Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia.

Cryptococcus neoformans causes lethal meningitis and accounts for approximately 10%-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this fungus invades the mammalian brain. To investigate the dynamics of C. neoformans tissue invasion, we mapped fungal localization and host cell interactions in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. We confirm high fungal burden in mouse upper airway after nasal inoculation. Yeast in turbinates were frequently titan cells, with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of the upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of C. neoformans, by finding viable fungi in the bloodstream of mice a few days after intranasal infection. As early as 24 h post systemic infection, the majority of C. neoformans cells traversed the blood-brain barrier, and were engulfed or in close proximity to microglia. Our work presents a new method for investigating microbial invasion, establishes that C. neoformans can breach multiple tissue barriers within the first days of infection, and demonstrates microglia as the first cells responding to C. neoformans invasion of the brain.IMPORTANCECryptococcal meningitis causes 10%-15% of AIDS-associated deaths globally. Still, brain-specific immunity to cryptococci is a conundrum. By employing innovative imaging, this study reveals what occurs during the first days of infection in brain and in airways. We found that titan cells predominate in upper airways and that cryptococci breach the upper airway mucosa, which implies that, at least in mice, the upper airways are a site for fungal dissemination. This would signify that mucosal immunity of the upper airway needs to be better understood. Importantly, we also show that microglia, the brain-resident macrophages, are the first responders to infection, and microglia clusters are formed surrounding cryptococci. This study opens the field to detailed molecular investigations on airway immune response, how fungus traverses the blood-brain barrier, how microglia respond to infection, and ultimately how microglia monitor the blood-brain barrier to preserve brain function.

Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia.

The fungus Cryptococcus neoformans causes lethal meningitis in humans with weakened immune systems and is estimated to account for 10-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this environmental fungus evades the immune system and invades the mammalian brain before the onset of overt symptoms. To investigate the dynamics of C. neoformans tissue invasion, we mapped early fungal localisation and host cell interactions at early times in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. Made possible by these techniques, we confirm high fungal burden in mouse upper airway turbinates after nasal inoculation. Surprisingly, most yeasts in turbinates were titan cells, indicating this microenvironment enables titan cell formation with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of C. neoformans, via imaging C. neoformans within blood vessels of mouse lungs and finding viable fungi in the bloodstream of mice a few days after intranasal infection, suggesting that bloodstream access can occur via lung alveoli. In a model of systemic cryptococcosis, we show that as early as 24 h post infection, majority of C. neoformans cells traversed the blood-brain barrier, and are engulfed or in close proximity to microglia. Our work establishes that C. neoformans can breach multiple tissue barriers within the first days of infection. This work presents a new method for investigating cryptococcal invasion mechanisms and demonstrates microglia as the primary cells responding to C. neoformans invasion.

Programmed Cell Death in Unicellular Versus Multicellular Organisms.

Programmed cell death (self-induced) is intrinsic to all cellular life forms, including unicellular organisms. However, cell death research has focused on animal models to understand cancer, degenerative disorders, and developmental processes. Recently delineated suicidal death mechanisms in bacteria and fungi have revealed ancient origins of animal cell death that are intertwined with immune mechanisms, allaying earlier doubts that self-inflicted cell death pathways exist in microorganisms. Approximately 20 mammalian death pathways have been partially characterized over the last 35 years. By contrast, more than 100 death mechanisms have been identified in bacteria and a few fungi in recent years. However, cell death is nearly unstudied in most human pathogenic microbes that cause major public health burdens. Here, we consider how the current understanding of programmed cell death arose through animal studies and how recently uncovered microbial cell death mechanisms in fungi and bacteria resemble and differ from mechanisms of mammalian cell death.

Similar evolutionary trajectories in an environmental Cryptococcus neoformans isolate after human and murine infection.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI-SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

Galleria mellonella immune melanization is fungicidal during infection.

A key component of the insect immune response is melanin production, including within nodules, or aggregations of immune cells surrounding microbes. Melanization produces oxidative and toxic intermediates that limit microbial infections. However, a direct fungicidal role of melanin during infection has not been demonstrated. We previously reported that the fungus Cryptococcus neoformans is encapsulated with melanin within nodules of Galleria mellonella hosts. Here we developed techniques to study melanin's role during C. neoformans infection in G. mellonella. We provided evidence that in vivo melanin-encapsulation was fungicidal. To further study immune melanization, we applied tissue-clearing techniques to visualize melanized nodules in situ throughout the larvae. Further, we developed a time-lapse microscopy protocol to visualize the melanization kinetics in extracted hemolymph following fungal exposure. Using this technique, we found that cryptococcal melanin and laccase enhance immune melanization. We extended this approach to study the fungal pathogens Candida albicans and Candida auris. We find that the yeast morphologies of these fungi elicited robust melanization responses, while hyphal and pseudohyphal morphologies were melanin-evasive. Approximately 23% of melanin-encapsulated C. albicans yeast can survive and breakthrough the encapsulation. Overall, our results provide direct evidence that immune melanization functions as a direct antifungal mechanism in G. mellonella.

