Measuring Stress Phenotypes in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human fungal pathogen capable of surviving in a wide range of environments and hosts. It has been developed as a model organism to study fungal pathogenesis due to its fully sequenced haploid genome and optimized gene deletion and mutagenesis protocols. These methods have greatly aided in determining the relationship between Cryptococcus genotype and phenotype. Furthermore, the presence of congenic mata and matα strains associated with a defined sexual cycle has helped further understand cryptococcal biology. Several in vitro stress conditions have been optimized to closely mimic the stress that yeast encounter in the environment or within the infected host. These conditions have proven to be extremely useful in elucidating the role of several genes in allowing yeast to adapt and survive in hostile external environments. This chapter describes various in vitro stress conditions that could be used to test the sensitivity of different mutant strains, as well as the protocol for preparing them. We have also included a list of mutants that could be used as a positive control strain when testing the sensitivity of the desired strain to a specific stress.

Induction of Dormancy in Cryptococcus neoformans In Vitro: The HypNOS Protocol.

Cryptococcus neoformans is the second major cause of death in patients with HIV. During a latent infection, this pathogenic fungus survives in the host for years without causing symptoms of active disease. Upon favorable conditions, such as immunosuppression due to HIV infection, or other conditions (steroid use or organ transplantation), the yeast may reactivate and cause active cryptococcosis. Hence, dormancy is an important phase in the pathogenesis of C. neoformans. Additionally, C. neoformans also persists during antifungal treatment and causes disease recurrence, which is a major medical problem, especially in low- and middle-income countries. To survive in the host, yeast cells must react to the stresses they are exposed to and generate a cellular response that is favorable for yeast survival. A prominent strategy used by C. neoformans to combat challenging surroundings is dormancy, which may translate into a viable, but nonculturable phenotype (VBNC). This chapter describes an in vitro protocol to generate and characterize dormant Cryptococci.

Measuring Replicative Lifespan in Cryptococcus neoformans.

Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic yeast Cryptococcus neoformans is essential because old yeast cells with age-related phenotypes accumulate during infection and are associated with increased virulence and antifungal tolerance. Microdissection and microfluidic devices are valuable tools for continuously tracking cells at the single-cell level. In this chapter, we describe the features of these two platforms and outline technical limitations and information to study aging mechanisms while assessing the lifespan of yeast cells.

In Vitro Titan Cell Generation in Cryptococcus neoformans and Automated Cell Size Measurements.

A special feature of the human fungal pathogen Cryptococcus neoformans is its morphological changes triggered by the interaction with the host. During infection, a specific increase in cell size is observed, particularly in lung tissue, from a typical cell size of 5-7 µm cells to cells larger than 10 µm, dubbed titan cells (TCs). However, the study of this specific cell subpopulation was, until now, only possible via recovery of TCs from lungs of mice during experimental infections where stable and reproducible generation of TCs occurs.The protocol described here generates TCs using in vitro conditions and measures cell size using a rapid, automated method. TC generation in vitro is robust and reproducible, generating yeast cells harboring the same characteristics of TCs generated in vivo.

Use of 2D minilungs from human embryonic stem cells to study the interaction of Cryptococcus neoformans with the respiratory tract.

Organoids can meet the needs between the use of cell culture and in vivo work, bringing together aspects of multicellular tissues, providing a more similar in vitro system for the study of various components, including host-interactions with pathogens and drug response. Organoids are structures that resemble organs in vivo, originating from pluripotent stem cells (PSCs) or adult stem cells (ASCs). There is great interest in deepening the understanding of the use of this technology to produce information about fungal infections and their treatments. This work aims the use 2D human lung organoid derived from human embryonic stem cells (hESCs), to investigate Cryptococcus neoformans-host interactions. C. neoformans is an opportunistic fungus acquired by inhalation that causes systemic mycosis mainly in immunocompromised individuals. Our work highlights the suitability of human minilungs for the study of C. neoformans infection (adhesion, invasion and replication), the interaction with the surfactant and induction of the host's alveolar pro-inflammatory response.

Histone acetyltransferase Gcn5-mediated histone H3 acetylation facilitates cryptococcal morphogenesis and sexual reproduction.

