Cuticular profiling of insecticide resistant Aedes aegypti.

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms. Contact-based insecticides are absorbed through the insect cuticle, which is comprised mainly of chitin polysaccharides, cuticular proteins, hydrocarbons, and phenolic biopolymers sclerotin and melanin. Cuticle interface alterations can slow or prevent insecticide penetration in a phenomenon referred to as cuticular resistance. Cuticular resistance characterization of the yellow fever mosquito, Aedes aegypti, is lacking. In the current study, we utilized solid-state nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and transmission electron microscopy to gain insights into the cuticle composition of congenic cytochrome P450 monooxygenase insecticide resistant and susceptible Ae. aegypti. No differences in cuticular hydrocarbon content or phenolic biopolymer deposition were found. In contrast, we observed cuticle thickness of insecticide resistant Ae. aegypti increased over time and exhibited higher polysaccharide abundance. Moreover, we found these local cuticular changes correlated with global metabolic differences in the whole mosquito, suggesting the existence of novel cuticular resistance mechanisms in this major disease vector.

Cuticular profiling of insecticide resistant Aedes aegypti.

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms. Contact-based insecticides are absorbed through the insect cuticle, which is comprised mainly of chitin polysaccharides, cuticular proteins, hydrocarbons, and phenolic biopolymers sclerotin and melanin. Cuticle interface alterations can slow or prevent insecticide penetration in a phenomenon referred to as cuticular resistance. Cuticular resistance characterization of the yellow fever mosquito, Aedes aegypti , is lacking. In the current study, we utilized solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy, gas chromatography/mass spectrometry (GC-MS), and transmission electron microscopy (TEM) to gain insights into the cuticle composition of congenic cytochrome P450 monooxygenase insecticide resistant and susceptible Ae. aegypti . No differences in cuticular hydrocarbon content or phenolic biopolymer deposition were found. In contrast, we observed cuticle thickness of insecticide resistant Ae. aegypti increased over time and exhibited higher polysaccharide abundance. Moreover, we found these local cuticular changes correlated with global metabolic differences in the whole mosquito, suggesting the existence of novel cuticular resistance mechanisms in this major disease vector.

Melanization of Candida auris Is Associated with Alteration of Extracellular pH.

Candida auris is a recently emerged global fungal pathogen, which causes life-threatening infections, often in healthcare settings. C. auris infections are worrisome because the fungus is often resistant to multiple antifungal drug classes. Furthermore, C. auris forms durable and difficult to remove biofilms. Due to the relatively recent, resilient, and resistant nature of C. auris, we investigated whether it produces the common fungal virulence factor melanin. Melanin is a black-brown pigment typically produced following enzymatic oxidation of aromatic precursors, which promotes fungal virulence through oxidative stress resistance, mammalian immune response evasion, and antifungal peptide and pharmaceutical inactivation. We found that certain strains of C. auris oxidized L-DOPA and catecholamines into melanin. Melanization occurred extracellularly in a process mediated by alkalinization of the extracellular environment, resulting in granule-like structures that adhere to the fungus' external surface. C. auris had relatively high cell surface hydrophobicity, but there was no correlation between hydrophobicity and melanization. Melanin protected the fungus from oxidative damage, but we did not observe a protective role during infection of macrophages or Galleria mellonella larvae. In summary, C. auris alkalinizes the extracellular medium, which promotes the non-enzymatic oxidation of L-DOPA to melanin that attaches to its surface, thus illustrating a novel mechanism for fungal melanization.

Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin.

Melanin is a major virulence factor in pathogenic fungi that enhances the ability of fungal cells to resist immune clearance. Cryptococcus neoformans is an important human pathogenic fungus that synthesizes melanin from exogenous tissue catecholamine precursors during infection, but the type of melanin made in cryptococcal meningoencephalitis is unknown. We analyzed the efficacy of various catecholamines found in brain tissue in supporting melanization using animal brain tissue and synthetic catecholamine mixtures reflecting brain tissue proportions. Solid-state NMR spectra of the melanin pigment produced from such mixtures yielded more melanin than expected if only the preferred constituent dopamine had been incorporated, suggesting uptake of additional catecholamines. Probing the biosynthesis of melanin using radiolabeled catecholamines revealed that C. neoformans melanization simultaneously incorporated more than one catecholamine, implying that the pigment was polytypic in nature. Nonetheless, melanin derived from individual or mixed catecholamines had comparable ability to protect C. neoformans against ultraviolet light and oxidants. Our results indicate that melanin produced during infection differs depending on the catecholamine composition of tissue and that melanin pigment synthesized in vivo is likely to accrue from the polymerization of a mixture of precursors. From a practical standpoint, our results strongly suggest that using dopamine as a polymerization precursor is capable of producing melanin pigment comparable to that produced during infection. On a more fundamental level, our findings uncover additional structural complexity for natural cryptococcal melanin by demonstrating that pigment produced during human infection is likely to be composed of polymerized moieties derived from chemically different precursors.

Unconventional Constituents and Shared Molecular Architecture of the Melanized Cell Wall of C. neoformans and Spore Wall of S. cerevisiae.

The fungal cell wall serves as the interface between the cell and the environment. Fungal cell walls are composed largely of polysaccharides, primarily glucans and chitin, though in many fungi stress-resistant cell types elaborate additional cell wall structures. Here, we use solid-state nuclear magnetic resonance spectroscopy to compare the architecture of cell wall fractions isolated from Saccharomyces cerevisiae spores and Cryptococcus neoformans melanized cells. The specialized cell walls of these two divergent fungi are highly similar in composition. Both use chitosan, the deacetylated derivative of chitin, as a scaffold on which a polyaromatic polymer, dityrosine and melanin, respectively, is assembled. Additionally, we demonstrate that a previously identified but uncharacterized component of the S. cerevisiae spore wall is composed of triglycerides, which are also present in the C. neoformans melanized cell wall. Moreover, we identify a tyrosine-derived constituent in the C. neoformans wall that, although it is not dityrosine, is a non-pigment constituent of the cell wall. The similar composition of the walls of these two phylogenetically distant species suggests that triglycerides, polyaromatics, and chitosan are basic building blocks used to assemble highly stress-resistant cell walls and the use of these constituents may be broadly conserved in other fungal species.

Tailoring NMR experiments for structural characterization of amorphous biological solids: A practical guide.

Many interesting solid-state targets for biological research do not form crystalline structures; these materials include intrinsically disordered proteins, plant biopolymer composites, cell-wall polysaccharides, and soil organic matter. The absence of aligned repeating structural elements and atomic-level rigidity presents hurdles to achieving structural elucidation and obtaining functional insights. We describe strategies for adapting several solid-state NMR methods to determine the molecular structures and compositions of these amorphous biosolids. The main spectroscopic problems in studying amorphous structures by NMR are over/under-sampling of the spin signals and spectral complexity. These problems arise in part because amorphous biosolids typically contain a mix of rigid and mobile domains, making it difficult to select a single experiment or set of acquisition conditions that fairly represents all nuclear spins in a carbon-based organic sample. These issues can be addressed by running hybrid experiments, such as using direct excitation alongside cross polarization-based methods, to develop a more holistic picture of the macromolecular system. In situations of spectral crowding or overlap, the structural elucidation strategy can be further assisted by coupling 13C spins to nuclei such as 15N, filtering out portions of the spectrum, highlighting individual moieties of interest, and adding a second or third spectral dimension to an NMR experiment in order to spread out the resonances and link them pairwise through space or through bonds. We discuss practical aspects and illustrations from the recent literature for 1D experiments that use cross or direct polarization and both homo- and heteronuclear 2D and 3D solid-state NMR experiments.

Solid-state NMR spectroscopy identifies three classes of lipids in Cryptococcus neoformans melanized cell walls and whole fungal cells.

