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L-3,4-dihydroxyphenylalanine (L-DOPA), a naturally occurring tyrosine derivative, is prevalent in environments that include mosquito habitats, potentially serving as part of their diet. Given its role as a precursor for melanin synthesis we investigated the effect of dietary L-DOPA on mosquito physiology and immunity to Plasmodium falciparum and Cryptococcus neoformans infection. Dietary L-DOPA was incorporated into mosquito melanin via a non-canonical pathway and had profound transcriptional effects that were associated with enhanced immunity, increased pigmentation, and reduced lifespan. Increased melanization resulted in an enhanced capacity to absorb electromagnetic radiation that affected mosquito temperatures. Bacteria in the mosquito microbiome were sources of dopamine, which is a substrate for melanization. Our results illustrate how an environmentally abundant amino acid analogue can affect mosquito physiology and suggest its potential usefulness as an environmentally friendly vector control agent to reduce malaria transmission, warranting further research and field studies.

Cryptococcus neoformans is a fungal pathogen that can cause life-threatening brain infections in immunocompromised individuals. Unlike other fungal pathogens, it possesses a protective polysaccharide capsule that is crucial for its virulence. During infections, Cryptococcus cells release copious amounts of extracellular polysaccharides (exo-PS) that interfere with host immune responses. Both exo-PS and capsular-PS play pivotal roles in Cryptococcus infections and serve as essential targets for disease diagnosis and vaccine development strategies. However, understanding their structure is complicated by their polydispersity, complexity, sensitivity to sample isolation and processing, and scarcity of methods capable of isolating and analyzing them while preserving their native structure. In this study, we employ small-angle neutron scattering (SANS) and ultra-small-angle neutron scattering (USANS) for the first time to investigate both fungal cell suspensions and extracellular polysaccharides in solution. Our data suggests that exo-PS in solution exhibits collapsed chain-like behavior and demonstrates mass fractal properties that indicate a relatively condensed pore structure in aqueous environments. This observation is also supported by scanning electron microscopy (SEM). The local structure of the polysaccharide is characterized as a rigid rod, with a length scale corresponding to 3-4 repeating units. This research not only unveils insights into exo-PS and capsular-PS structures but also demonstrates the potential of USANS for studying changes in cell dimensions and the promise of contrast variation in future neutron scattering studies.

Understanding the mechanisms of antibody-mediated neutralization of SARS-CoV-2 is critical in combating the COVID-19 pandemic. Based on previous reports of antibody catalysis, we investigated the proteolysis of spike (S) by antibodies in COVID-19 convalescent plasma (CCP) and its contribution to viral neutralization. Quenched fluorescent peptides were designed based on S epitopes to sensitively detect antibody-mediated proteolysis. We observed epitope cleavage by CCP from different donors which persisted when plasma was heat-treated or when IgG was isolated from plasma. Further, purified CCP antibodies proteolyzed recombinant S domains, as well as authentic viral S. Cleavage of S variants suggests CCP antibody-mediated proteolysis is a durable phenomenon despite antigenic drift. We differentiated viral neutralization occurring via direct interference with receptor binding from that occurring by antibody-mediated proteolysis, demonstrating that antibody catalysis enhanced neutralization. These results suggest that antibody-catalyzed damage of S is an immunologically relevant function of neutralizing antibodies against SARS-CoV-2., Competing Interests: Declaration of interests None declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Practical experiments drive important scientific discoveries in biology, but theory-based research studies also contribute novel-sometimes paradigm-changing-findings. Here, we appraise the roles of theory-based approaches focusing on the experiment-dominated wet-biology research areas of microbial growth and survival, cell physiology, host-pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome-sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most-direct and most-scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non-mainstream; and the importance of theory-based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more-recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID-19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology-related papers and-although we ourselves are involved in laboratory experiments and practical fieldwork-also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Fungi play essential roles in global health, ecology, and economy, but their thermal biology is relatively unexplored. Mushrooms, the fruiting body of mycelium, were previously noticed to be colder than surrounding air through evaporative cooling. Here, we confirm those observations using infrared thermography and report that this hypothermic state is also observed in mold and yeast colonies. The relatively colder temperature of yeasts and molds is also mediated via evaporative cooling and associated with the accumulation of condensed water droplets on plate lids above colonies. The colonies appear coldest at their center and the surrounding agar appears warmest near the colony edges. The analysis of cultivated Pleurotus ostreatus mushrooms revealed that the hypothermic feature of mushrooms can be observed throughout the whole fruiting process and at the level of mycelium. The mushroom's hymenium was coldest, and different areas of the mushroom appear to dissipate heat differently. We also constructed a mushroom-based air-cooling prototype system capable of passively reducing the temperature of a semiclosed compartment by approximately 10 °C in 25 min. These findings suggest that the fungal kingdom is characteristically cold. Since fungi make up approximately 2% of Earth's biomass, their evapotranspiration may contribute to cooler temperatures in local environments.

