Loss of the scavenger receptor MARCO results in uncontrolled vomocytosis of fungi from macrophages.

Vomocytosis, also known as nonlytic exocytosis, is a process whereby fully phagocytosed microbes are expelled from phagocytes without discernible damage to either the phagocyte or microbe. Although this phenomenon was first described in the opportunistic fungal pathogen Cryptococcus neoformans in 2006, to date, mechanistic studies have been hampered by an inability to reliably stimulate or inhibit vomocytosis. Here we present the fortuitous discovery that macrophages lacking the scavenger receptor MAcrophage Receptor with COllagenous domain (MARCO), exhibit near-total vomocytosis of internalised cryptococci within a few hours of infection. Marco-/- macrophages also showed elevated vomocytosis of a yeast-locked C. albicans strain, suggesting this to be a broadly relevant observation. We go on to show that MARCO's role in modulating vomocytosis is independent of its role as a phagocytic receptor, suggesting that this protein may play an important and hitherto unrecognised role in modulating macrophage behaviour.

Microglia are not protective against cryptococcal meningitis.

Microglia provide protection against a range of brain infections including bacteria, viruses and parasites, but how these glial cells respond to fungal brain infections is poorly understood. We investigated the role of microglia in the context of cryptococcal meningitis, the most common cause of fungal meningitis in humans. Using a series of transgenic- and chemical-based microglia depletion methods we found that, contrary to their protective role during other infections, loss of microglia did not affect control of Cryptococcus neoformans brain infection which was replicated with several fungal strains. At early time points post-infection, we found that microglia depletion lowered fungal brain burdens, which was related to intracellular residence of C. neoformans within microglia. Further examination of extracellular and intracellular fungal populations revealed that C. neoformans residing in microglia were protected from copper starvation, whereas extracellular yeast upregulated copper transporter CTR4. However, the degree of copper starvation did not equate to fungal survival or abundance of metals within different intracellular niches. Taken together, these data show how tissue-resident myeloid cells may influence fungal phenotype in the brain but do not provide protection against this infection, and instead may act as an early infection reservoir.

Toll-like receptor 4 and macrophage scavenger receptor 1 crosstalk regulates phagocytosis of a fungal pathogen.

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4-/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4-/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.

Species- and strain-specific differences in the phagocytosis of Prototheca: insights from live-cell imaging.

The genus Prototheca is an extremely unusual group of achlorophyllic, obligately heterotrophic algae. Six species have been identified as pathogens of vertebrates, including cattle and humans. In cattle, P. bovis is the main infectious pathogen and is associated with bovine mastitis. In contrast, human infections typically involve P. wickerhamii and are associated with a spectrum of varying clinical presentations. Prototheca spp. enter the host from the environment and are therefore likely to be initially recognized by cells of the innate immune system. However, little is known about the nature of the interaction between Prototheca spp. and host phagocytes. In the present study, we adopt a live-cell imaging approach to investigate these interactions over time. Using environmental and clinical strains, we show that P. bovis cells are readily internalized and processed by macrophages, whereas these immune cells struggle to internalize P. wickerhamii. Serum opsonization of P. wickerhamii only marginally improves phagocytosis, suggesting that this species (but not P. bovis) may have evolved mechanisms to evade phagocytosis. Furthermore, we show that inhibition of the kinases Syk or PI3K, which are both critical for innate immune signaling, drastically reduces the uptake of P. bovis. Finally, we show that genetic ablation of MyD88, a signaling adaptor critical for Toll-like receptor signaling, has little impact on uptake but significantly prolongs phagosome maturation once P. bovis is internalized. Together, our data suggest that these two pathogenic Prototheca spp. have very different host-pathogen interactions which have potential therapeutic implications for the treatment of human and animal disease.

Cryptococcus neoformans Secretes Small Molecules That Inhibit IL-1ß Inflammasome-Dependent Secretion.

Cryptococcus neoformans is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule. Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of C. neoformans by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing proper inflammasome function. In this context, we analyzed the impact of molecules secreted by C. neoformans B3501 strain and its acapsular mutant Δcap67 in inflammasome activation in an in vitro model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome-dependent events (i.e., IL-1ß secretion and LDH release via pyroptosis) more strongly than conditioned media from Δcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens, and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) and DL-p-Hydroxyphenyllactic acid (HPLA) were present in B3501's conditioned media and that ILA alone or with HPLA is involved in the regulation of inflammasome activation by C. neoformans. These results were confirmed by in vivo experiments, where exposure to conditioned media led to higher fungal burdens in Acanthamoeba castellanii culture as well as in higher fungal loads in the lungs of infected mice. Overall, the results presented show that conditioned media from a wild-type strain can inhibit a vital recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival in vitro and in vivo and suggesting that secretion of aromatic metabolites, such as ILA, during cryptococcal infections fundamentally impacts pathogenesis.

Viral infection triggers interferon-induced expulsion of live Cryptococcus neoformans by macrophages.

Cryptococcus neoformans is an opportunistic human pathogen, which causes serious disease in immunocompromised hosts. Infection with this pathogen is particularly relevant in HIV+ patients, where it leads to around 200,000 deaths per annum. A key feature of cryptococcal pathogenesis is the ability of the fungus to survive and replicate within the phagosome of macrophages, as well as its ability to be expelled from host cells via a novel non-lytic mechanism known as vomocytosis. Here we show that cryptococcal vomocytosis from macrophages is strongly enhanced by viral coinfection, without altering phagocytosis or intracellular proliferation of the fungus. This effect occurs with distinct, unrelated human viral pathogens and is recapitulated when macrophages are stimulated with the anti-viral cytokines interferon alpha or beta (IFNα or IFNß). Importantly, the effect is abrogated when type-I interferon signalling is blocked, thus underscoring the importance of type-I interferons in this phenomenon. Lastly, our data help resolve previous, contradictory animal studies on the impact of type I interferons on cryptococcal pathogenesis and suggest that secondary viral stimuli may alter patterns of cryptococcal dissemination in the host.

Pyrifenox, an ergosterol inhibitor, differentially affects Cryptococcus neoformans and Cryptococcus gattii.

Cryptococcosis is a life-threatening fungal infection. New therapeutic approaches are necessary to combat cryptococcosis, as the currently available therapeutic protocols are expensive and generally result in deleterious side effects. Pyrifenox is an antifungal compound that affects phytopathogens by inhibiting the biosynthesis of ergosterol. In this study, we investigated the effects of pyrifenox on Cryptococcus neoformans and Cryptococcus gattii growth, capsule architecture and export of the major capsule component, glucuroxylomannan (GXM). Pyrifenox inhibited the growth of C. neoformans, but was significantly less effective against C. gattii. The resistance of C. gattii to pyrifenox was associated with the expression of efflux pump genes, particularly AFR1 and AFR2, since mutant cells lacking expression of these genes became sensitive to pyrifenox. Analysis of the cryptococcal capsule by India ink counterstaining, immunofluorescence, and scanning electron microscopy showed that pyrifenox affected capsular dimensions in both species. However, GXM fibers were shorter and uniformly distributed in C. neoformans, whereas in C. gattii the number of fibers was reduced. Pyrifenox-treated C. gattii developed unusually long chains of undivided cells. The secretion of GXM was markedly reduced in both species after treatment with pyrifenox. Altogether, the results indicated that pyrifenox differently affects C. neoformans and C. gattii. In addition, it highlights a potential role for pyrifenox as an inhibitor of GXM export in experimental models involving pathogenic cryptococci.

Vomocytosis: What we know so far.

Vomocytosis, or nonlytic exocytosis, has been reported for Cryptococcus neoformans since 2006. Since then, the repertoire of vomocytosing pathogens and host cells has increased and so have the molecular components linked to vomocytosis occurrence. Nonetheless, the mechanism underlying this phenomenon, whether it is triggered by the host or the pathogen, and how it affects disease progression are still unresolved. This review contains a summary of the main findings regarding vomocytosis and the outstanding questions puzzling scientists to this day.

Extracellular vesicles of human pathogenic fungi.

Extracellular vesicles play a significant role in many aspects of cellular life including cell-to-cell communication, pathogenesis and cancer progression. However very little is known about their role in fungi and we are just at the beginning of understanding their influence on fungal pathophysiology and host-pathogen interactions. Recent findings have revealed a role for fungal vesicles in triggering anti-microbial activities as well as in modulating virulence strategies, suggesting potential new avenues for antifungal therapies. In this review, we summarize our current understanding of fungal extracellular vesicles, including their biogenesis, secretion and size variation, and discuss how they may influence the human immune response and some key questions that remain unanswered.