IMPORTANCE: Eukaryotic gene transcription is typically regulated by a series of histone modifications, which play a crucial role in adapting to complex environmental stresses. In the ubiquitous human fungal pathogen Cryptococcus neoformans, sexual life cycle is a continuous intracellular differentiation process that strictly occurs in response to mating stimulation. Despite the comprehensive identification of the regulatory factors and genetic pathways involved in its sexual cycle, understanding of the epigenetic modifications involved in this process remains quite limited. In this research, we found that histone acetyltransferase Gcn5-mediated histone H3 acetylation plays a crucial role in completing the cryptococcal sexual cycle, including yeast-hyphae morphogenesis and the subsequent sexual reproduction. Furthermore, we demonstrated that Gcn5 participates in this process primarily through regulating the key morphogenesis regulator Znf2 and its targets. This study thus provided a comprehensive understanding of how histone acetylation modification impacts sexual life cycle in a high-risk human pathogenic fungus.

Mouse Organotypic Brain Slice Cultures: A Novel Model for Studying Neuroimmune Responses to Cryptococcal Brain Infections.

Cryptococcal meningitis affects millions of people worldwide and is especially prevalent in regions with a high burden of HIV/AIDS. The study of the pathophysiology of this often fatal disease has been significantly hindered by the lack of reliable experimental models, especially at the level of the brain, which is the main organ of injury. Here we outline our novel protocol for the use of hippocampal organotypic brain slice cultures (HOCs) to study the host-fungal interactions during cryptococcal infections of the brain. HOCs are a powerful platform for investigating neuroimmune interactions as they allow for the preservation of all innate neuroglial cells including microglia, astrocytes, and neurons, all of which maintain their three-dimensional architecture and functional connectivity. We made HOCs from neonatal mice and infected these with a fluorescent strain of Cryptococcus neoformans for 24 h. Using immunofluorescent staining, we confirmed the presence and morphology of microglia, astrocytes, and neurons in HOCs prior to infection. Using fluorescent and light microscopy, we also confirmed that Cryptococcus neoformans encapsulates and buds in vitro, as it would in a host. Finally, we demonstrate that infection of HOCs with Cryptococcus neoformans results in close association of the fungal cells with host microglial cells. Our results demonstrate the utility of HOCs as a model to study the pathophysiology and host neuroimmune responses in neurocryptococcosis, which may assist in improving our collective understanding of the pathogenesis of this disease.

Vomocytosis of Cryptococcus neoformans cells from murine, bone marrow-derived dendritic cells.

Cryptococcus neoformans (CN) cells survive within the acidic phagolysosome of macrophages (MΦ) for extended times, then escape without impacting the viability of the host cell via a phenomenon that has been coined 'vomocytosis'. Through this mechanism, CN disseminate throughout the body, sometimes resulting in a potentially fatal condition-Cryptococcal Meningitis (CM). Justifiably, vomocytosis studies have focused primarily on MΦ, as alveolar MΦ within the lung act as first responders that ultimately expel this fungal pathogen. Herein, we hypothesize that dendritic cells (DCs), an innate immune cell with attributes that include phagocytosis and antigen presentation, can also act as 'vomocytes'. Presciently, this report shows that vomocytosis of CN indeed occurs from murine, bone marrow-derived DCs. Primarily through time-lapse microscopy imaging, we show that rates of vomocytosis events from DCs are comparable to those seen from MΦ and further, are independent of the presence of the CN capsule and infection ratios. Moreover, the phagosome-altering drug bafilomycin A inhibits this phenomenon from DCs. Although DC immunophenotype does not affect the total number of vomocytic events, we observed differences in the numbers of CN per phagosome and expulsion times. Interestingly, these observations were similar in murine, bone marrow-derived MΦ. This work not only demonstrates the vomocytic ability of DCs, but also investigates the complexity of vomocytosis regulation in this cell type and MΦ under multiple modulatory conditions. Understanding the vomocytic behavior of different phagocytes and their phenotypic subtypes is needed to help elucidate the full picture of the dynamic interplay between CN and the immune system. Critically, deeper insight into vomocytosis could reveal novel approaches to treat CM, as well as other immune-related conditions.

A comparative study of IL-33 and its receptor ST2 in a C57BL/6 J mouse model of pulmonary Cryptococcus neoformans infection.