Melanin is a complex pigment that is found in various fungal species and is associated with a multitude of protective functions against environmental stresses. In Cryptococcus neoformans, melanin is synthesized from exogenous substrate and deposited in the cell wall. Although melanin is often cited as a protector against mechanical stress, there is a paucity of direct experimental data supporting this claim. To probe whether melanin enhances cellular strength, we used ultrasonic cavitation and French cell press pressure to stress cryptococcal cells and then measured changes in cellular morphology and fragmentation for melanized and nonmelanized C. neoformans cells. Melanized yeast cells exhibited lower rates of fragmentation and greater cell areas than did nonmelanized yeast cells after sonication or French press passage. When subjected to French press passage, both melanized and nonmelanized cells exhibited responses that were dependent on their culture age. Our results indicate that melanization protects against some of the morphological changes, such as fragmentation and cellular shrinkage, that are initiated by mechanical energy derived from either sonic cavitation or French press passage, thus supporting the notion that this pigment provides mechanical strength for fungal cell walls. IMPORTANCE Melanin was shown in prior microbiological experiments to be associated with protection against environmental stressors, and it has often been cited as being associated with mechanical stress protection. However, there is a lack of direct experimentation to confirm this claim. We examined the responses of melanized and nonmelanized C. neoformans cells to sonication and French press passage, and we report differences in outcomes depending not only on melanization status but also on culture age. Such findings have important implications for the design and interpretation of laboratory experiments involving C. neoformans. In addition, the elucidation of some of the mechanical properties of melanin promotes further research into fungal melanin applications in health care and industry.

As human activity in space continues to increase, understanding how biological assets respond to spaceflight conditions is becoming more important. Spaceflight conditions include exposure to ionizing radiation, microgravity, spacecraft vibrations and hypervelocity; all of which can affect the viability of biological organisms. Previous studies have shown that melanin-producing fungi are capable of surviving the vacuum of space and Mars-simulated conditions in Low Earth Orbit. This survival has been associated in part with the protective effects of melanin, but a comparison of fungal viability in the presence or absence of melanin following spaceflight has never been tested. In this study, we evaluated the protective effects of melanin by comparing the viability of melanized and non-melanized clones of Cryptococcus neoformans yeasts following a roundtrip to the International Space Station. Yeast colonies were placed inside two MixStix silicone tubes; one stayed on Earth and the other was transported inside for 29 days before returning to Earth. Post-flight analysis based on colony-forming unit numbers shows that melanized yeast viability was 50% higher than non-melanized yeasts, while no difference was observed between the Earth-bound control samples. The results suggest that fungal melanin could increase the lifespan of biological assets in space., (© 2022 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall-associated polysaccharide aggregates that we call "capsule ghosts", implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones., Competing Interests: Conflict of interest The authors have no conflicts of interest to report., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Candida auris is a recently described multidrug-resistant pathogenic fungus that is increasingly responsible for health care-associated outbreaks across the world. Bloodstream infections of this fungus cause death in up to 70% of cases. Aggravating this scenario, the disease-promoting mechanisms of C. auris are poorly understood. Fungi release extracellular vesicles (EVs) that carry a broad range of molecules, including proteins, lipids, carbohydrates, pigments, and RNA, many of which are virulence factors. Here, we carried out a comparative molecular characterization of C. auris and Candida albicans EVs and evaluated their capacity to modulate effector mechanisms of host immune defense. Using proteomics, lipidomics, and transcriptomics, we found that C. auris released EVs with payloads that were significantly different from those of EVs released by C. albicans. EVs released by C. auris potentiated the adhesion of this yeast to an epithelial cell monolayer, while EVs from C. albicans had no effect. C. albicans EVs primed macrophages for enhanced intracellular yeast killing, whereas C. auris EVs promoted survival of the fungal cells. Moreover, EVs from both C. auris and C. albicans induced the activation of bone marrow-derived dendritic cells. Together, our findings show distinct profiles and properties of EVs released by C. auris and by C. albicans and highlight the potential contribution of C. auris EVs to the pathogenesis of this emerging pathogen. IMPORTANCE Candida auris is a recently described multidrug-resistant pathogenic fungus that is responsible for outbreaks across the globe, particularly in the context of nosocomial infections. Its virulence factors and pathogenesis are poorly understood. Here, we tested the hypothesis that extracellular vesicles (EVs) released by C. auris are a disease-promoting factor. We describe the production of EVs by C. auris and compare their biological activities against those of the better-characterized EVs from C. albicans. C. auris EVs have immunoregulatory properties, of which some are opposite those of C. albicans EVs. We also explored the cargo and structural components of those vesicles and found that they are remarkably distinct compared to EVs from C. auris's phylogenetic relative Candida albicans.

