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

Abstract

The limulus test is a well-established method for the diagnosis of both gram (-) sepsis and invasive fungal infection. To diagnose deep-seated fungal infections, a (1-->3)-beta-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. It is suggested that the limulus reactive substance was released from the fungi to the blood, however, 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 fraction, 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 concentrations as low as 100 ng/ml. Limulus factor G reactivity of CAWS was sensitive to (1-->3)-beta-glucanase, zymolyase and was, at least in part, bound to ConA-agarose. The ConA-bound fraction also reacted with anti-beta-glucan antibody. CAWS is mainly composed of mannan and (1-->6)-beta-glucan, in addition to protein, assessed by 1H-NMR spectroscopy. CAWS also reacted with typing sera of Candida spp., specific for cell wall mannan. Chemical, immunochemical and biochemical analyses of CAWS strongly suggested that the limulus factor G-activating substance was a mannan-beta-glucan complex, present within the architecture of the yeast cell wall.

Saccharomyces cerevisiae‐ and Candida albicans‐Derived Mannan Induced Production of Tumor Necrosis Factor Alpha by Human Monocytes in a CD14‐ and Toll‐Like Receptor 4‐Dependent Manner

“…The S. cerevisiae mannan is the neutral fraction, and neither phosphorus nor nitrogen was detected in the specimens, indicating that mannoprotein was not present in the specimens. Mannan fraction from C. albicans IFO 1385 was prepared as described previously (48). Glucan from S. cerevisiae (G-5011) and LPS from Escherichia coli O55:B5 were purchased from Sigma (St. Louis, Mo., U.S.A.).…”

“…To examine the contamination of these fungal cell wall preparations with extraneous LPS, a colorimetric Limulus test (Endospecy test; Seikagaku Co., Ltd., Tokyo) (27) were carried out. Possible contamination of mannan fractions with endogeneous β(1-3)-glucan was examined by Gluspecy test (Seikagaku Co.) (18,48). These results are shown in Table 1.…”

Chemistry and Biology of Angiitis Inducer, Candida albicans Water‐Soluble Mannoprotein‐β‐Glucan Complex (CAWS)

“…We first cultured C. albicans in a completely synthetic medium in order to obtain water-soluble limulus factor G activating substance that is released from its cells, and obtained a water-soluble polysaccharide fraction released by it into the culture supernatant (C. albicans water-soluble fraction: CAWS), which is thought to be similar to the free β-1,3-D-glucan actually present in patient blood (52). Since the microorganism changes its appearance considerably depending on the culture conditions, observation of the cell wall structure by electron microscopy confirmed that it adopted a structure similar to natural media ( Fig.…”

Administration of human immunoglobulin suppresses development of murine systemic vasculitis induced with Candida albicans water-soluble fraction: an animal model of Kawasaki disease

“…This is a useful animal model of KD, since it exhibits many histological similarities to KD [6,7]. On the other hand, Ohno et al reported unique biologic characteristics for substances obtained from the C. albicans water-soluble fraction (CAWS) [8][9][10]. Studies in mice demonstrated that CAWS induced vasculitis at a clearly higher incidence than CADS, and that the vasculitis was histologically similar to that induced by CADS [11,12].…”