Strain variation in phagocytosis of Cryptococcus neoformans: dissociation of susceptibility to phagocytosis from activation and binding of opsonic fragments of C3

Abstract

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 efficacy of complement‐mediated phagocytosis of Cryptococcus neoformans is dependent on the location of C3 in the polysaccharide capsule and involves both direct and indirectC3‐mediated interactions

“…Almost two decades ago it was reported that C. neoformans strains differed in their ability to be phagocytozed in the presence of serum but this phenomenon remains unexplained 27–29. In this study we demonstrate an inverse association between capsule volume and complement‐mediated phagocytosis.…”

Molecular architecture of the Cryptococcus neoformans capsule

“…Incubation of encapsulated cryptococci in normal human serum leads to activation of the alternative complement system and deposition of potentially opsonic fragments of C3 at the capsular surface and in the capsule interior (Kozel et al ., 1988; 1989). In an effort to assess the extent to which C3 penetrates to the capsule interior, encapsulated cryptococci were incubated for 30 min in 40% NHS, washed with PBS, and the sites of C3 binding were determined by immunofluorescence using fluorescein isothiocyanate (FITC)‐labelled Fab fragments of polyclonal C3 antibody.…”

Monoclonal antibodies can affect complement deposition on the capsule of the pathogenic fungus Cryptococcus neoformans by both classical pathway activation and steric hindrance

“…When C3 localizes deep inside the capsule, as occurs for cryptococcal cells with large capsules, C3 is not accessible to the receptor and the process is inefficient in promoting phagocytosis (Zaragoza et al ., 2003). Consequently, the efficiency of C‐mediated phagocytosis is critically dependent on the size of the capsule of the C. neoformans strain (Mitchell and Friedman, 1972; Kozel et al ., 1988b; Small and Mitchell, 1989; Yasuoka et al ., 1994; Zaragoza et al ., 2003). Recent studies show that another factor that influences C3 localization is the source of serum, with serum from different mammalian species in different regions of the capsule (Gates and Kozel, 2006).…”