Chemical and immunochemical characterization of limulus factor G-activating substance of Candida spp.

The limulus test is a well-established method for the diagnosis of both Gra m (-) sepsis and invasive fungal infection. To diagnose deep-seated fungal infections, a (1-->3)-beta-D-glucan-specific chromogenic kit (Fungitec G te st MK) has been developed and applied clinically. It is suggested that the limulus reactive substance was released from the fungi to the blood, howeve r, its chemical properties were not precisely examined in detail because of the limited quantity available. In this study, we used chemically defined liquid medium to culture Candida spp. and collected the water soluble fract ion, CAWS. The yield of CAWS was circa 100 mg/l, independent of the strain of Candida. CAWS reacted with limulus factor G (Fungitec G test MK) at conc entrations as low as 100 ng/ml. Limulus factor G reactivity of CAWS was sen sitive to (1 --> 3)-beta-glucanase, zymolyase and was, at least in part, bo und to ConA-agarose. The ConA-bound fraction also reacted with anti-beta-gl ucan antibody. CAWS is mainly composed of mannan and (1 --> 6)-beta-glucan, in addition to protein, assessed by H-1-NMR spectroscopy. CAWS also reacte d with typing sera of Candida spp., specific for cell wall mannan. Chemical , immunochemical and biochemical analyses of CAWS strongly suggested that t he limulus factor G-activating substance was a mannan-beta-glucan complex, present within the architecture of the yeast cell wall. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.