Yeast cell death pathway requiring AP-3 vesicle trafficking leads to vacuole/lysosome membrane permeabilization.

Unicellular eukaryotes have been suggested as undergoing self-inflicted destruction. However, molecular details are sparse compared with the mechanisms of programmed/regulated cell death known for human cells and animal models. Here, we report a molecular cell death pathway in Saccharomyces cerevisiae leading to vacuole/lysosome membrane permeabilization. Following a transient cell death stimulus, yeast cells die slowly over several hours, consistent with an ongoing molecular dying process. A genome-wide screen for death-promoting factors identified all subunits of the AP-3 complex, a vesicle trafficking adapter known to transport and install newly synthesized proteins on the vacuole/lysosome membrane. To promote cell death, AP-3 requires its Arf1-GTPase-dependent vesicle trafficking function and the kinase Yck3, which is selectively transported to the vacuole membrane by AP-3. Video microscopy revealed a sequence of events where vacuole permeability precedes the loss of plasma membrane integrity. AP-3-dependent death appears to be conserved in the human pathogenic yeast Cryptococcus neoformans.

The Dark Side of Estrogen Stops Translation to Induce Apoptosis.

The role of hormones in triggering cell death has been controversial. In this issue of Molecular Cell, Li et al. (2019) have defined a molecular pathway where an unexpected estrogen receptor, phosphodiesterase 3A, allows its partner Schlafen-12 to inhibit survival pathways, ultimately leading to apoptosis.

KCTD: A new gene family involved in neurodevelopmental and neuropsychiatric disorders.

The underlying molecular basis for neurodevelopmental or neuropsychiatric disorders is not known. In contrast, mechanistic understanding of other brain disorders including neurodegeneration has advanced considerably. Yet, these do not approach the knowledge accrued for many cancers with precision therapeutics acting on well-characterized targets. Although the identification of genes responsible for neurodevelopmental and neuropsychiatric disorders remains a major obstacle, the few causally associated genes are ripe for discovery by focusing efforts to dissect their mechanisms. Here, we make a case for delving into mechanisms of the poorly characterized human KCTD gene family. Varying levels of evidence support their roles in neurocognitive disorders (KCTD3), neurodevelopmental disease (KCTD7), bipolar disorder (KCTD12), autism and schizophrenia (KCTD13), movement disorders (KCTD17), cancer (KCTD11), and obesity (KCTD15). Collective knowledge about these genes adds enhanced value, and critical insights into potential disease mechanisms have come from unexpected sources. Translation of basic research on the KCTD-related yeast protein Whi2 has revealed roles in nutrient signaling to mTORC1 (KCTD11) and an autophagy-lysosome pathway affecting mitochondria (KCTD7). Recent biochemical and structure-based studies (KCTD12, KCTD13, KCTD16) reveal mechanisms of regulating membrane channel activities through modulation of distinct GTPases. We explore how these seemingly varied functions may be disease related.

Whi2: a new player in amino acid sensing.

A critical function of human, yeast, and bacterial cells is the ability to sense and respond to available nutrients such as glucose and amino acids. Cells must also detect declining nutrient levels to adequately prepare for starvation conditions by inhibiting cell growth and activating autophagy. The evolutionarily conserved protein complex TORC1 regulates these cellular responses to nutrients, and in particular to amino acid availability. Recently, we found that yeast Whi2 (Saccharomyces cerevisiae) and a human counterpart, KCTD11, that shares a conserved BTB structural domain, are required to suppress TORC1 activity under low amino acid conditions. Using yeast, the mechanisms were more readily dissected. Unexpectedly, Whi2 suppresses TORC1 activity independently of the well-known SEACIT-GTR pathway, analogous to the GATOR1-RAG pathway in mammals. Instead, Whi2 requires the plasma membrane-associated phosphatases Psr1 and Psr2, which were known to bind Whi2, although their role was unknown. Yeast WHI2 was previously reported to be involved in regulating several fundamental cellular processes including cell cycle arrest, general stress responses, the Ras-cAMP-PKA pathway, autophagy, and mitophagy, and to be frequently mutated in the yeast knockout collections and in genome evolution studies. Most of these observations are likely explained by the ability of Whi2 to inhibit TORC1. Thus, understanding the function of yeast Whi2 will provide deeper insights into the disease-related KCTD family proteins and the pathogenesis of plant and human fungal infections.

Targeting intrinsic cell death pathways to control fungal pathogens.

