Brigitte Cadieux

Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box

Cryptococcus neoformans is generally considered to be an opportunistic fungal pathogen because of its tendency to infect immunocompromised individuals, particularly those infected with HIV. However, this view has been challenged by the recent discovery of specialized interactions between the fungus and its mammalian hosts, and by the emergence of the related species Cryptococcus gattii as a primary pathogen of immunocompetent populations. In this Review, we highlight features of cryptococcal pathogens that reveal their adaptation to the mammalian environment. These features include not only remarkably sophisticated interactions with phagocytic cells to promote intracellular survival, dissemination to the central nervous system and escape, but also surprising morphological and genomic adaptations such as the formation of polyploid giant cells in the lung.

Adaptation of Cryptococcus neoformans to mammalian hosts: integrated regulation of metabolism and virulence.

ABSTRACT The basidiomycete fungus Cryptococcus neoformans infects humans via inhalation of desiccated yeast cells or spores from the environment. In the absence of effective immune containment, the initial pulmonary infection often spreads to the central nervous system to result in meningoencephalitis. The fungus must therefore make the transition from the environment to different mammalian niches that include the intracellular locale of phagocytic cells and extracellular sites in the lung, bloodstream, and central nervous system. Recent studies provide insights into mechanisms of adaptation during this transition that include the expression of antiphagocytic functions, the remodeling of central carbon metabolism, the expression of specific nutrient acquisition systems, and the response to hypoxia. Specific transcription factors regulate these functions as well as the expression of one or more of the major known virulence factors of C. neoformans. Therefore, virulence factor expression is to a large extent embedded in the regulation of a variety of functions needed for growth in mammalian hosts. In this regard, the complex integration of these processes is reminiscent of the master regulators of virulence in bacterial pathogens.

The Mannoprotein Cig1 supports iron acquisition from heme and virulence in the pathogenic fungus Cryptococcus neoformans.

Iron acquisition is critical for virulence of the human pathogenic fungus Cryptococcus neoformans. The cryptococcal transcript for the extracellular mannoprotein Cig1 is highly regulated by iron and abundant in iron-starved cells, suggesting a role in iron acquisition. Indeed, loss of Cig1 resulted in delayed growth on heme at physiological pH. Expression of CIG1 is regulated by the pH-responsive transcription factor Rim101, and loss of Rim101 also impaired growth on heme. A cig1Δ mutant was less susceptible than the wild-type strain to noniron metalloporphyrins, further indicating a role for Cig1 in heme uptake. Recombinant Cig1 exhibited the absorbance spectrum of a heme-binding protein upon heme titration, and Cig1 may therefore function as a hemophore at the cell surface. Cig1 contributed to virulence in a mouse model of cryptococcosis but only in a mutant that also lacked the high-affinity iron uptake system. Overall, Cig1-mediated heme uptake is a potential therapeutic target in C. neoformans.

Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme, for Capsule Formation, and for Virulence

ABSTRACT Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans.

A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs.

The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD(+)/NADH and down-regulation of Fe-S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C. neoformans.

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

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.

Pathogenic yeasts deploy cell surface receptors to acquire iron in vertebrate hosts.

