Development of a chitin assay for the quantification of fungus

Abstract

Chitin, a unique structural polysaccharide found in fungi and arthropods, is not produced by vertebrates. Thus, the potential applications of a specific and sensitive assay for chitin are numerous, including the evaluation of the extent of fungal keratitis. Chitin is a homopolymer of beta (1, 4) linked D-N-acetylglucosamine. We have developed a simple and reproducible assay for chitin and applied it to Candida albicans cultures. The assay involves homogenization of the culture and treatment with 21.1 M KOH to remove soluble materials, including proteins. This base treatment also deacetylates the chitin to the glucosamine polymer, chitosan. Chitosan is hydrolyzed by 0.5 M H2SO4 to glucosamine monomers which are then deaminated by the addition of NaNO2 to the acid solution. The resulting 2,5-anhydromannose is reduced by NaB[3H]4 to 1-[3H] 2,5-anhydromannitol. This radiolabelled sugar is isolated by paper chromatography and quantified via liquid scintillation. The sensitivity of this assay is assessed by comparison of colony forming units (CFU's) with a glucosamine standard. A typical run of the assay detects 53.1 CFU/c.p.m., and 356,000 c.p.m. per nanomole of N-acetylglucosamine. The specificity of the assay is very high because of the unique nature of chitin. This method of chitin determination may be a useful alternative method for future investigations involving the study of fungal infections in mammalian tissues.

Antifungal activity of well-defined chito-oligosaccharide preparations against medically relevant yeasts

“…Members of this class of antifungals are fungistatic rather than fungicidal. When fungi are exposed to fungistatic agents, cells enter a non-growth-survival phase in which viability is not affected but new growth is inhibited [34]. In general, fungistatic substances limit the growth by interfering with protein production, DNA replication, and other aspects of cellular metabolism.…”

Candida africana sp. nov., a new human pathogen or a variant of Candida albicans?

“…The strains described here were unable to switch the cellular phenotype and could not assimilate amino sugars. N ‐Acetylglucosamine is known as an important inducer for the process of yeast‐mycelial conversion [15], germ‐tube generation [16], synthesis of chitin, which represents a homopolymer of β‐(1,4)‐linked d‐ N ‐acetylglucosamine [17], and chlamydospore formation [16]. A mutant of C. albicans has been isolated that was defective in production of β‐ N ‐acetylglucosaminidase but was still pathogenetic [18].…”

Fungal and Parasitic Infections of the Eye

“…Similarly, the use of a battery of fluorescein-conjugated lectins has been shown to be useful in detection of ocular mycoses (218). The use of such tests as a chitin assay (151) or PCR (192) may prove useful, but these assays currently suffer from a cumbersome technique in the former and lack of detection across fungal genera in the latter.…”