No abstract

Serum obtained from normal human subjects contains antibodies reactive in an enzyme-linked immunosorbent assay with the glucuronoxylomannan (GXM) of Cryptococcus neoformans. The frequency of occurrence of class-specific antibodies among normal subjects was 28% for immunoglobulin G (IgG), 98% for IgM, and 3% for IgA. Anti-GXM antibodies with kappa light chains occurred in 98% of normal subjects, while the occurrence of lambda light chains was 28%. Each of five subjects with high levels of anti-GXM IgG antibodies had readily detectable antibodies of the IgG2 isotype; two of the five subjects had readily detectable IgGl antibody. An examination of sera from human immunodeficiency virus-infected patients showed that human immunodeficiency virus infection was accompanied by a significant decrease in the occurrence of IgM antibodies and anti-GXM antibodies with kappa light chains; these decreases occurred early in infection when CD4 counts were still .500 cells per ,ul. A slight but not statistically significant decrease in the occurrence of anti-GXM IgG antibodies was seen only in patients with CD4 levels of <200 cells per ,lI. Sera from normal subjects with high levels of anti-GXM IgG antibodies were examined to identify any contribution of the antibodies to complement activation or to opsonization of the yeast cells. An analysis of the kinetics for activation and binding of C3 to the yeast cell showed no pattern of quantitative or qualitative differences between sera with high or low levels of anti-GXM IgG antibodies. Phagocytosis studies showed that the naturally occurring IgG antibodies did not contribute to opsonization of the yeast cells.

Phagocytosis of Cryptococcus neoformans is markedly influenced by the presence of a polysaccharide capsule.We examined activation and binding of C3 fragments to eight isolates of C. neqformans. All isolates were shown to have capsules by light and electron microscopy. These strains differed in susceptibility to phagocytosis by neutrophils. Yeast cells were opsonized by incubation in normal human serum. Five strains were resistant to ingestion, two strains showed intermediate levels of resistance to ingestion, and one strain was quite sensitive to phagocytosis. Yeast cells opsonized with heat-inactivated serum (56°C for 30 min) neither attached nor were ingested by neutrophils. A quantitative estimate of the amount of C3 bound to the yeast cells was determined by use of normal human serum containing 1251-labeled C3. The results showed approximately 5 x 106 to 10 x 106 C3 molecules per yeast cell regardless of whether the yeast cells were sensitive or resistant to phagocytosis. Bound C3 was eluted from the yeast cells by treatment with 0.1 M NH2OH (pH 10), and the eluted fragments were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Results of this analysis showed that little of the C3 was in the form of C3b, and there was substantial decay to iC3b, the inactive decay product of C3b. This pattern of decay was similar with all strains. Immunoelectron microscopy was used to assess the ultrastructural location of the C3 fragments bound to the yeast cells. C3 fragments were bound to the perimeter of the capsule regardless of whether the isolate was sensitive or resistant to phagocytosis. Thus, phagocytosis-sensitive and phagocytosis-resistant isolates were similar with regard to the amount, molecular form, and ultrastructural location of C3 fragments bound to the cryptococcal capsule. These results further indicate that activation of the complement cascade is necessary but not sufficient for phagocytosis of the yeast cell. 2794 on August 1, 2020 by guest http://iai.asm.org/ Downloaded from on August 1, 2020 by guest http://iai.asm.org/ Downloaded from

The complement system plays a key role in resistance to cryptococcosis. In the present study, we examined several factors that influence the binding of C3 cleavage fragments to Cryptococcus neoformans. Binding of C3 was determined by using normal human serum supplemented with 12_l-labeled C3. Incubation of encapsulated cryptococci in 20% serum led to the binding of approximately 3.2 x 106 molecules of C3 to each cell. The binding of C3 was markedly inhibited by heating the serum at 56°C for 30 min or by chelation of the serum with EDTA. Chelation of the serum with EGTA [ethylene glycol-bis(O-aminoethyl ether)-N,N,N',N'-tetraacetic acid] reduced binding of C3 by 37%. These results indicated that activation of C3 cleavage fragments and their binding to C. neoformans was primarily dependent upon the alternative pathway. Bound C3 could be removed by incubation with 1.0 M hydroxylamine (pH1 10) but not by incubation with 3.5 M NaSCN or with phosphate-buffered saline containing 0.1% sodium dodecyl sulfate. These results suggested that C3 fragments were bound to C. neoformans by ester bonds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C3 fragments eluted from the yeast showed the presence of protein bands consistent with the presence of iC3b. C3b was not detected on the yeast after incubation with serum for time intervals as short as 2.5 min, indicating a rapid conversion of cell-bound C3b to iC3b. These results indicate that iC3b is the ligand which most likely interacts with the phagoctye C3 receptors involved in the phagocytosis of C. neoformans.

Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding-of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 107 to 5 x 107 C3 molecules to the yeast cells. The kinetics * Corresponding author.