Immunoglobulin constant regions provide stabilization to the paratope and enforce epitope specificity.

Constant domains in antibody molecules at the level of the Fab (CH1 and CL) have long been considered to be simple scaffolding elements that physically separate the paratope-defining variable (V) region from the effector function-mediating constant (C) regions. However, due to recent findings that C domains of different isotypes can modulate the fine specificity encoded in the V region, elucidating the role of C domains in shaping the paratope and influencing specificity is a critical area of interest. To dissect the relative contributions of each C domain to this phenomenon, we generated antibody fragments with different C regions omitted, using a set of antibodies targeting capsular polysaccharide from the fungal pathogen, Cryptococcus neoformans. Antigen specificity mapping and functional activity measurements revealed that V region-only antibody fragments exhibited poly-specificity to antigenic variants and extending to recognition of self-antigens, while measurable hydrolytic activity of the capsule was greatly attenuated. To better understand the mechanistic origins of the remarkable loss of specificity that accompanies the removal of C domains from identical paratopes, we performed molecular dynamics simulations which revealed increased paratope plasticity in the scFv relative to the corresponding Fab. Together, our results and provide insight into how the remarkable specificity of immunoglobulins is governed and maintained at the level of the Fab through the enforcement of structural restrictions on the paratope by CH1 domains.

In Vivo Modeling of Cryptococcus neoformans Infection and Collection of Murine Samples.

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease.

Biolistic Transformation of Cryptococcus neoformans.

Biolistic transformation of Cryptococcus neoformans is used as a molecular tool to genetically alter or delete targeted genes. The DNA is introduced into the yeast on DNA-coated gold beads by a helium shock wave produced using a biolistic particle system. The procedure often involves insertion of a dominant selectable marker into the desired site by homologous recombination. To increase the likelihood of homologous recombination, large fragments of overlapping DNA are used. The two most used dominant selectable markers are nourseothricin and Geneticin. With the need to generate multiple gene deletions in the same strain, there are recyclable marker systems, such as the bacteriophage P1 Cre-loxP system or CRISPR that provide additional useful molecular tools. While newer strategies exist to generate deletions and introduce markers and other gene modifications, biolistic transformation has remained a viable tool to facilitate the construction of genetically modified yeast strains. This chapter provides a working protocol on how to delete and restore a gene in C. neoformans.

Models for Inducing Experimental Cryptococcosis in Mice.

Cryptococcus neoformans and Cryptococcus gattii are the predominant etiological agents of cryptococcosis, a particularly problematic disease in immunocompromised individuals. The increased clinical use of immunosuppressive drugs, the inherent ability of Cryptococcus species to suppress and evade host immune responses, and the emergence of drug-resistant yeast support the need for model systems that facilitate the design of novel immunotherapies and antifungals to combat disease progression. The mouse model of cryptococcosis is a widely used system to study Cryptococcus pathogenesis and the efficacy of antifungal drugs in vivo. In this chapter, we describe three commonly used strategies to establish cryptococcosis in mice: intranasal, intratracheal, and intravenous inoculations. Also, we discuss the methodology for delivering drugs to mice via intraperitoneal injection.

Electron Microscopy of Cryptococcus neoformans: Processing Challenges to Avoid Artifacts.

This chapter describes methodological details for preparing specimens of Cryptococcus neoformans (although it can be applied to any species of the genus) and their subsequent analysis by scanning and transmission electron microscopy. Adaptations to conventional protocols for better preservation of the sample, as well as to avoid artifacts, are presented. The protocols may be used to examine both the surface ultrastructure and the interior of this pathogenic fungus in detail.

Assessing Phagocytosis of Cryptococcus neoformans Cells in Human Monocytes or the J774 Murine Macrophage Cell Line.

