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.

Comparative Analysis of Capsular and Secreted Polysaccharides Produced by Rhodotorula mucilaginosa and Cryptococcus neoformans.

Fungal infections are a global public health challenge, especially among immunocompromised patients. Basidiomycetous yeasts, such as Rhodotorula mucilaginosa, have emerged as opportunistic pathogens, but have received less attention than Cryptococcus neoformans. This study aimed to characterize the polysaccharides of R. mucilaginosa and compare them with those of C. neoformans, analyzing their clinical implications. Comprehensive physicochemical, mechanical, and ultrastructural analyses of polysaccharides from both species were performed, revealing correlations with virulence and pathogenicity. R. mucilaginosa cells are surrounded by a capsule smaller than that produced by C. neoformans, but with similar polysaccharides. Those polysaccharides are also secreted by R. mucilaginosa. Cross-reactivity with R. mucilaginosa was observed in a diagnostic C. neoformans antigen test, using both in vitro and in vivo samples, highlighting the need for more reliable tests. Some R. mucilaginosa strains exhibited virulence comparable to that of C. neoformans in an invertebrate experimental model (Tenebrio molitor). This study contributes to a deeper understanding of yeast pathogenicity and virulence, highlighting the need for more accurate diagnostic tests to improve the differential diagnosis of infections caused by basidiomycetous yeasts.

Clinical Challenges of Emerging and Re-Emerging Yeast Infections in the Context of the COVID-19 Pandemic.

During the geological eras, some fungi, through adaptation and/or environmental/ecological pressure, interacted directly and indirectly with humans, through occasionally harmful interaction interdependent on the individual's immunological condition. Infections caused by yeasts are underreported, subjugated, and underdiagnosed, and treatment is restricted to a few drugs, even after the significant progress of medicine and pharmacology. In the last centuries, antagonistically, there has been an exponential increase of immunocompromised individuals due to the use of immunosuppressive drugs such as corticosteroids, increased cases of transplants, chemotherapeutics, autoimmune diseases, neoplasms, and, more recently, coronavirus disease 2019 (COVID-19). This review aims to survey emerging and re-emerging yeast infections in the current clinical context. Currently, there is an immense clinical challenge for the rapid and correct diagnosis and treatment of systemic mycoses caused by yeasts due to the terrible increase in cases in the current context of COVID-19.

Ultrastructural Study of Cryptococcus neoformans Surface During Budding Events.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. It is surrounded by three concentric structures that separate the cell from the extracellular space: the plasma membrane, the cell wall and the polysaccharide (PS) capsule. Although several studies have revealed the chemical composition of these structures, little is known about their ultrastructural organization and remodeling during C. neoformans budding events. Here, by combining the latest and most accurate light and electron microscopy techniques, we describe the morphological remodeling that occurs among the capsule, cell wall and plasma membrane during budding in C. neoformans. Our results show that the cell wall deforms to generate a specialized region at one of the cell's poles. This region subsequently begins to break into layers that are slightly separated from each other and with thick tips. We also observe a reorganization of the capsular PS around the specialized regions. While daughter cells present their PS fibers aligned in the direction of budding, mother cells show a similar pattern but in the opposite direction. Also, daughter cells form multilamellar membrane structures covering the continuous opening between both cells. Together, our findings provide compelling ultrastructural evidence for C. neoformans surface remodeling during budding, which may have important implications for future studies exploring these remodeled specialized regions as drug-targets against cryptococcosis.

