Role of clathrin-mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans

Abstract

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life‐threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non‐iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin‐mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin‐containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.

Iron Assimilation during Emerging Infections Caused by Opportunistic Fungi with emphasis on Mucorales and the Development of Antifungal Resistance

“…Mutants of LAS17 were unable to utilise iron from haem and showed increased survival in preliminary in vivo mouse models. As such, these results indicate that the CME pathway may have an important role in haem utilisation, growth and virulence of C. neoformans in vivo [100]. Although haem/haemoglobin utilisation has yet to be demonstrated in Mucorales, putative haem oxygenase genes have been identified in R. arrhizus and L corymbifera [101,102].…”

“…Other proteins involved in haem utilisation include Vps22, and Vps20/Snf7, which are components of the cytosolic protein complexes ESCRT-II and ESCRT-III, respectively [96,98,99]. Recently, Bairwa et al, 2019 confirmed additional proteins involved in the clathrin-mediated endocytosis (CME) of haem/haemoglobin by C. neoformans [100]. Their work strongly suggested that the clathrin heavy chain (Chc1) protein (a component of CME), may have a central role in the uptake and trafficking of haem/haemoglobin.…”

“…This was demonstrated by the impaired ability of strains lacking the CHC1 gene to internalise haemoglobin. Additionally, CHC1 mutants were unable to grow in medium containing haemin or haemoglobin as the sole iron source [100]. Furthermore, the loss of CHC1 abolishes growth at 37 • C, which is a key virulence determinant for C. neoformans infection.…”

A Cytoplasmic Heme Sensor Illuminates the Impacts of Mitochondrial and Vacuolar Functions and Oxidative Stress on Heme-Iron Homeostasis in Cryptococcus neoformans

“…Most iron in vertebrate hosts is present in heme, and heme-containing proteins such as hemoglobin and the haptoglobin-hemoglobin complex are potentially abundant sources of iron for fungal pathogens during disease (18). The mechanisms for acquisition and use of heme-iron sources are starting to be discovered in some fungi, including Aspergillus fumigatus, Candida albicans, Candida glabrata, C. neoformans, Schizosaccharomyces pombe, and Paracoccidioides brasiliensis (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). For example, studies in C. neoformans revealed important roles in heme use for endocytosis and endomembrane trafficking machinery, including the ESCRT proteins Vps22, Vps23, and Snf7, and proteins for clathrin-mediated endocytosis such as Chc1 (clathrin heavy chain 1), Las17 (a nucleation᎑promoting factor for actin assembly), and Rvs161/Rvs167 (vesicle scission proteins; amphiphysins) (25,26,28).…”

“…Interestingly, the CFEM hemophore proteins in C. albicans constitute a relay network that has recently been shown to mediate heme acquisition from human serum albumin (36). For C. neoformans, defects in cellular trafficking for heme uptake result in attenuated virulence, highlighting the importance of heme iron in pathogenesis (25,26,35). Many of the iron acquisition functions in C. neoformans are regulated by iron regulatory proteins such as the monothiol glutaredoxin Grx4 and the transcription factors Cir1 and HapX (37)(38)(39).…”