Cryptococcosis is the leading invasive fungal infection in the world today. Over the past century, the causative agents, Cryptococcus neoformans and Cryptococcus gattii, have risen from the status of medical curiosities to common but life-threatening central nervous system pathogens. In an elegant experimental pathobiology study of these two organisms carried out by Ngamskulrungroj et al., there are three matters that merit further discussion. First is the question of whether there is a variable specific pathobiology for each yeast strain. Does it make biological and clinical sense to designate C. neoformans and C. gattii as two separate species? Second is the matter of how the organisms differ pathologically at the site of infection. Finally, there is the possibility that the human immune system responds differently to each species. Although no single study can provide definitive mechanistic answers to the important questions, this experimental pathology study and its discussion clearly frame the issues to be dissected.
Cryptococcus neoformans is a ubiquitous human fungal pathogen. This pathogen can undergo morphotype transition between the yeast and the filamentous form and such morphological transition has been implicated in virulence for decades. Morphotype transition is typically observed during mating, which is governed by pheromone signaling. Paradoxically, components specific to the pheromone signaling pathways play no or minimal direct roles in virulence. Thus, the link between morphotype transition and virulence and the underlying molecular mechanism remain elusive. Here, we demonstrate that filamentation can occur independent of pheromone signaling and mating, and both mating-dependent and mating-independent morphotype transition require the transcription factor Znf2. High expression of Znf2 is necessary and sufficient to initiate and maintain sex-independent filamentous growth under host-relevant conditions in vitro and during infection. Importantly, ZNF2 overexpression abolishes fungal virulence in murine models of cryptococcosis. Thus, Znf2 bridges the sex-independent morphotype transition and fungal pathogenicity. The impacts of Znf2 on morphological switch and pathogenicity are at least partly mediated through its effects on cell adhesion property. Cfl1, a Znf2 downstream factor, regulates morphogenesis, cell adhesion, biofilm formation, and virulence. Cfl1 is the first adhesin discovered in the phylum Basidiomycota of the Kingdom Fungi. Together with previous findings in other eukaryotic pathogens, our findings support a convergent evolution of plasticity in morphology and its impact on cell adhesion as a critical adaptive trait for pathogenesis.
The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of human immunodeficiency virus (HIV)-infected patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche that also renders them relatively insensitive to antifungal agents. Here we report an attempt to address this limitation by using a fluorescence-based drug screening method to identify potential inhibitors of intracellular proliferation of C. neoformans. The Prestwick Chemical Library(®) of FDA-approved small molecules was screened for compounds that limit the intracellular replication of a fluorescently-tagged C. neoformans reference strain (H99-GFP) in macrophages. Preliminary screening revealed 19 of 1200 compounds that could significantly reduce intracellular growth of the pathogen. Secondary screening and host cell cytotoxicity assays highlighted fendiline hydrochloride as a potential drug candidate for the development of future anticryptococcal therapies. Live cell imaging demonstrated that this Ca(2+) channel blocker strongly enhanced phagosome maturation in macrophages leading to improved fungal killing and reduced intracellular replication. Whilst the relatively high dose of fendiline hydrochloride required renders it unfit for clinical deployment against cryptococcosis, this study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards the development of novel antifungal therapies for this neglected disease.
Cryptococcus neoformans is a fatal fungal pathogen of humans that efficiently parasitises macrophages. Birds can be colonised by cryptococci and can transmit cryptococcosis to humans via inhalation of inoculated bird excreta. However, colonisation of birds appears to occur in the absence of symptomatic infection. Here, using a pure population of primary bird macrophages, we demonstrate a mechanism for this relationship. We find that bird macrophages are able to suppress the growth of cryptococci seen in mammalian cells despite C. neoformans being able to grow at bird body temperature, and are able to escape from bird macrophages by vomocytosis. A small subset of cryptococci are able to adapt to the inhibitory intracellular environment of bird macrophages, exhibiting a large cell phenotype that rescues growth suppression. Thus, restriction of intracellular growth combined with survival at bird body temperature explains the ability of birds to efficiently spread C. neoformans in the environment whilst avoiding systemic disease.
The Pacific Northwest outbreak of cryptococcosis, caused by a near-clonal lineage of the fungal pathogen Cryptococcus gattii, represents the most significant cluster of life-threatening fungal infections in otherwise healthy human hosts currently known. The outbreak lineage has a remarkable ability to grow rapidly within human white blood cells, using a unique ‘division of labour’ mechanism within the pathogen population, where some cells adopt a dormant behaviour to support the growth of neighbouring cells. Here we demonstrate that pathogenic ‘division of labour’ can be triggered over large cellular distances and is mediated through the release of extracellular vesicles by the fungus. Isolated vesicles released by virulent strains are taken up by infected host macrophages and trafficked to the phagosome, where they trigger the rapid intracellular growth of non-outbreak fungal cells that would otherwise be eliminated by the host. Thus, long distance pathogen-to-pathogen communication via extracellular vesicles represents a novel mechanism to control complex virulence phenotypes in Cryptococcus gattii and, potentially, other infectious species.
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.
An understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogen Ustilago maydis. However, mfe2 mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in the had1 gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog, had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of an mfe2Δ mutant. We also show that short-chain fatty acids induce cell death in U. maydis and that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms by U. maydis that includes potential metabolic contributions to proliferation in planta and an effect on virulence-related morphogenesis.
The human fungal pathogen Cryptococcus neoformans is characterized by its ability to induce a distinct polysaccharide capsule in response to a number of host-specific environmental stimuli. The induction of capsule is a complex biological process encompassing regulation at multiple steps, including the biosynthesis, transport, and maintenance of the polysaccharide at the cell surface. By precisely regulating the composition of its cell surface and secreted polysaccharides, C. neoformans has developed intricate ways to establish chronic infection and dormancy in the human host. The plasticity of the capsule structure in response to various host conditions also underscores the complex relationship between host and parasite. Much of this precise regulation of capsule is achieved through the transcriptional responses of multiple conserved signaling pathways that have been coopted to regulate this C. neoformans-specific virulence-associated phenotype. This review focuses on specific host stimuli that trigger the activation of the signal transduction cascades and on the downstream transcriptional responses that are required for robust encapsulation around the cell.
Cryptococcus gattii is responsible for an expanding epidemic of serious infections in Western Canada and the Northwestern United States (NW), some patients with these infections respond poorly to azole antifungals, and high azole MICs have been reported in NW C. gattii. In this study, 25 NW C. gattii had higher MICs for multiple azoles (but not amphotericin B) than did 34 non-NW C. gattii or 20 Cryptococcus neoformans strains. We therefore examined the roles in azole resistance of overexpression of or mutations in the gene (ERG11) encoding the azole target enzyme. ERG11/ACT1 mRNA ratios were higher in C. gattii than in C. neoformans, but these ratios did not differ in NW and non-NW C. gattii strains, nor did they correlate with fluconazole MICs within any group. Three NW C. gattii strains with low azole MICs and 2 with high azole MICs had deduced Erg11p sequences that differed at one or more position from that of the fully-sequenced NW C. gattii strain R265. However, the azole MICs of conditional S. cerevisiae erg11 mutants expressing the 5 variant ERG11s were within 2-fold of the azole MICs of S. cerevisiae expressing the ERG11s from C. gattii R265, non-NW C. gattii strain WM276, or C. neoformans strains H99 or JEC21. We conclude that neither ERG11 overexpression nor variations in ERG11 coding sequences was responsible for the high azole MICs observed in the NW C. gattii strains we studied.
Browning is one of the most common postharvest changes in button mushrooms, which often results in economic losses. Phenolic compounds, which are associated with browning, were extracted from the non-bruised and bruised skin tissue of various button mushrooms with a sulphite containing solution and analysed with UHPLC-PDA-MS. In total, 34 phenolic compounds were detected. Only small differences in the total phenolic content between bruising-tolerant and sensitive strains were observed. The contents of γ-L-glutaminyl-4-hydroxybenzene (GHB) and γ-L-glutaminyl-3,4-dihydroxybenzene (GDHB) correlated with bruising-sensitivity, for example a R(2) of 0.85 and 0.98 was found for non-bruised brown strains, respectively. In non-bruised skin tissue of the strains with brown caps, the GHB and GDHB contents in sensitive strains were on average 20 times and 15 times higher, respectively, than in tolerant strains. GHB and GDHB likely participate in the formation of brown coloured GHB-melanin, which seemed the predominant pathway in bruising-related discoloration of button mushrooms.