It has been reported that IL-33 receptor ST2 deficiency mitigates Cryptococcus neoformans (C. neoformans) pulmonary infection in BALB/c mice. IL-33 may modulate immune responses in ST2-dependent and ST2-independent manners. The host genetic background (i.e., BALB/c, C57BL/6 J) influences immune responses against C. neoformans. In the present study, we aimed to explore the roles of IL-33 and ST2 in pulmonary C. neoformans-infected mice on a C57BL/6 J genetic background. C. neoformans infection increased IL-33 expression in lung tissues. IL-33 deficiency but not ST2 deficiency significantly extended the survival time of C. neoformans-infected mice. In contrast, either IL-33 or ST2 deficiency reduced fungal burdens in lung, spleen and brain tissues from the mice following C. neoformans intratracheal inoculation. Similarly, inflammatory responses in the lung tissues were more pronounced in both the IL-33-/- and ST2-/- infected mice. However, mucus production was decreased in IL-33-/- infected mice alone, and the level of IL-5 in bronchoalveolar lavage fluid (BALF) was substantially decreased in the IL-33-/- infected mice but not ST2-/- infected mice. Moreover, IL-33 deficiency but not ST2 deficiency increased iNOS-positive macrophages. At the early stage of infection, the reduced pulmonary fungal burden in the IL-33-/- and ST2-/- mice was accompanied by increased neutrophil infiltration. Collectively, IL-33 regulated pulmonary C. neoformans infection in an ST2-dependent and ST2-independent manner in C57BL/6 J mice.

Macrophage Mediated Immunomodulation During Cryptococcus Pulmonary Infection.

Macrophages are key cellular components of innate immunity, acting as the first line of defense against pathogens to modulate homeostatic and inflammatory responses. They help clear pathogens and shape the T-cell response through the production of cytokines and chemokines. The facultative intracellular fungal pathogen Cryptococcus neoformans has developed a unique ability to interact with and manipulate host macrophages. These interactions dictate how Cryptococcus infection can remain latent or how dissemination within the host is achieved. In addition, differences in the activities of macrophages have been correlated with differential susceptibilities of hosts to Cryptococcus infection, highlighting the importance of macrophages in determining disease outcomes. There is now abundant information on the interaction between Cryptococcus and macrophages. In this review we discuss recent advances regarding macrophage origin, polarization, activation, and effector functions during Cryptococcus infection. The importance of these strategies in pathogenesis and the potential of immunotherapy for cryptococcosis treatment is also discussed.

Ionizing radiation and chemical oxidant exposure impacts on Cryptococcus neoformans transfer RNAs.

Cryptococcus neoformans is a fungus that is able to survive abnormally high levels of ionizing radiation (IR). The radiolysis of water by IR generates reactive oxygen species (ROS) such as H2O2 and OH-. C. neoformans withstands the damage caused by IR and ROS through antioxidant production and enzyme-catalyzed breakdown of ROS. Given these particular cellular protein needs, questions arise whether transfer ribonucleic acids molecules (tRNAs) undergo unique chemical modifications to maintain their structure, stability, and/or function under such environmental conditions. Here, we investigated the effects of IR and H2O2 exposure on tRNAs in C. neoformans. We experimentally identified the modified nucleosides present in C. neoformans tRNAs and quantified changes in those modifications upon exposure to oxidative conditions. To better understand these modified nucleoside results, we also evaluated tRNA pool composition in response to the oxidative conditions. We found that regardless of environmental conditions, tRNA modifications and transcripts were minimally affected. A rationale for the stability of the tRNA pool and its concomitant profile of modified nucleosides is proposed based on the lack of codon bias throughout the C. neoformans genome and in particular for oxidative response transcripts. Our findings suggest that C. neoformans can rapidly adapt to oxidative environments as mRNA translation/protein synthesis are minimally impacted by codon bias.

Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans.

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications.

The Cyclin Cln1 Controls Polyploid Titan Cell Formation following a Stress-Induced G2 Arrest in Cryptococcus.

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

miR­4792 regulates inflammatory responses in Cryptococcus neoformans­infected microglia.

