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Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a fungal pathogen, which may have wider implications for human health as aneuploidy is proposed to promote tumourigenesis.

Invasive fungal pathogens are major causes of human mortality and morbidity 1 , 2 . Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans , the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages 3 – 8 . Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses 9 – 12 . We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease. Cryptococcus neoformans secretes CPL1 protein, which induces alternative activation of macrophages via Toll-like receptor 4 in mice and is essential for fungal virulence.

A common feature shared by systemic fungal pathogens of environmental origin, such as Cryptococcus neoformans , is their ability to adapt to mammalian core body temperature. In C. neoformans , this adaptation is accompanied by Ccr4-mediated decay of ribosomal protein mRNAs. Here we use the related, but thermo-intolerant species Cryptococcus amylolentus to demonstrate that this response contributes to host-temperature adaptation and pathogenicity of cryptococci. In a C. neoformans ccr4 Δ mutant, stabilized ribosomal protein mRNAs are retained in the translating pool, and stress-induced transcriptomic changes are reduced in comparison with the wild type strain, likely due to ineffective translation of transcription factors. In addition, the mutant displays increased exposure of cell wall glucans, and recognition by Dectin-1 results in increased phagocytosis by lung macrophages, linking mRNA decay to adaptation and immune evasion. The fungal pathogen Cryptococcus neoformans can adapt to mammalian core body temperature. Here, Bloom et al. show that Ccr4-mediated decay of ribosomal protein mRNAs is important for thermotolerance and immune evasion by promoting masking of cell wall glucans.

Developmental transitions between single-cell yeast and multicellular filaments underpin virulence of diverse fungal pathogens. For the leading human fungal pathogen Candida albicans, filamentation is thought to be required for immune cell escape via induction of an inflammatory programmed cell death. Here we perform a genome-scale analysis of C. albicans morphogenesis and identify 102 negative morphogenetic regulators and 872 positive regulators, highlighting key roles for ergosterol biosynthesis and N-linked glycosylation. We demonstrate that C. albicans filamentation is not required for escape from host immune cells; instead, macrophage pyroptosis is driven by fungal cell-wall remodelling and exposure of glycosylated proteins in response to the macrophage phagosome. The capacity of killed, previously phagocytized cells to drive macrophage lysis is also observed with the distantly related fungal pathogen Cryptococcus neoformans . This study provides a global view of morphogenetic circuitry governing a key virulence trait, and illuminates a new mechanism by which fungi trigger host cell death. Several pathogenic fungi such as Candida albicans undergo transitions between single-celled forms and multicellular filaments. Here the authors perform a genome-scale analysis of C. albicans and show that, contrary to common belief, filamentation is not required for escape from host immune cells.

Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulating Cryptococcus neoformans and Candida albicans , thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture of C. neoformans . Internalization of C. neoformans by KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth of C. neoformans is inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions. Patients with liver diseases are at increased risk of fungal infections. Here the authors show that Kupffer cells are critical for the filtration of fungi out of the blood and thereby for liver-mediated protection against disseminating fungal infection.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian β-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C . neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C . neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C . neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host. The protein Galectin-3 modulates host immunity and plays roles during infections. Here, Almeida et al. show that this protein contributes to host defence against infection with the fungal pathogen Cryptococcus neoformans by inhibiting fungal growth and inducing lysis of fungal extracellular vesicles.