Monocyte/macrophage cells play a central role in innate immunity against C. neoformans and C. gattii, species known to cause human disease. Cryptococcus is the only fungal genus known to possess such a large extracellular polysaccharide capsule, which impacts interactions of innate cells with the yeast. This interaction results in different fates, such as phagocytosis and intracellular proliferation and, as the interaction progresses, vomocytosis, cell-to-cell transfer, lysis of macrophages, or yeast killing. Differentiating internalized versus external Cryptococcus cells is thus essential to evaluate monocyte-macrophage phagocytosis. We describe here a protocol that allows quantification of Cryptococcus spp. phagocytosis using quantitative flow cytometry in human monocytes and a murine macrophage cell line (J774).

Proteomic Profiling of Samples Derived from a Murine Model Following Cryptococcus neoformans Infection.

Proteomic profiling provides in-depth information about the regulation of diverse biological processes, activation of and communication across signaling networks, and alterations to protein production, modifications, and interactions. For infectious disease research, mass spectrometry-based proteomics enables detection of host defenses against infection and mechanisms used by the pathogen to evade such responses. In this chapter, we outline protein extraction from organs, tissues, and fluids collected following intranasal inoculation of a murine model with the human fungal pathogen Cryptococcus neoformans. We describe sample preparation, followed by purification, processing on the mass spectrometer, and a robust bioinformatics analysis. The information gleaned from proteomic profiling of fungal infections supports the detection of novel biomarkers for diagnostic and prognostic purposes.

Two Methods of Measuring Cryptococcus neoformans Fungal Burden in Macrophages.

The ability of C. neoformans to survive and replicate within host phagocytes enables it to evade the immune system and allows for persistence of the infection. As such, measuring fungal burden of C. neoformans strains-and indeed how drug treatments can influence fungal burden-provides important information about C. neoformans pathogenesis. In this chapter, we describe two methods that may be used to appraise fungal burden: a standard end-point colony-formation assay for calculating the average number of yeast per host cell and a fluorescence microscopy-based method that may be used to measure changes in fungal burden in individual living macrophages in real time.

Quantification of C. neoformans Capsule Diameter.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

Interaction Between Macrophages and Cryptococcus neoformans: Distinguishing Phagocytosed Versus External Fungi.

The interaction between macrophages and Cryptococcus neoformans is crucial in the pathogenesis of cryptococcosis. These phagocytes are important immune effectors, but also a niche in which facultative intracellular parasites, such as C. neoformans, thrive. Consequently, phagocytosis of cryptococcal cells and its outcomes are very frequently studied. One major issue with several of the tests used for this, however, is that macrophage-C. neoformans interaction does not always result in phagocytosis, as fungi may be attached to the external surface of the phagocyte. The most used methodologies to study phagocytosis of cryptococcal cells have varying degrees of precision in separating fungi that are truly internalized from those that are outside macrophages. Here we describe two assays to measure phagocytosis that can differentiate internal from external C. neoformans cells.

Measuring Laccase Activity and Melanin Production in Cryptococcus neoformans.

Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.

Antibody Isolation in C. neoformans.

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

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.

Vaccine Strategies for Cryptococcus neoformans.

Cryptococcus neoformans infections are a major worldwide concern as current treatment strategies are becoming less effective in alleviating the infection. The most extreme and fatal cases are those of immunocompromised individuals. Clinical treatments for cryptococcosis are limited to a few classes of approved drugs, and due to a rise in drug resistance, these drugs are becoming less effective. Therefore, it is essential to develop innovative ways to control this infection. Vaccinations have emerged as a safe, viable, and cost-effective solution to treat a number of diseases over the years. Currently, there are no clinically available vaccines to treat cryptococcal infections, but a number of studies have shown promising results in animal models. Here, we present step-by-step experimental protocols using live-attenuated or heat-killed C. neoformans cells as a vaccination strategy in a preventive or in a therapeutic murine model of cryptococcosis.

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.