Melanin is a complex multifunctional pigment found in all kingdoms of life, including fungi. The complex chemical structure of fungal melanins, yet to be fully elucidated, lends them multiple unique functions ranging from radioprotection and antioxidant activity to heavy metal chelation and organic compound absorption. Given their many biological functions, fungal melanins present many possibilities as natural compounds that could be exploited for human use. This review summarizes the current discourse and attempts to apply fungal melanin to enhance human health, remove pollutants from ecosystems, and streamline industrial processes. While the potential applications of fungal melanins are often discussed in the scientific community, they are successfully executed less often. Some of the challenges in the applications of fungal melanin to technology include the knowledge gap about their detailed structure, difficulties in isolating melanotic fungi, challenges in extracting melanin from isolated species, and the pathogenicity concerns that accompany working with live melanotic fungi. With proper acknowledgment of these challenges, fungal melanin holds great potential for societal benefit in the coming years.

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans ( Cn ), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides , and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo . In this work, we characterized the ability of Hc to produce cellular-attached (C-gly- Hc ) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly- Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly- Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly- Hc and E-gly- Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly- Hc and E-gly- Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly- Hc and E-gly- Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella . These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gonçalves, Rodriguez de La Noval, Ferreira, Honorato, Araújo, Frases, Pizzini, Nosanchuk, Cordero, Rodrigues, Peralta, Nimrichter and Guimarães.)

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-1 β 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., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Pedro Henrique Bürgel et al.)

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence., (© 2020 Crawford et al.)

Melanins are a unique class of pigments found throughout the biosphere with a wide variety of functions, structures, and presentations. Cordero and Casadevall highlight the wide range of places melanins are found and the diverse functions they play in nature., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans , the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes., (© 2020 Chrissian et al.)

The fungal human pathogen Cryptococcus neoformans undergoes melanization in response to nutrient starvation and exposure to exogenous melanin precursors. Melanization protects the fungus against host defense mechanisms such as oxidative damage and other environmental stressors (e.g., heat/cold stress, antimicrobial compounds, ionizing radiation). Conversely, the melanization process generates cytotoxic intermediates, and melanized cells are potentially susceptible to overheating and to certain melanin-binding drugs. Despite the importance of melanin in C. neoformans biology, the signaling mechanisms regulating its synthesis are poorly understood. The recent report by D. Lee, E.-H. Jang, M. Lee, S.-W. Kim, et al. [mBio 10(5):e02267-19, 2019, https://doi.org/10.1128/mBio.02267-19] provides new insights into how C. neoformans regulates melanization. The authors identified a core melanin regulatory network consisting of transcription factors and kinases required for melanization under low-nutrient conditions. The redundant and epistatic connections of this melanin-regulating network demonstrate that C. neoformans melanization is complex and carefully regulated at multiple levels. Such complex regulation reflects the multiple functions of melanin in C. neoformans biology., (Copyright © 2020 Cordero et al.)