Investigating the factors that influence the inflammatory response of microglial cells is crucial for understanding the pathogenesis of cryptococcal meningitis (CM). MicroRNAs (miRNAs/miRs) play an important role in inducing host defenses and activating the immune response during microbial infection; however, the regulatory mechanisms of miRNAs in cryptococcal meningitis remain poorly defined. In a previous study, the authors assessed the miRNA profiles of THP­1 (human acute monocytic leukemia cells) cells following Cryptococcus neoformans (C. neoformans) infection. In the present study, it was found that miR­4792 expression was downregulated in BV2 cells infected with C. neoformans, whilst that of its target gene, epidermal growth factor receptor (EGFR), was upregulated. Infected cells in which miR­4792 was overexpressed exhibited a decreased EGFR transcript expression, reduced mitogen­activated protein kinase (MAPK) signaling and a decreased secretion of inflammatory cytokines. In addition, following antifungal treatment in patients with cryptococcal meningitis, the levels of miR­4792 in the cerebrospinal fluid significantly increased, whilst the expression of EGFR significantly decreased. In addition, receiver operator characteristic analysis revealed miR­4792 (AUCROC=0.75) and EGFR (AUCROC=0.79) as potential diagnostic markers in patients with cryptococcal meningitis.

Macrophage-Derived Osteopontin Influences the Amplification of Cryptococcus neoformans-Promoting Type 2 Immune Response.

A multifunctional glycoprotein, osteopontin (OPN), can modulate the function of macrophages, resulting in either protective or deleterious effects in various inflammatory diseases and infection in the lungs. Although macrophages play the critical roles in mediating host defenses against cryptococcosis or cryptococcal pathogenesis, the involvement of macrophage-derived OPN in pulmonary infection caused by fungus Cryptococcus has not been elucidated. Thus, our current study aimed to investigate the contribution of OPN to the regulation of host immune response and macrophage function using a mouse model of pulmonary cryptococcosis. We found that OPN was predominantly expressed in alveolar macrophages during C. neoformans infection. Systemic treatment of OPN during C. neoformans infection resulted in an enhanced pulmonary fungal load and an early onset of type 2 inflammation within the lung, as indicated by the increase of pulmonary eosinophil infiltration, type 2 cytokine production, and M2-associated gene expression. Moreover, CRISPR/Cas9-mediated OPN knockout murine macrophages had enhanced ability to clear the intracellular fungus and altered macrophage phenotype from pathogenic M2 to protective M1. Altogether, our data suggested that macrophage-derived OPN contributes to the elaboration of C. neoformans-induced type 2 immune responses and polarization of M2s that promote fungal survival and proliferation within macrophages.

The prevalence and mortality of cryptococcal meningitis in patients with autoimmune diseases: a systematic review and meta-analysis.

Growing evidence suggests that autoimmune diseases (AIDs) are risk factors for cryptococcal meningitis (CM). Therefore, understanding the epidemiological and clinical profile of CM in patients with AIDs is important. This meta-analysis assessed the prevalence, clinical profiles, and clinical outcomes of CM in AIDs. Studies on CM in patients with AIDs were searched for in PubMed, EMBASE, Web of Science, and China National Knowledge Infrastructure, and meta-analyses were performed using the statistical program of R. Nineteen studies with 36,631 patients with AIDs were analyzed. The overall pooled CM prevalence was 0.4% (95% confidence interval [CI], 0.3-0.6%), 90.7% of which occurred in female patients. Thirteen studies with 77 patients with AIDs diagnosed with CM were analyzed, and the mortality rate was 26.7% (95% CI, 9.5-47.2%). Of patients with systemic lupus erythematosus, 30.1% of CM cases were initially misdiagnosed (95% CI, 0-65.6%). The primary symptom of CM with AIDs was headache (99.4%; 95% CI, 92.1-100%), followed by fever (93.7%; 95% CI, 82.8-100%) and vomiting (37.2%; 95% CI, 13.2-61.2%). The prevalence of CM infections among patients with AIDs should not be underestimated despite non-specific clinical presentations as there were fatal outcomes. Our results suggest that more research is needed to understand the relationship between AIDs and CM, and clinical trials are necessary to improve treatment strategies.

Vph1 is associated with the copper homeostasis of Cryptococcus neoformans serotype D.

We clarified the roles of VPH1 in Cryptococcus neoformans serotype D by examining the detailed phenotypes of VPH1-deficient cells (Δvph1) in terms of their capability to grow in acidic and alkaline pH, at a high temperature, and under high osmotic conditions, in addition to the involvement of VPH1 in copper (Cu) homeostasis and the expression of some C. neoformans virulence factors. Δvph1 could grow well on minimal medium (YNB) but exhibited hypersensitivity to 20 µM Cu due to the failure to induce Cu-detoxifying metallothionein genes (CMT1 and CMT2). In contrast, Δvph1 exhibited defective growth on rich medium (YPD), and the induction of Cu transporter genes (CTR1 and CTR4) did not occur in this medium, implying that this strain was incapable of the uptake of Cu ions for growth. However, the addition of excess Cu promoted CTR gene expression and supported Δvph1 growth. These results suggested that the lack of the VPH1 gene disturbed Cu homeostasis in C. neoformans. Moreover, the loss of Vph1 function influenced the urease activity of C. neoformans.