A primary virulence-associated trait of the opportunistic fungal pathogen Cryptococcus neoformans is the production of melanin pigments that are deposited into the cell wall and interfere with the host immune response. Previously, our solid-state NMR studies of isolated melanized cell walls (melanin "ghosts") revealed that the pigments are strongly associated with lipids, but their identities, origins, and potential roles were undetermined. Herein, we exploited spectral editing techniques to identify and quantify the lipid molecules associated with pigments in melanin ghosts. The lipid profiles were remarkably similar in whole C. neoformans cells, grown under either melanizing or nonmelanizing conditions; triglycerides (TGs), sterol esters (SEs), and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and nonmelanized cells, melanin ghosts were relatively enriched in SEs and PPs. In contrast to lipid structures reported during early stages of fungal growth in nutrient-rich media, variants found herein could be linked to nutrient stress, cell aging, and subsequent production of substances that promote chronic fungal infections. The fact that TGs and SEs are the typical cargo of lipid droplets suggests that these organelles could be connected to C. neoformans melanin synthesis. Moreover, the discovery of PPs is intriguing because dolichol is a well-established constituent of human neuromelanin. The presence of these lipid species even in nonmelanized cells suggests that they could be produced constitutively under stress conditions in anticipation of melanin synthesis. These findings demonstrate that C. neoformans lipids are more varied compositionally and functionally than previously recognized.

Melanin deposition in two Cryptococcus species depends on cell-wall composition and flexibility.

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.

The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans.

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.

The melanization road more traveled by: Precursor substrate effects on melanin synthesis in cell-free and fungal cell systems.

Natural brown-black eumelanin pigments confer structural coloration in animals and potently block ionizing radiation and antifungal drugs. These functions also make them attractive for bioinspired materials design, including coating materials for drug-delivery vehicles, strengthening agents for adhesive hydrogel materials, and free-radical scavengers for soil remediation. Nonetheless, the molecular determinants of the melanin "developmental road traveled" and the resulting architectural features have remained uncertain because of the insoluble, heterogeneous, and amorphous characteristics of these complex polymeric assemblies. Here, we used 2D solid-state NMR, EPR, and dynamic nuclear polarization spectroscopic techniques, assisted in some instances by the use of isotopically enriched precursors, to address several open questions regarding the molecular structures and associated functions of eumelanin. Our findings uncovered: 1) that the identity of the available catecholamine precursor alters the structure of melanin pigments produced either in Cryptococcus neoformans fungal cells or under cell-free conditions; 2) that the identity of the available precursor alters the scaffold organization and membrane lipid content of melanized fungal cells; 3) that the fungal cells are melanized preferentially by an l-DOPA precursor; and 4) that the macromolecular carbon- and nitrogen-based architecture of cell-free and fungal eumelanins includes indole, pyrrole, indolequinone, and open-chain building blocks that develop depending on reaction time. In conclusion, the availability of catecholamine precursors plays an important role in eumelanin development by affecting the efficacy of pigment formation, the melanin molecular structure, and its underlying scaffold in fungal systems.

N-acetylglucosamine affects Cryptococcus neoformans cell-wall composition and melanin architecture.

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 15N-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.

Uncovering potential 'herbal probiotics' in Juzen-taiho-to through the study of associated bacterial populations.

Juzen-taiho-to (JTT) is an immune-boosting formulation of ten medicinal herbs. It is used clinically in East Asia to boost the human immune functions. The active factors in JTT have not been clarified. But, existing evidence suggests that lipopolysaccharide (LPS)-like factors contribute to the activity. To examine this possibility, JTT was subjected to a series of analyses, including high resolution mass spectrometry, which suggested the presence of structural variants of LPS. This finding opened a possibility that JTT contains immune-boosting bacteria. As the first step to characterize the bacteria in JTT, 16S ribosomal RNA sequencing was carried out for Angelica sinensis (dried root), one of the most potent immunostimulatory herbs in JTT. The sequencing revealed a total of 519 bacteria genera in A. sinensis. The most abundant genus was Rahnella, which is widely distributed in water and plants. The abundance of Rahnella appeared to correlate with the immunostimulatory activity of A. sinensis. In conclusion, the current study provided new pieces of evidence supporting the emerging theory of bacterial contribution in immune-boosting herbs.