Zinc and Iron Homeostasis: Target-Based Drug Screening as New Route for Antifungal Drug Development.

The incidence of fungal diseases is on the rise and the number of fatalities is still unacceptably high. While advances into antifungal drug development have been made there remains an urgent need to develop novel antifungal agents targeting as-yet unexploited pathways, such as metal ion homeostasis. Here we report such an approach by developing a metal sensor screen in the opportunistic human fungal pathogen Candida albicans. Using this reporter strain, we screened a library of 1,200 compounds and discovered several active compounds not previously described as chemical entities with antifungal properties. Two of these, artemisinin and pyrvinium pamoate, have been further characterized and their interference with metal homeostasis and potential as novel antifungal compounds validated. Lastly, we demonstrate that the same strain can be used to report on intracellular conditions within host phagocytes, paving the way toward the development of novel screening platforms that could identify compounds with the potential to perturb ion homeostasis of the pathogen specifically within host cells.

15-keto-prostaglandin E2 activates host peroxisome proliferator-activated receptor gamma (PPAR-γ) to promote Cryptococcus neoformans growth during infection.

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.

A Glucuronoxylomannan Epitope Exhibits Serotype-Specific Accessibility and Redistributes towards the Capsule Surface during Titanization of the Fungal Pathogen Cryptococcus neoformans.

Disseminated infections with the fungal species Cryptococcus neoformans or, less frequently, Cryptococcus gattii are an important cause of mortality in immunocompromised individuals. Central to the virulence of both species is an elaborate polysaccharide capsule that consists predominantly of glucuronoxylomannan (GXM). Due to its abundance, GXM is an ideal target for host antibodies, and several monoclonal antibodies (mAbs) have previously been derived using purified GXM or whole capsular preparations as antigens. In addition to their application in the diagnosis of cryptococcosis, anti-GXM mAbs are invaluable tools for studying capsule structure. In this study, we report the production and characterization of a novel anti-GXM mAb, Crp127, that unexpectedly reveals a role for GXM remodeling during the process of fungal titanization. We show that Crp127 recognizes a GXM epitope in an O-acetylation-dependent, but xylosylation-independent, manner. The epitope is differentially expressed by the four main serotypes of Cryptococcus neoformans and C. gattii, is heterogeneously expressed within clonal populations of C. gattii serotype B strains, and is typically confined to the central region of the enlarged capsule. Uniquely, however, this epitope redistributes to the capsular surface in titan cells, a recently characterized morphotype where haploid 5-µm cells convert to highly polyploid cells of >10 µm with distinct but poorly understood capsular characteristics. Titan cells are produced in the host lung and critical for successful infection. Crp127 therefore advances our understanding of cryptococcal morphological change and may hold significant potential as a tool to differentially identify cryptococcal strains and subtypes.

In Fungal Intracellular Pathogenesis, Form Determines Fate.

For pathogenic microbes to survive ingestion by macrophages, they must subvert powerful microbicidal mechanisms within the phagolysosome. After ingestion, Candida albicans undergoes a morphological transition producing hyphae, while the surrounding phagosome exhibits a loss of phagosomal acidity. However, how these two events are related has remained enigmatic. Now Westman et al. (mBio 9:e01226-18, 2018, https://doi.org/10.1128/mBio.01226-18) report that phagosomal neutralization results from disruption of phagosomal membrane integrity by the enlarging hyphae, directly implicating the morphological transition in physical damage that promotes intracellular survival. The C. albicans intracellular strategy shows parallels with another fungal pathogen, Cryptococcus neoformans, where a morphological changed involving capsular enlargement intracellularly is associated with loss of membrane integrity and death of the host cell. These similarities among distantly related pathogenic fungi suggest that morphological transitions that are common in fungi directly affect the outcome of the fungal cell-macrophage interaction. For this class of organisms, form determines fate in the intracellular environment.

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.

Characterizing the Mechanisms of Nonopsonic Uptake of Cryptococci by Macrophages.