The capsule of Cryptococcus neoformans is its dominant virulence factor and plays a key role in the biology of this fungus. In this essay, we focus on the capsule as a cellular structure and note the limitations inherent in the current methodologies available for its study. Given that no single method can provide the structure of the capsule, our notions of what is the cryptococcal capsule must be arrived at by synthesizing information gathered from very different methodological approaches including microscopy, polysaccharide chemistry and physical chemistry of macromolecules. The emerging picture is one of a carefully regulated dynamic structure that is constantly rearranged as a response to environmental stimulation and cellular replication. In the environment, the capsule protects the fungus against desiccation and phagocytic predators. In animal hosts the capsule functions in both offensive and defensive modes, such that it interferes with immune responses while providing the fungal cell with a defensive shield that is both antiphagocytic and capable of absorbing microbicidal oxidative bursts from phagocytic cells. Finally, we delineate a set of unsolved problems in the cryptococcal capsule field that could provide fertile ground for future investigations.

Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ∼200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ∼30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans' melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms., (© 2019 Camacho et al.)

Cryptococcus neoformans is an environmental pathogenic fungus with a worldwide geographical distribution that is responsible for hundreds of thousands of human cryptococcosis cases each year. During infection, the yeast undergoes a morphological transformation involving capsular enlargement that increases microbial volume. To understand the factors that play a role in environmental dispersal of C. neoformans and C. gattii , we evaluated the cell density of Cryptococcus using Percoll isopycnic gradients. We found differences in the cell densities of strains belonging to C. neoformans and C. gattii species complexes. The buoyancy of C. neoformans strains varied depending on growth medium. In minimal medium, the cryptococcal capsule made a major contribution to the cell density such that cells with larger capsules had lower density than those with smaller capsules. Removing the capsule, by chemical or mechanical methods, increased the C. neoformans cell density and reduced buoyancy. Melanization of the C. neoformans cell wall, which also contributes to virulence, produced a small but consistent increase in cell density. Encapsulated C. neoformans sedimented much more slowly in seawater as its density approached the density of water. Our results suggest a new function for the capsule whereby it can function as a flotation device to facilitate transport and dispersion in aqueous fluids. IMPORTANCE The buoyancy of a microbial cell is an important physical characteristic that may affect its transportability in fluids and interactions with tissues during infection. The polysaccharide capsule surrounding C. neoformans is required for infection and dissemination in the host. Our results indicate that the capsule has a significant effect on reducing cryptococcal cell density, altering its sedimentation in seawater. Modulation of microbial cell density via encapsulation may facilitate dispersal for other important encapsulated pathogens., (Copyright © 2018 Vij et al.)

Phagosomal acidification is a critical cellular mechanism for the inhibition and killing of ingested microbes by phagocytic cells. The acidic environment activates microbicidal proteins and creates an unfavorable environment for the growth of many microbes. Consequently, numerous pathogenic microbes have developed strategies for countering phagosomal acidification through various mechanisms that include interference with phagosome maturation. The human-pathogenic fungus Cryptococcus neoformans resides in acidic phagosomes after macrophage ingestion that actually provides a favorable environment for replication, since the fungus replicates faster at acidic pH. We hypothesized that the glucuronic acid residues in the capsular polysaccharide had the capacity to affect phagosomal acidity through their acid-base properties. A ratiometric fluorescence comparison of imaged phagosomes containing C. neoformans to phagosomes containing beads showed that the latter were significantly more acidic. Similarly, phagosomes containing nonencapsulated C. neoformans cells were more acidic than those containing encapsulated cells. Acid-base titrations of isolated C. neoformans polysaccharide revealed that it behaves as a weak acid with maximal buffering capacity around pH 4 to 5. We interpret these results as indicating that the glucuronic acid residues in the C. neoformans capsular polysaccharide can buffer phagosomal acidification. Interference with phagosomal acidification represents a new function for the cryptococcal capsule in virulence and suggests the importance of considering the acid-base properties of microbial capsules in the host-microbe interaction for other microbes with charged residues in their capsules. IMPORTANCE Cryptococcus neoformans is the causative agent of cryptococcosis, a devastating fungal disease that affects thousands of individuals worldwide. This fungus has the capacity to survive inside phagocytic cells, which contributes to persistence of infection and dissemination. One of the major antimicrobial mechanisms of host phagocytes is to acidify the phagosomal compartment after ingestion of microbes. This study shows that the capsule of C. neoformans can interfere with full phagosomal acidification by serving as a buffer., (Copyright © 2018 De Leon-Rodriguez et al.)

