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J Biol Chem ; 295(16): 5362-5376, 2020 04 17.
Article in English | MEDLINE | ID: mdl-32132174

ABSTRACT

ß-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 ß-1,6-glucan, another primary ß-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for ß-1,6-glucan using a modified recombinant endo-ß-1,6-glucanase having diminished glucan hydrolase activity. The purified ß-1,6-glucanase derivative bound to the ß-1,6-glucan pustulan with a KD of 16.4 nm We validated the specificity of this ß-1,6-glucan probe by demonstrating its ability to detect cell wall ß-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 ß-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 ß-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 ß-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 ß-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.


Subject(s)
Biosensing Techniques/methods , Fungal Polysaccharides/chemistry , Glycoside Hydrolases/metabolism , Polysaccharides/analysis , beta-Glucans/analysis , Animals , Candida/metabolism , Enzyme Assays/methods , Female , Fungal Polysaccharides/metabolism , Glycoside Hydrolases/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Polysaccharides/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , beta-Glucans/metabolism
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