Heparinase gene homolog in Cryptococcus neoformans is involved in diazonium blue B reaction and capsule formation

Diazobenzoic acid B (DBB) staining stains basidiomycete fungi, and is used to distinguish basidiomycetous yeast from ascomycetous yeast. In this study, we investigated the mechanisms underlying DBB staining, by screening mutants of the basidiomycetous yeast Cryptococcus neoformans that stained DBB negative. We identified a heparinase homolog responsible for DBB staining and capsule formation in C. neoformans. We observed lower transcript levels of this gene after 16 h of culture than after 24 hours, consistent with older cells being more suitable for DBB staining. Moreover, capsule thickness significantly decreased in a strain with disruption of wherein the heparinase gene homolog was disrupted, and the mutant strain showed considerable cell enlargement compared to the wild-type strain. Furthermore, the mutant did not grow in the presence of 0.01% SDS. A His-tagged heparinase gene homolog-complemented strain was constructed and the heparinase gene homolog protein was detected in the culture supernatant, indicating extracellular secretion.,DBB undergoes a diazo coupling reaction with naphthol, producing a red color, which is attributed to the formation of a long conjugated double bond between DBB and naphthol. Similar long conjugated double bonds may also be generated during the DBB staining of basidiomycete yeasts. Thus, the extracellular heparinase gene homolog protein can cleave the bond between the polysaccharide and the cell wall or membrane, leading to the formation of a diazo compound that, stains the cell red.