Pigmentation is a fundamental characteristic of living organisms that is used to absorb radiation energy and to regulate temperature. Since darker pigments absorb more radiation than lighter ones, they stream more heat, which can provide an adaptive advantage at higher latitudes and a disadvantage near the Tropics, because of the risk of overheating. This intuitive process of color-mediated thermoregulation, also known as the theory of thermal melanism (TTM), has been only tested in ectothermic animal models [1-8]. Here, we report an association between yeast pigmentation and their latitude of isolation, with dark-pigmented isolates being more frequent away from the Tropics. To measure the impact of microbial pigmentation in energy capture from radiation, we generated 20 pigmented variants of Cryptococcus neoformans and Candida spp. Infrared thermography revealed that dark-pigmented yeasts heated up faster and reached higher temperatures (up to 2-fold) than lighter ones following irradiation. Melanin-pigmented C. neoformans exhibited a growth advantage relative to non-melanized yeasts when incubated under the light at 4°C but increased thermal susceptibility at 25°C ambient temperatures. Our results extend the TTM to microbiology and suggest pigmentation as an ancient adaptation mechanism for gaining thermal energy from radiation. The contribution of microbial pigmentation in heat absorption is relevant to microbial ecology and for estimating global temperatures. The color variations available in yeasts provide new opportunities in chromatology to quantify radiative heat transfer and validate biophysical models of heat flow [9] that are not possible with plants or animals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Melanization is an intrinsic characteristic of many fungal species, but details of this process are poorly understood because melanins are notoriously difficult pigments to study. While studying the binding of cell-wall dyes, Eosin Y or Uvitex, to melanized and non-melanized Cryptococcus neoformans cells we noted that melanization leads to reduced fluorescence intensity, suggesting that melanin interfered with dye binding to the cell wall. The growth of C. neoformans in melanizing conditions with either of the cell-wall dyes resulted in an increase in supernatant-associated melanin, consistent with blockage of melanin attachment to the cell wall. This effect provided the opportunity to characterize melanin released into culture supernatants. Released melanin particles appeared mostly as networked structures having dimensions consistent with previously described extracellular vesicles. Hence, dye binding to the cell wall created conditions that resembled the 'leaky melanin' phenotype described for certain cell-wall mutants. In agreement with earlier studies on fungal melanins biosynthesis, our observations are supportive of a model whereby C. neoformans melanization proceeds by the attachment of melanin nanoparticles to the cell wall through chitin, chitosan, and various glucans.

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 μm cells and large titan cells (> 10 μm and up to 100 μm). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 μm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways., Competing Interests: The authors have declared that no competing interests exist.

Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15 N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother-daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In summary, GlcNAc supplementation had pleiotropic effects on cell-wall and melanin architectures, and thus established its capacity to perturb these structures, a property that could prove useful for metabolic tracking studies.

Microbial melanins provide a biocompatible and scalable approach for bioremediation and radioprotection technologies due to their physicochemical properties., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Melanin pigments are found in many diverse fungal species, where they serve a variety of functions that promote fitness and cell survival. Melanotic fungi inhabit some of the most extreme habitats on earth such as the damaged nuclear reactor at Chernobyl and the highlands of Antarctica, both of which are high-radiation environments. Melanotic fungi migrate toward radioactive sources, which appear to enhance their growth. This phenomenon, combined with the known capacities of melanin to absorb a broad spectrum of electromagnetic radiation and transduce this radiation into other forms of energy, raises the possibility that melanin also functions in harvesting such energy for biological usage. The ability of melanotic fungi to harness electromagnetic radiation for physiological processes has enormous implications for biological energy flows in the biosphere and for exobiology, since it provides new mechanisms for survival in extraterrestrial conditions. Whereas some features of the way melanin-related energy transduction works can be discerned by linking various observations and circumstantial data, the mechanistic details remain to be discovered.

Science is international by nature. Scientific exchange and international mobility are essential for training young scientists in general, and international collaboration has been directly linked to high-quality science and innovation. In this article, we present evidence showing that international mobility has a direct and beneficial impact on scientific discovery, career development and cultural maturity, especially for students and young scientists., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)