Brown et al. (2012) [1] recently highlighted the growing threat that fungal pathogens pose for humans, as well as the pressing need for additional antifungal drugs and efficacious vaccines. In this context, the process of iron acquisition presents compelling opportunities to prevent or treat fungal diseases because iron is an essential nutrient for pathogen proliferation in vertebrate hosts. Fungi and other pathogens must deploy competitive uptake mechanisms to steal iron from host sources and overcome the iron sequestration associated with nutritional immunity [2]. These extracellular and surface uptake functions may provide readily accessible targets for drugs to block iron uptake, and iron transporters might also be exploited to introduce antifungal agents into fungal cells [3], [4]. Additionally, extracellular or exposed transporters may be useful vaccine targets to block iron uptake and pathogen proliferation. Mechanisms of iron acquisition have been well characterized in many microbial pathogens, and information is rapidly accumulating for fungal pathogens [5]–[9]. Fungi generally acquire iron by several mechanisms including: 1) the production and uptake of siderophores; 2) the use of a ferroxidase-iron permease complex for high-affinity uptake; 3) the transport of ferrous iron; and 4) the acquisition of iron from heme and hemoglobin [7]–[9]. Reductases in the plasma membrane and secreted reductants facilitate the reduction of ferric iron to ferrous iron for high- or low-affinity uptake [7]–[9]. The exploration of these mechanisms in fungal pathogens has revealed intriguing connections between cell surface molecules (cell wall and secreted proteins, capsular polysaccharide, and biofilms) and functions for iron acquisition from vertebrate sources (Figure 1A, B). Here we highlight these connections for the pathogenic yeasts Candida albicans and Cryptococcus neoformans. We also discuss the extent to which the newly identified functions add depth to our understanding of iron acquisition by fungi and illustrate the complex integration of iron sensing and virulence. Figure 1 Cell surface functions for iron acquisition in C. albicans and C. neoformans. Cell Surface Proteins Link the Use of Ferritin, Heme, and Hemoglobin with Biofilm Formation in C. albicans The adhesin Als3 and the CFEM domain protein Rbt5 are two notable examples of proteins that link cell surface activities related to morphogenesis and/or biofilm formation with iron acquisition in C. albicans (Figure 1C). Als3 is a hypha-specific surface protein that functions as an adhesin for epithelial and endothelial cells, and that also mediates adherence to extracellular matrix proteins (reviewed in [10]). The ability of C. albicans to switch its morphology between yeast and hyphal forms is important for virulence, as is the ability of the fungus to form biofilms on implanted medical devices. Als3 contributes to virulence in mouse models of candidiasis, although the impact of the protein depends on the method of inoculation and the immune status of the host [10]. Expression of ALS3 is transcriptionally controlled in a complex manner by regulators of the yeast-hyphal transition, by the major regulator of biofilm formation Bcr1, and by the alkaline response transcription factor Rim101 [10]–[14]. Als3 is an interesting multifunctional protein because, in addition to binding to and provoking endocytosis of the fungus by host cells, it also plays a role in biofilm formation and it binds the host iron-storage protein ferritin. Specifically, Almeida et al. (2008) [15] found that C. albicans can use ferritin as an iron source at physiological pH, and that Als3 is required for this process. Ferritin is bound by hyphal cells that express Als3 (but not by yeast cells), and deletion of the ALS3 alleles eliminates both binding and growth on ferritin. The ferritin binding by hyphae is also observed when these cells invade epithelial cells in vitro, and Als3 is required for C. albicans to damage these cells [15]. Overall, Als3 functionally links biofilm formation, the yeast-hyphal transition, and the use of a specific host iron source. The second example of the connection between biofilm formation, morphology, and C. albicans iron acquisition involves Rbt5, an O-mannosylated, glycosylphosphatidylinositol (GPI)-anchored protein (Figure 1C) [16]–[18]. Rbt5 is located in the plasma membrane and the cell wall, the protein binds heme and hemoglobin, and deletion of RBT5 reduces the use of these host iron sources by C. albicans [16]–[18]. RBT5 expression is induced by iron limitation and negatively regulated by the Tup1 regulator of morphology [17], [19]. Related genes encoding CFEM domain proteins are present in C. albicans, and these include RBT51/PGA10, CSA1, CSA2, and PGA7. Rbt51 also participates in hemoglobin binding and, in fact, the protein was originally identified in a screen for C. albicans genes that allowed S. cerevisiae to use iron from hemoglobin [17]. Weissman et al. (2008) [18] exploited this property of Rbt51 to identify mutations in S. cerevisiae that blocked hemoglobin use. The identified functions included subunits of the vacuolar ATPase, components of the ESCRT system, HOPS complex proteins and t-SNAREs, and several other functions. Notably, the ESCRT and HOPS complexes, and the t-SNAREs, contribute to endocytosis. An examination of C. albicans mutants with defects in the corresponding genes confirmed that many of these functions played roles in iron use from hemoglobin (e.g., Vma11, Vps41, ESCRT complex components, Myo5). Overall, these studies revealed an endocytic pathway for heme/hemoglobin internalization and trafficking to the vacuole for processing. Similar to Als3, mutants lacking Rbt5, Rbt51, or the related protein Csa1 form thinner and more fragile biofilms with less extracellular matrix compared with the wild-type strain [20], [21]. These mutants also displayed changes in their cell surface as examined microscopically and by measurements of cell surface hydrophobicity. Interestingly, conserved transcriptional regulation by the biofilm regulator Bcr1 is observed for three CFEM genes in C. albicans (RBT5, PGA7, and CSA1) and for three genes (CFEM2, CFEM3, and CFEM6) in the related pathogen Candida parapsilosis [22]. However, these proteins have divergent roles in that the three CFEM proteins are not involved in biofilm formation in C. parapsilosis [22].

The ESCRT machinery influences haem uptake and capsule elaboration in Cryptococcus neoformans

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.

The endosomal sorting complex required for transport machinery influences haem uptake and capsule elaboration inCryptococcus neoformans: ESCRT machinery and virulence inCryptococcus neoformans

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.