The pathogenic fungus Cryptococcus enters the human host via inhalation into the lung and is able to reside in a niche environment that is serum- (opsonin) limiting. Little is known about the mechanism by which nonopsonic phagocytosis occurs via phagocytes in such situations. Using a combination of soluble inhibitors of phagocytic receptors and macrophages derived from knockout mice and human volunteers, we show that uptake of nonopsonized Cryptococcus neoformans and C. gattii via the mannose receptor is dependent on macrophage activation by cytokines. However, although uptake of C. neoformans is via both dectin-1 and dectin-2, C. gattii uptake occurs largely via dectin-1. Interestingly, dectin inhibitors also blocked phagocytosis of unopsonized Cryptococci in wax moth (Galleria mellonella) larvae and partially protected the larvae from infection by both fungi, supporting a key role for host phagocytes in augmenting early disease establishment. Finally, we demonstrated that internalization of nonopsonized Cryptococci is not accompanied by the nuclear translocation of NF-κB or its concomitant production of proinflammatory cytokines such as TNF-α. Thus, nonopsonized Cryptococci are recognized by mammalian phagocytes in a manner that minimizes proinflammatory cytokine production and potentially facilitates fungal pathogenesis.

Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii.

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.

Quantifying donor-to-donor variation in macrophage responses to the human fungal pathogen Cryptococcus neoformans.

Cryptococcosis remains the leading cause of fungal meningitis worldwide, caused primarily by the pathogen Cryptococcus neoformans. Symptomatic cryptococcal infections typically affect immunocompromised patients. However, environmental exposure to cryptococcal spores is ubiquitous and most healthy individuals are thought to harbor infections from early childhood onwards that are either resolved, or become latent. Since macrophages are a key host cell for cryptococcal infection, we sought to quantify the extent of individual variation in this early phagocyte response within a small cohort of healthy volunteers with no reported immunocompromising conditions. We show that rates of both intracellular fungal proliferation and non-lytic expulsion (vomocytosis) are remarkably variable between individuals. However, we demonstrate that neither gender, in vitro host inflammatory cytokine profiles, nor polymorphisms in several key immune genes are responsible for this variation. Thus the data we present serve to quantify the natural variation in macrophage responses to this important human pathogen and will hopefully provide a useful "benchmark" for the research community.

Variability in innate host immune responses to cryptococcosis

Cryptococcosis is an invasive fungal disease caused by Cryptococcus neoformans and the closely related species C. gattii. The severe form of the disease, cryptococcal meningitis (CM), is rapidly fatal without treatment. Although typically a disease of immunocompromised (especially HIV-positive) individuals, there is growing awareness of cryptococcal disease amongst non-immunocompromised patients. Whilst substantial progress has been made in understanding the pathogenicity of C. neoformans in HIV patients, prospective data on cryptococcosis outside the context of HIV remains lacking. Below we review how innate immune responses vary between hosts depending on immunological status, and discuss risk factors and predictors of disease outcome in different groups.

Vomocytosis of live pathogens from macrophages is regulated by the atypical MAP kinase ERK5.

Vomocytosis, or nonlytic extrusion, is a poorly understood process through which macrophages release live pathogens that they have failed to kill back into the extracellular environment. Vomocytosis is conserved across vertebrates and occurs with a diverse range of pathogens, but to date, the host signaling events that underpin expulsion remain entirely unknown. We use a targeted inhibitor screen to identify the MAP kinase ERK5 as a critical suppressor of vomocytosis. Pharmacological inhibition or genetic manipulation of ERK5 activity significantly raises vomocytosis rates in human macrophages, whereas stimulation of the ERK5 signaling pathway inhibits vomocytosis. Lastly, using a zebrafish model of cryptococcal disease, we show that reducing ERK5 activity in vivo stimulates vomocytosis and results in reduced dissemination of infection. ERK5 therefore represents the first host signaling regulator of vomocytosis to be identified and a potential target for the future development of vomocytosis-modulating therapies.

Using Flow Cytometry to Analyze Cryptococcus Infection of Macrophages.

Flow cytometry is a powerful analytical technique, which is increasingly being used to study the interaction between host cells and intracellular pathogens. Flow cytometry is capable of measuring a greater number of infected cells within a sample compared to alternative techniques such as fluorescence microscopy. This means that robust quantification of rare events during infection is possible. Our lab and others have developed flow cytometry methods to study interactions between host cells and intracellular pathogens, such as Cryptococcus neoformans, to quantify phagocytosis, intracellular replication, and non-lytic expulsion or "vomocytosis" from the phagosome. Herein we describe these methods and how they can be applied to the study of C. neoformans as well as other similar intracellular pathogens.