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Development of a novel ?-1,6-glucan-specific detection system using functionally-modified recombinant endo-?-1,6-glucanase.

ABSTRACT: ?-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring ?-1,6-glucan, another primary ?-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for ?-1,6-glucan using a modified recombinant endo-?-1,6-glucanase having diminished glucan hydrolase activity. The purified ?-1,6-glucanase derivative bound to the ?-1,6-glucan pustulan with a KD of 16.4 nm We validated the specificity of this ?-1,6-glucan probe by demonstrating its ability to detect cell wall ?-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long ?-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular ?-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro We also used this assay to measure ?-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for ?-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

SUBMITTER: Yamanaka D

PROVIDER: S-EPMC7170528 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Development of a novel β-1,6-glucan-specific detection system using functionally-modified recombinant endo-β-1,6-glucanase.

Yamanaka Daisuke D Takatsu Kazushiro K Kimura Masahiro M Swamydas Muthulekha M Ohnishi Hiroaki H Umeyama Takashi T Oyama Fumitaka F Lionakis Michail S MS Ohno Naohito N

The Journal of biological chemistry 20200304 16

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan ...[more]

PMID: 32132174

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Project description:UnlabelledThe fungal pathogen Histoplasma capsulatum parasitizes host phagocytes. To avoid antimicrobial immune responses, Histoplasma yeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. Pathogenic Histoplasma yeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show that Histoplasma Eng1 is a glucanase that hydrolyzes ?-(1,3)-glycosyl linkages but is not required for Histoplasma growth in vitro or for cell separation. However, Histoplasma yeasts lacking Eng1 function have attenuated virulence in vivo, particularly during the cell-mediated immunity stage. Histoplasma yeasts deficient for Eng1 show increased exposure of cell wall ?-glucans, which results in enhanced binding to the Dectin-1 ?-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-?) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed ?-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In ?-glucan-producing Histoplasma strains, Eng1 acts in concert with ?-glucan to minimize ?-glucan exposure: ?-glucan provides a masking function by covering the ?-glucan-rich cell wall, while Eng1 removes any remaining exposed ?-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed ?-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes.ImportanceThe success of Histoplasma capsulatum as an intracellular pathogen results, in part, from an ability to minimize its detection by receptors on phagocytic cells of the immune system. In this study, we showed that Histoplasma pathogenic yeast cells, but not avirulent mycelia, secrete a ?-glucanase, Eng1, which reduces recognition of fungal cell wall ?-glucans. We demonstrated that the Eng1 ?-glucanase promotes Histoplasma virulence by reducing levels of surface-exposed ?-glucans on yeast cells, thereby enabling Histoplasma yeasts to escape detection by the host ?-glucan receptor, Dectin-1. As a consequence, phagocyte recognition of Histoplasma yeasts is reduced, leading to less proinflammatory cytokine production by phagocytes and less control of Histoplasma infection in vivo Thus, Histoplasma yeasts express two mechanisms to avoid phagocyte detection: masking of cell wall ?-glucans by ?-glucan and enzymatic removal of exposed ?-glucans by the Eng1 ?-glucanase.

Dataset Information

Development of a novel ?-1,6-glucan-specific detection system using functionally-modified recombinant endo-?-1,6-glucanase.

Publications

Development of a novel β-1,6-glucan-specific detection system using functionally-modified recombinant endo-β-1,6-glucanase.

Similar Datasets

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