Dexamethasone and Methylprednisolone Promote Cell Proliferation, Capsule Enlargement, and in vivo Dissemination of C. neoformans.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, who often have some inflammatory condition and, therefore, end up using glucocorticoids, such as dexamethasone and methylprednisolone. Although the effects of this class of molecules during cryptococcosis have been investigated, their consequences for the biology of C. neoformans is less explored. Here, we studied the effects of dexamethasone and methylprednisolone on the metabolism and on the induction of virulence factors in C. neoformans. Our results showed that both glucocorticoids increased fungal cell proliferation and surface electronegativity but reduced capsule and secreted polysaccharide sizes, as well as capsule compaction, by decreasing the density of polysaccharide fibers. We also tested whether glucocorticoids could affect the fungal virulence in Galleria mellonella and mice. Although the survival rate of Galleria larvae increased, those from mice showed a tendency to decrease, with infected animals dying earlier after glucocorticoid treatments. The pathogenesis of spread of cryptococcosis and the interleukin secretion pattern were also assessed for lungs and brains of infected mice. While increases in the spread of the fungus to lungs were observed after treatment with glucocorticoids, a significant difference in brain was observed only for methylprednisolone, although a trend toward increasing was also observed for dexamethasone. Moreover, increases in both pulmonary and cerebral IL-10 production, reduction of IL-6 production but no changes in IL-4, IL-17, and INF-γ were also observed after glucocorticoid treatments. Finally, histopathological analysis confirmed the increase in number of fungal cells in lung and brain tissues of mice previously subjected to dexamethasone or methylprednisolone treatments. Together, our results provide compelling evidence for the effects of dexamethasone and methylprednisolone on the biology of C. neoformans and may have important implications for future clinical treatments, calling attention to the risks of using these glucocorticoids against cryptococcosis or in immunocompromised individuals.

Membrane Elastic Properties During Neural Precursor Cell Differentiation.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.

The mechanical properties of microbial surfaces and biofilms.

Microbes can modify their surface structure as an adaptive mechanism for survival and dissemination in the environment or inside the host. Altering their ability to respond to mechanical stimuli is part of this adaptive process. Since the 1990s, powerful micromanipulation tools have been developed that allow mechanical studies of microbial cell surfaces, exploring little known aspects of their dynamic behavior. This review concentrates on the study of mechanical and rheological properties of bacteria and fungi, focusing on their cell surface dynamics and biofilm formation.

The environmental yeast Cryptococcus liquefaciens produces capsular and secreted polysaccharides with similar pathogenic properties to those of C. neoformans.

Invasive fungal infections, including cryptococcosis, are a growing threat to immunocompromised patients. Although Cryptococcus neoformans and Cryptococcus gattii are the main agents of human cryptococcosis, opportunistic infections by environmental species, such as C. liquefaciens, have been observed recently. The main Cryptococcus virulence factor is the production and secretion of polysaccharides (PS). Previously, we showed that both species produce PS of similar composition. Here, we examined the ultrastructure and biological activity of capsular and secreted PS from C. liquefaciens, and yeast pathogenicity to an invertebrate host, in comparison with C. neoformans. Ultrastructural analysis by high-resolution microscopy showed that both species produce large and complex capsules. PS from both species had indistinguishable effects on phagocytosis levels, NO production and the secretion of a variety of immune mediators. Challenge with C. liquefaciens or C. neoformans led to complete lethality of G. mellonella larvae. Treatment with C. liquefaciens PS could not protect mice against infection with C. neoformans. We conclude that polysaccharides of the environmental yeast C. liquefaciens have strikingly similar ultrastructural and biological properties to those of C. neoformans, highlighting the importance of monitoring the emergence of new fungal pathogens for which thermotolerance may be an important transitional step towards pathogenesis in humans.

Cryptococcus neoformans capsular polysaccharides form branched and complex filamentous networks viewed by high-resolution microscopy.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. Its main virulence factor is an extracellular polysaccharide capsule whose structure, assembly and dynamics remain poorly understood. In this study, we apply improved protocols for sample preparation and recently-developed scanning microscopy techniques to visualize the ultrastructure of the C. neoformans capsule at high-resolution (up to 1 nm) and improved structural preservation. Although most capsule structures in nature consist of linear polymers, we show here that the C. neoformans capsule is a 'microgel-like' structure composed of branched polysaccharides. Moreover, we imaged the capsule-to-cell wall link, which is formed by thin fibers that branch out of thicker capsule filaments, and have one end firmly embedded in the cell wall structure. Together, our findings provide compelling ultrastructural evidence for a branched and complex capsule conformation, which may have important implications for the biological activity of the capsule as a virulence factor.