The \u3ci\u3eC. neoformans\u3c/i\u3e Cell Wall: A Scaffold for Virulence

Cryptococcus neoformans is a globally distributed opportunistic fungal pathogen and the causative agent of life threatening cryptococcal meningoencephalitis in immunocompromised individuals, resulting in ~180,000 deaths each year worldwide. A primary virulence-associated trait of this organism is the production of melanin. Melanins are a class of diverse pigments produced via the oxidation and polymerization of aromatic ring compounds that have a characteristically complex, heterogenous, and amorphous structure. They are synthesized by representatives of all biological kingdoms and share a multitude of remarkable properties such as the ability to absorb ultraviolet (UV) light and protect against ionizing radiation. Melanin production in fungi contributes to pathogenicity by conferring resistance to environmental assault and interfering with the host immune response. In C. neoformans, melanin pigments are extruded into the cell wall and form strong associations with various key constituents that are essential for its accumulation and in turn, its ability to function as a virulence factor. The most well-established components of this melanization “scaffold” are the polysaccharides chitin and chitosan; despite a body of evidence suggesting that perturbing the biosynthesis of these polysaccharides disrupts melanization and significantly diminishes virulence, elucidating the underlying molecular interactions between chitin/chitosan and the melanin pigment has proven challenging due to the complex and incompletely-defined structures of the resulting macromolecular fungal assemblies. In this work we use solid-state NMR (ssNMR), a technique uniquely suited to study complex and heterogeneous composite materials, to obtain a molecular level characterization of the cellular “scaffold” on which C. neoformans pigments are deposited. We demonstrate that augmenting the relative or total amount of chitin and chitin modulates cell wall architecture and influences both pigment deposition and retention. We additionally illustrate that perturbing chitin/chitosan biosynthesis unfavorably affects cell-wall flexibility and prevents the accumulation of melanin. Moreover, we identify lipids as an additional group of cellular constituents that strongly bind to the melanin pigment and thus could play a role in the melanization process. In sum, our findings support a model in which it is not the formation of melanin pigments that serves as a virulence factor in C. neoformans, but rather the deposition and retention of the pigments within the cell wall, and thus it is the melanized cell wall that is important rather than the melanin itself

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

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

Use of endotracheal stent for hemostasis of a bleeding glomus tumor.

Glomus tumor is a rare benign neoplasm, commonly present in the dermis but cases have been reported at the gastrointestinal and respiratory tracts. We present a case of a tracheal glomus tumor that developed persistent bleeding after bronchoscopic mechanical debulking/biopsy, that required placement of endobronchial stent for hemostasis. Case A 52 year-old, nonsmoker, female presented with dry persistent cough and shortness of breath. CT scan showed a lower tracheal tumor measuring 1.5 cm. Bronchoscopy revealed an endotracheal tumor at the distal third of the trachea. Endobronchial biopsy at outside hospital was non-diagnostic and patient was referred to our service for diagnosis. Rigid bronchoscopy revealed an endotracheal sessile tumor, causing 60% obstruction of the tracheal lumen. Mechanical debulking was performed with the apple-coring technique. Debulking of 80% of the tumor was achieved, however it was complicated by persistent bleeding from the mass. Mechanical compression at the base of the tumor using the bronchoscope temporarily controlled the bleeding. A 16mm balloon was inflated for tamponading with partial results. It was decided to deploy an endobronchial stent to achieve permanent hemostasis. A self-expanding metal stent 18×60mm (Aero Merit Medical, Utah, USA) was deployed to cover the bed of the tumor completely, which caused an immediate and sustained hemostasis. Pathology report showed a Glomus tumor with benign features. Two weeks later the stent was removed and the patient was referred for surgical evaluation. Discussion Glomus tumor is a rare, benign neoplasm with male: female ratio of 7:1. Grossly it is a pink vascular nodule. Histologically, it is composed of glomus cells with prominent vascular and smooth muscle components. Preoperative linear Doppler ultrasonography should be done to specify the size, depth and anatomic criteria. Transthoracic segmental resection with primary reconstruction is the treatment of choice yet bronchoscopic resection has been used to relieve obstruction prior to the definitive operation. Submucosal invasion is common so bronchoscopic resection should be reserved for the surgical high risk population given the risk of recurrence. Criteria for malignancy include deep location and a size \u3e2, or atypical mitotic figures, or high nuclear grade and \u3e5 mitotic figures per HPF. Our case illustrates a Glomus tumor that was partially resected with minimal invasion using rigid bronchoscopy, post resection bleeding was successfully managed by placing a stent that successfully achieved hemostasis. Conclusion Self-expanding metal stent could be used to achieve hemostasis for bleeding endotracheal tumors, like Glomus tumors

Restenosis Following Bronchoscopic Airway Stenting for Complex Tracheal Stenosis

BACKGROUND: Nonsurgical patients with complex postintubation tracheal stenosis (PITS) and tracheostomy-associated tracheal stenosis (PTTS) often require airway stenting. However, the optimal approach is unknown. Identifying patients at higher risk for restenosis after stent removal may allow the treating physician to individualize the vigilance and duration of airway stenting, and help optimize outcomes. METHODS: This was a single-center retrospective analysis of prospectively collected data on all patients with complex PITS and/or PTTS treated with protocolized bronchoscopic airway stenting over a consecutive 16-year period. The primary outcome analyzed was restenosis rate at 1 year after stent removal. Predictors for restenosis and factors influencing risk for death during stent therapy were also assessed. RESULTS: Of the 181 subjects treated with silicone airway stenting, 128 were available for analysis of the primary outcome. Restenosis by 1 year after stent removal occurred in 58%. Independent predictors for restenosis were coexisting diabetes [odd ratio (OR)=3.10, 95% confidence interval (CI)=1.04-9.24; P=0.04], morbid obesity (OR=3.13, 95% CI=1.20-8.17; P=0.02), and occurrence of stent-associated complications requiring bronchoscopic management (OR=2.13, 95% CI=1.12-4.03; P=0.02). The overall mortality during the initial stenting period was 14%, and a silicone Y-stent was associated with a higher risk of death (OR=3.58, 95% CI=1.40-9.14; P=0.008). CONCLUSION: Tracheal restenosis after silicone stent therapy for complex PITS and PTTS is common and more likely to occur in patients with diabetes, morbid obesity, and frequent stent-associated complications. Mortality risk during stent therapy is not negligible, and a Y-stent should be utilized only after careful consideration. These findings may be incorporated into the approach to bronchoscopic airway stenting in these patients