Biology subjects

Stark, R. E.

Publications and source records attributed to Stark, R. E..

7 recordsLinked to original sources

bioRxiv2025

Polymorphic α-Glucans as Structural Scaffolds in Cryptococcus Cell Walls for Chitin, Capsule, and Melanin: Insights from 13C and 1H Solid-State NMR

Cryptococcus species are major fungal pathogens responsible for life-threatening infections in approximately a million individuals globally each year, with alarmingly high mortality rates. These fungi are distinguished by a distinctive cell wall architecture further reinforced by two virulence-associated layers, melanin and capsule, rendering them insensitive to antifungal agents targeting the cell wall, such as echinocandins. The molecular interplay between these three biomolecular layers remains poorly understood. Here we employ solid-state NMR spectroscopy to examine intact cells of both wild-type and capsule-deficient strains of C. neoformans, along with its melanized cells. High-resolution 13C and 1H data revealed five distinct structural forms of -1,3-glucans that play versatile roles in forming the rigid cell wall scaffold by interacting with chitin microfibrils and chitosan, and in stabilizing the mobile matrix by associating with {beta}-1,6-glucan and a small fraction of {beta}-1,3-glucan. Two primary forms of -1,3-glucans were distributed throughout the cell wall, hosting melanin deposition in the inner domain and capsule attachment on the cell surface. These findings offer a paradigm shift in understanding the cryptococcal cell wall and its interaction with two key virulence factors on opposite sides, raising critical biochemical questions that could inform the development of more effective antifungal treatments for cryptococcosis.

biochemistry↗

bioRxiv2024

New Insights Into The Melanin Structure Of Lomentospora prolificans

Lomentospora prolificans is a filamentous fungus with a global distribution, manifesting particularly higher prevalence in human-impacted environments. This organism is associated with a wide spectrum of human infections, especially in immunosuppressed individuals, for whom it causes severe and debilitating illnesses with high morbidity and mortality that are compounded by its pan-resistant profile with respect to antifungal drugs. Melanin is a ubiquitous pigment among fungi with a broad range of actions that include promoting fungal virulence. Although melanin is one of the most studied virulence factors in pathogenic fungi, relatively little is known about the chemistry of this pigment in L. prolificans. In the current study we characterized L. prolificans-associated melanin using chemical, biological, biophysical and structural techniques, also assessing the impact of inhibitors of distinct melanization pathways. Our results reveal that this pathogenic fungus makes multiple types of melanin pigments and suggests the possibility of a new type of melanin, which is synthesized together with a mixture of DHN-, DOPA- and pyomelanin types. These insights enhance our understanding of L. prolificans virulence mechanisms, paving the way for potential therapeutic interventions.

biochemistry↗

bioRxiv2024

Dietary L-3,4-dihydroxyphenylalanine (L-DOPA) augments cuticular melanization in Anopheles mosquitos while reducing their lifespan and malaria parasite burden

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 investigate the effect of dietary L-DOPA on mosquito physiology and immunity to Plasmodium falciparum and Cryptococcus neoformans infection. Dietary L-DOPA is incorporated into mosquito melanin via a non-canonical pathway and has a profound transcriptional effect associated with enhanced immunity, increased pigmentation, and reduced lifespan. Increased melanization results in an enhanced capacity to absorb electromagnetic radiation that affects mosquito temperatures. Bacteria in the mosquito microbiome act as sources of dopamine, 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.

microbiology↗

bioRxiv2023

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.

biochemistry↗

bioRxiv2021

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.

microbiology↗

bioRxiv2020

Solid-state NMR spectroscopy identifies three classes of lipids in C. 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 non-melanizing conditions; triglycerides (TGs), sterol esters (SEs) and polyisoprenoids (PPs) were the major constituents. Although no quantitative differences were found between melanized and non-melanized 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 non-melanized 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.

biophysics↗

bioRxiv2019

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-asociated 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 of [~]200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres of [~]30 nm in diameter, the 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.

microbiology↗