Fungal brain infection modelled in a human-neurovascular-unit-on-a-chip with a functional blood-brain barrier.

The neurovascular unit, which consists of vascular cells surrounded by astrocytic end-feet and neurons, controls cerebral blood flow and the permeability of the blood-brain barrier (BBB) to maintain homeostasis in the neuronal milieu. Studying how some pathogens and drugs can penetrate the human BBB and disrupt neuronal homeostasis requires in vitro microphysiological models of the neurovascular unit. Here we show that the neurotropism of Cryptococcus neoformans-the most common pathogen causing fungal meningitis-and its ability to penetrate the BBB can be modelled by the co-culture of human neural stem cells, brain microvascular endothelial cells and brain vascular pericytes in a human-neurovascular-unit-on-a-chip maintained by a stepwise gravity-driven unidirectional flow and recapitulating the structural and functional features of the BBB. We found that the pathogen forms clusters of cells that penetrate the BBB without altering tight junctions, suggesting a transcytosis-mediated mechanism. The neurovascular-unit-on-a-chip may facilitate the study of the mechanisms of brain infection by pathogens, and the development of drugs for a range of brain diseases.

Cryptococcal infection of the colon in a patient without concurrent human immunodeficiency infection: a case report and literature review.

Cryptococcosis is a fungal infection that is rarely reported in patients without human immunodeficiency virus (HIV) infection, especially when the central nervous system (CNS) or pulmonary system is not involved. We report a case of isolated colonic cryptococcosis without disseminated disease in a 64-year-old immunocompetent woman without HIV infection who presented with chronic diarrhea and no episodes of fever or weight loss. The diagnosis was based on histopathology examination. Furthermore, we performed a literature review showing that few reports have been published so far and in the case of colonic cryptococcal infection, the prognosis is favorable among HIV-uninfected patients.

Cryptococcus neoformans-Infected Macrophages Release Proinflammatory Extracellular Vesicles: Insight into Their Components by Multi-omics.

Cryptococcus neoformans causes deadly mycosis in immunocompromised individuals. Macrophages are key cells fighting against microbes. Extracellular vesicles (EVs) are cell-to-cell communication mediators. The roles of EVs from infected host cells in the interaction with Cryptococcus remain uninvestigated. Here, EVs from viable C. neoformans-infected macrophages reduced fungal burdens but led to shorter survival of infected mice. In vitro, EVs induced naive macrophages to an inflammatory phenotype. Transcriptome analysis showed that EVs from viable C. neoformans-infected macrophages activated immune-related pathways, including p53 in naive human and murine macrophages. Conserved analysis demonstrated that basic cell biological processes, including cell cycle and division, were activated by infection-derived EVs from both murine and human infected macrophages. Combined proteomics, lipidomics, and metabolomics of EVs from infected macrophages showed regulation of pathways such as extracellular matrix (ECM) receptors and phosphatidylcholine. This form of intermacrophage communication could serve to prepare cells at more distant sites of infection to resist C. neoformans infection.IMPORTANCECryptococcus neoformans causes cryptococcal meningitis, which is frequent in patients with HIV/AIDS, especially in less-developed countries. The incidence of cryptococcal meningitis is close to 1 million each year globally. Macrophages are key cells that protect the body against microbes, including C. neoformans Extracellular vesicles are a group of membrane structures that are released from cells such as macrophages that modulate cell activities via the transfer of materials such as proteins, lipids, and RNAs. In this study, we found that Cryptococcus neoformans-infected macrophages produce extracellular vesicles that enhance the inflammatory response in Cryptococcus-infected mice. These Cryptococcus neoformans-infected macrophage vesicles also showed higher fungicidal biological effects on inactivated macrophages. Using omics technology, unique protein and lipid signatures were identified in these extracellular vesicles. Transcriptome analysis showed that these vesicles activated immune-related pathways like p53 in naive macrophages. The understanding of this intermacrophage communication could provide potential targets for the design of therapeutic agents to fight this deadly mycosis.