Invasive fungal infections kill more than 1.5 million people each year, with limited treatment options. There is no vaccine available in clinical use to prevent and control fungal infections. Our recent studies showed that a mutant of the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase in Cryptococcus neoformans , elicited superior protective Th1 host immunity. Here, we demonstrate that the heat-killed fbp1 Δ cells (HK-fbp1) can be harnessed to confer protection against a challenge by the virulent parental strain, even in animals depleted of CD4 + T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that HK-fbp1 vaccination induces significant cross-protection against challenge with diverse invasive fungal pathogens. Thus, our data suggest that HK-fbp1 has the potential to be a broad-spectrum vaccine candidate against invasive fungal infections in both immunocompetent and immunocompromised populations. Cryptococcus neoformans is a fungal pathogen that infects the lungs and then often disseminates to the central nervous system, causing meningitis. How Cryptococcus is able to suppress host immunity and escape the antifungal activity of macrophages remains incompletely understood. We reported that the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase, promotes Cryptococcus virulence by regulating host- Cryptococcus interactions. Our recent studies demonstrated that the fbp1 Δ mutant elicited superior protective Th1 host immunity in the lungs and that the enhanced immunogenicity of heat-killed fbp1 Δ yeast cells can be harnessed to confer protection against a subsequent infection with the virulent parental strain. We therefore examined the use of heat-killed fbp1 Δ cells in several vaccination strategies. Interestingly, the vaccine protection remains effective even in mice depleted of CD4 + T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that vaccinating mice with heat-killed fbp1 Δ induces significant cross-protection against challenge with diverse invasive fungal pathogens, including C. neoformans , C. gattii , and Aspergillus fumigatus , as well as partial protection against Candida albicans . Thus, our data suggest that the heat-killed fbp1Δ strain has the potential to be a suitable vaccine candidate against cryptococcosis and other invasive fungal infections in both immunocompetent and immunocompromised populations. IMPORTANCE Invasive fungal infections kill more than 1.5 million people each year, with limited treatment options. There is no vaccine available in clinical use to prevent and control fungal infections. Our recent studies showed that a mutant of the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase in Cryptococcus neoformans , elicited superior protective Th1 host immunity. Here, we demonstrate that the heat-killed fbp1 Δ cells (HK-fbp1) can be harnessed to confer protection against a challenge by the virulent parental strain, even in animals depleted of CD4 + T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that HK-fbp1 vaccination induces significant cross-protection against challenge with diverse invasive fungal pathogens. Thus, our data suggest that HK-fbp1 has the potential to be a broad-spectrum vaccine candidate against invasive fungal infections in both immunocompetent and immunocompromised populations.

Cryptococcus neoformans is a major fungal pathogen that disseminates to the central nervous system (CNS) to cause fatal meningoencephalitis, but little is known about immune responses within this immune-privileged site. CD4 + T cells have demonstrated roles in anticryptococcal defenses, but increasing evidence suggests that they may contribute to clinical deterioration and pathology in both HIV-positive (HIV+) and non-HIV patients who develop immune reconstitution inflammatory syndrome (IRIS) and post-infectious inflammatory response syndrome (PIIRS), respectively. Here we report a novel murine model of cryptococcal meningoencephalitis and a potential damaging role of T cells in disseminated cryptococcal CNS infection. In this model, fungal burdens plateaued in the infected brain by day 7 postinfection, but activation of microglia and accumulation of CD45 hi leukocytes was significantly delayed relative to fungal growth and did not peak until day 21. The inflammatory leukocyte infiltrate consisted predominantly of gamma interferon (IFN-γ)-producing CD4 + T cells, conventionally believed to promote fungal clearance and recovery. However, more than 50% of mice succumbed to infection and neurological dysfunction between days 21 and 35 despite a 100-fold reduction in fungal burdens. Depletion of CD4 + cells significantly impaired IFN-γ production, CD8 + T cell and myeloid cell accumulation, and fungal clearance from the CNS but prevented the development of clinical symptoms and mortality. These findings conclusively demonstrate that although CD4 + T cells are necessary to control fungal growth, they can also promote significant immunopathology and mortality during CNS infection. The results from this model may provide important guidance for development and use of anti-inflammatory therapies to minimize CNS injury in patients with severe cryptococcal infections. IMPORTANCE CNS infection with the fungal pathogen Cryptococcus neoformans often results in debilitating brain injury and has a high mortality rate despite antifungal treatment. Treatment is complicated by the fact that immune responses needed to eliminate infection are also thought to drive CNS damage in a subset of both HIV+ and non-HIV patients. Thus, physicians need to balance efforts to enhance patients’ immune responses and promote microbiological control with anti-inflammatory therapy to protect the CNS. Here we report a novel model of cryptococcal meningoencephalitis demonstrating that fungal growth within the CNS does not immediately cause symptomatic disease. Rather, accumulation of antifungal immune cells critically mediates CNS injury and mortality. This model demonstrates that antifungal immune responses in the CNS can cause detrimental pathology and addresses the urgent need for animal models to investigate the specific cellular and molecular mechanisms underlying cryptococcal disease in order to better treat treat patients with CNS infections. CNS infection with the fungal pathogen Cryptococcus neoformans often results in debilitating brain injury and has a high mortality rate despite antifungal treatment. Treatment is complicated by the fact that immune responses needed to eliminate infection are also thought to drive CNS damage in a subset of both HIV+ and non-HIV patients. Thus, physicians need to balance efforts to enhance patients’ immune responses and promote microbiological control with anti-inflammatory therapy to protect the CNS. Here we report a novel model of cryptococcal meningoencephalitis demonstrating that fungal growth within the CNS does not immediately cause symptomatic disease. Rather, accumulation of antifungal immune cells critically mediates CNS injury and mortality. This model demonstrates that antifungal immune responses in the CNS can cause detrimental pathology and addresses the urgent need for animal models to investigate the specific cellular and molecular mechanisms underlying cryptococcal disease in order to better treat patients with CNS infections.

Cryptococcus neoformans ( Cn ) is a fungal pathogen responsible for cryptococcal meningitis, which accounts for 15% of AIDS-related deaths. Recent studies have shown that the absence of sterol β-glucosidase (EGCrP2, also known as Sgl1) in Cn significantly attenuates its virulence in a mouse infection model. However, the mechanisms underlying this virulence attenuation remain unclear. In this study, we observed a significant increase in dead cells after 3 days of culture of SGL1 -deficient Cn ( sgl1 Δ, KO) at 37°C, compared with wild-type (WT) and SGL1 -reconstituted Cn ( sgl1 Δ:: SGL1 , RE). qPCR analysis of WT, KO, and RE strains indicated that autophagy-related genes ( ATG s) were significantly downregulated in KO strain. Atg8-dependent GFP translocation to the vacuole was significantly delayed in KO strain under starvation conditions. This autophagy dysfunction was identified as the primary cause of the increased cell death observed in KO strain under nitrogen starvation conditions at 37°C. EGCrP2/Sgl1 is predominantly localized in the vacuoles of Cn , and its deletion results in the accumulation of not only ergosterol β-glucoside (EG), as previously reported, but also acylated EGs (AEGs). AEGs were much more potent than EG in activating the C-type lectin receptor Mincle in mice, rats, and humans. AEGs were released from KO strain via extracellular vesicles (EVs). Chemically synthesized 18:1-EG and EVs derived from KO strain, but not WT or RE strains, enhanced cytokine production in murine and human dendritic cells. AEG-dependent cytokine production was markedly reduced in dendritic cells from Mincle-deficient mice, and the number of KO strain in lung tissue from Mincle-deficient mice was substantially higher than wild-type mice on day 3 after infection. Intranasal administration of acylated sitosterol β-glucoside increased Mincle expression and cytokine production and reduced the Cn burden in lung tissue of Cn -infected mice. These findings suggest that autophagy dysfunction in KO strain and the host innate immune response via the AEG-dependent Mincle activation are critical in reducing Cn virulence in mice.

Cryptococcal meningitis (CM) causes approximately 1,200,000 deaths annually in people living with HIV and is also a threat to individuals with non-HIV-associated immune-compromising conditions, such as organ transplant recipients and other patients receiving immunosuppressants. Prior work has shown that normal human immunoglobulins (Igs) bind Cryptococcus neoformans (Cn) and that plasma levels of IgM, IgG, and IgA differ as a function of CM status. We investigated how human IgM, IgG, and IgA affect Cn growth, morphology, and protein synthesis. We found that IgA has major effects on these aspects of Cn biology and lends plausibility to the hypothesis that previously reported reductions in IgA levels in HIV-associated CM may influence Cn pathogenesis. Overall, our findings show that antibody immunity to Cn is more complex than previously thought.