Blap-6, a Novel Antifungal Peptide from the Chinese Medicinal Beetle Blaps rhynchopetera against Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans) is a pathogenic fungus that can cause life-threatening meningitis, particularly in individuals with compromised immune systems. The current standard treatment involves the combination of amphotericin B and azole drugs, but this regimen often leads to inevitable toxicity in patients. Therefore, there is an urgent need to develop new antifungal drugs with improved safety profiles. We screened antimicrobial peptides from the hemolymph transcriptome of Blaps rhynchopetera (B. rhynchopetera), a folk Chinese medicine. We found an antimicrobial peptide named blap-6 that exhibited potent activity against bacteria and fungi. Blap-6 is composed of 17 amino acids (KRCRFRIYRWGFPRRRF), and it has excellent antifungal activity against C. neoformans, with a minimum inhibitory concentration (MIC) of 0.81 µM. Blap-6 exhibits strong antifungal kinetic characteristics. Mechanistic studies revealed that blap-6 exerts its antifungal activity by penetrating and disrupting the integrity of the fungal cell membrane. In addition to its direct antifungal effect, blap-6 showed strong biofilm inhibition and scavenging activity. Notably, the peptide exhibited low hemolytic and cytotoxicity to human cells and may be a potential candidate antimicrobial drug for fungal infection caused by C. neoformans.

Impact of Nitric Oxide-Release Kinetics on Antifungal Activity.

Pathogenic fungi are an increasing health threat due to the rise in drug resistance. The limited number of antifungals currently available and growing incidence of multi-drug-resistant fungi has caused rising healthcare costs and a decreased quality of life for patients with fungal infections. Nitric oxide (NO) has previously been shown to act as an antimicrobial agent, albeit with a limited understanding of the effects of the NO-release kinetics against pathogenic fungi. Herein, the antifungal effects of four nitric oxide-releasing small molecules were studied against the pathogenic fungi Candida albicans, Candida auris, Cryptococcus neoformans, and Aspergillus fumigatus, to demonstrate the broad-spectrum antifungal activity of NO. A bolus dose of NO was found to eradicate fungi after 24 h, where nitric oxide donors with shorter half-lives achieved antifungal activity at lower concentrations and thus had wider selectivity indexes. Each NO donor was found to cause a severe surface destruction of fungi, and all NO donors exhibited compatibility with currently prescribed antifungals against several different fungi species.

Disseminated Cryptococcosis Post Eculizumab Therapy: A Case Report and Literature Review.

Eculizumab is a biologic medication used for the treatment of complement-related disorders including anti-acetylcholine receptor antibody-positive generalized myasthenia gravis. It targets C5 complement, preventing its cleavage into active terminal components. Thus, vaccination against encapsulated organisms is advised before starting this treatment. C5 also has a critical role against Cryptococcus neoformans infection. Here, we present a case of a 34-year-old man with a history of myasthenia gravis who was treated with prednisone and azathioprine in addition to eculizumab that was added to his regimen about a year ago, and who came to the hospital with headache, and was found to have Cryptococcus meningitis with disseminated cryptococcosis. The patient was negative for human immunodeficiency virus. He was treated with antifungal medications, and his condition improved. Although rarely reported, it is important to have a low threshold for diagnosis of cryptococcosis in patients on eculizumab given its complement inhibition mechanism of action.

A fungal protein organizes both glycogen and cell wall glucans.

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and ß-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.

Human plasma-like medium (HPLM) induces Cryptococcus neoformans in vivo cell morphologies.

The fungal pathogen Cryptococcus neoformans (C. neoformans) forms yeast cells of different sizes and morphological characteristics during infection. These features are usually not seen in standard laboratory in vitro conditions. Here, we describe in vivo cell morphologies when C. neoformans is grown in human plasma-like medium at 37°C, 5% CO2. We observed mixed-size populations of cells less than 1 µm up to 16.8 µm in cell diameter, increased capsule size, high chitin, and DNA content in larger cells. Our findings show that serum is not required for human plasma-like medium (HPLM)-induced C. neoformans cellular heterogeneity. Thus, this new method offers an opportunity to investigate factors of C. neoformans that mediate pathogenesis or host-pathogen interactions in a physiologically relevant setting.IMPORTANCEWe provide a description of new in vitro culture condition using the human plasma-like medium that supports the formation of the full range of in vivo cell morphologies of C. neoformans.