Fungal pathogens pose an increasing threat to public health. Limited clinical drug regimens and emerging drug-resistant isolates challenge infection control. The global burden of human fungal pathogens is estimated at 1 billion infections and 1.5 million deaths annually. In addition, plant fungal pathogens increasingly threaten global food resources. Novel strategies are needed to combat emerging fungal diseases and pan-resistant fungi. An untapped mechanistically novel approach is to pharmacologically activate the intrinsic cell death pathways encoded by pathogenic fungi. This strategy is analogous to new anti-cancer therapeutics now entering the clinic. Here we summarize the best understood examples of cell death mechanisms encoded by pathogenic fungi, contrast these to mammalian cell death pathways, and highlight the gaps in knowledge towards identifying potential death effectors as druggable targets.

Overview of BCL-2 Family Proteins and Therapeutic Potentials.

BCL-2 family proteins interact in a network that regulates apoptosis. The BH3 amino acid sequence motif serves to bind together this conglomerate protein family, both literally and figuratively. BH3 motifs are present in antiapoptotic and proapoptotic BCL-2 homologs, and in a separate group of unrelated BH3-only proteins often appended to the BCL-2 family. BH3-containing helices mediate many of their physical interactions to determine cell death versus survival, leading to the development of BH3 mimetics as therapeutics. Here we provide an overview of BCL-2 family interactions, their relevance in health and disease, and the progress toward regulating their interactions therapeutically.

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect.

OBJECTIVE: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. METHODS: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. RESULTS: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. INTERPRETATION: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.

Do Fungi Undergo Apoptosis-Like Programmed Cell Death?

This question of whether fungi undergo apoptosis-like programmed cell death can be separated into two questions. One question is about applying the term "apoptosis" to fungi, and the other is a more challenging question of whether fungi have evolved mechanisms that inflict self-injury. The answers to both questions depend on the definitions applied to "apoptosis" and "programmed cell death." Considering how these and other cell death terms originated and are currently defined for animals, some confusion arises when the terms are applied to fungi. While it is difficult to defend the concept of fungal apoptosis, the more interesting issue is whether fungi will eventually be found to encode programmed or extemporaneous self-destructive processes, as suggested by intriguing new findings.

Whi2 is a conserved negative regulator of TORC1 in response to low amino acids.

Yeast WHI2 was originally identified in a genetic screen for regulators of cell cycle arrest and later suggested to function in general stress responses. However, the function of Whi2 is unknown. Whi2 has predicted structure and sequence similarity to human KCTD family proteins, which have been implicated in several cancers and are causally associated with neurological disorders but are largely uncharacterized. The identification of conserved functions between these yeast and human proteins may provide insight into disease mechanisms. We report that yeast WHI2 is a new negative regulator of TORC1 required to suppress TORC1 activity and cell growth specifically in response to low amino acids. In contrast to current opinion, WHI2 is dispensable for TORC1 inhibition in low glucose. The only widely conserved mechanism that actively suppresses both yeast and mammalian TORC1 specifically in response to low amino acids is the conserved SEACIT/GATOR1 complex that inactivates the TORC1-activating RAG-like GTPases. Unexpectedly, Whi2 acts independently and simultaneously with these established GATOR1-like Npr2-Npr3-Iml1 and RAG-like Gtr1-Gtr2 complexes, and also acts independently of the PKA pathway. Instead, Whi2 inhibits TORC1 activity through its binding partners, protein phosphatases Psr1 and Psr2, which were previously thought to only regulate amino acid levels downstream of TORC1. Furthermore, the ability to suppress TORC1 is conserved in the SKP1/BTB/POZ domain-containing, Whi2-like human protein KCTD11 but not other KCTD family members tested.

Whi2 signals low leucine availability to halt yeast growth and cell death.

Cells are exquisitely tuned to environmental ques. Amino acid availability is rapidly sensed, allowing cells to adjust molecular processes and implement short or long-term metabolic shifts accordingly. How levels of most individual amino acids may be sensed and subsequently signaled to inform cells of their nutrient status is largely unknown. We made the unexpected observation that small changes in the levels of specific amino acids can have a profound effect on yeast cell growth, leading to the identification of yeast Whi2 as a negative regulator of cell growth in low amino acids. Although Whi2 was originally thought to be fungi-specific, Whi2 appears to share a conserved structural domain found in a family of 25 largely uncharacterized human genes encoding the KCTD (potassium channel tetramerization domain) protein family. Insights gained from yeast Whi2 are likely to be revealing about human KCTDs, many of which have been implicated or demonstrated to cause disease when mutated. Here we report new evidence that Whi2 responds to specific amino acids in the medium, particularly low leucine levels. We also discuss the known pathways of amino acid signaling and potential points of regulation by Whi2 in nutrient signaling in yeast and mammals.

Comment on "Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death".

Shlezinger et al (Reports, 8 September 2017, p. 1037) report that the common fungus Aspergillus fumigatus, a cause of aspergillosis, undergoes caspase-dependent apoptosis-like cell death triggered by lung neutrophils. However, the technologies they used do not provide reliable evidence that fungal cells die via a protease signaling cascade thwarted by a fungal caspase inhibitor homologous to human survivin.

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins.

The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes.