem, thus suggesting a Rim101-independent contribution to haem uptake. We also demonstrated that the ESCRT machinery acts downstream of the cAMP/protein kinase A pathway to influence capsule elaboration. Defects in the ESCRT components also attenuated virulence in macrophage survival assays and a mouse model of cryptococcosis to a greater extent than reported for loss of Rim101. Overall, these results indicate that the ESCRT complexes function in capsule elaboration, haem uptake and virulence via Rim101-dependent and independent mechanisms.

Iron acquisition in fungal pathogens of humans

“…88 An additional study showed that other ESCRT proteins, specifically Vps22 (ESCRT-II) and Snf7/ Vps20 (ESCRT-III), also contribute to heme use in C. neoformans . 89 Similar to C. albicans , a putative hemophore, Cig1, has also been described in C. neoformans that is involved in heme binding, uptake and virulence. 90 The Cig1 protein does not contain a CFEM domain and therefore must interact with heme via a different mechanism than the C. albicans proteins.…”

The cAMP/protein kinase A signaling pathway in pathogenic basidiomycete fungi: Connections with iron homeostasis

“…Reduced transcript levels of these genes were well correlated with the growth deficiency of the rim101 Δ mutant in low-iron media (O’Meara et al ., 2010). Furthermore, a series of recent studies showed that both the cig1 Δ mutant and the rim101 Δ mutant were defective in heme uptake and that Rim101 plays a role in heme utilization via the ESCRT machinery, although evidence for a Rim101-independent heme uptake pathway was also obtained (Cadieux et al ., 2013; Hu et al ., 2015). …”

“…A screen for functions needed for growth on heme revealed that proteins of the endosomal sorting complex required for transport (ESCRT) participate in iron acquisition, as well as in capsule formation and virulence (Hu et al ., 2013, 2015). Interestingly, ESCRT functions are also required in fungi for the activation of Rim101 (Selvig and Alspaugh, 2011; Ost et al ., 2015).…”

“…Interestingly, ESCRT functions are also required in fungi for the activation of Rim101 (Selvig and Alspaugh, 2011; Ost et al ., 2015). In this context, mutants defective in ESCRT functions share a number of phenotypes with a rim101 Δ mutant including impaired growth on heme as the sole iron source and a reduced capsule size (Hu et al ., 2013, 2015). Most of the impact of the ESCRT machinery on capsule occurs through participation with PKA in the activation of Rim101.…”

“…Also, the cAMP/PKA pathway regulates the expression of genes for capsule production (Pukkila-Worley et al ., 2005). For iron acquisition, the ESCRT machinery make a contribution both through an influence of Rim101 activation and, independently, perhaps through endocytosis of heme (Hu et al ., 2015). …”