Kozel T R, Pfrommer G S, Guerlain A S, Highison B A, Highison G J
Department of Microbiology, School of Medicine, University of Nevada-Reno 89557.
Infect Immun. 1988 Nov;56(11):2794-800. doi: 10.1128/iai.56.11.2794-2800.1988.
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. neoformans. 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 degrees 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 125I-labeled C3. The results showed approximately 5 X 10(6) to 10 X 10(6) 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.
新型隐球菌的吞噬作用受到多糖荚膜存在的显著影响。我们检测了C3片段与八株新型隐球菌的激活及结合情况。通过光学显微镜和电子显微镜观察，所有分离株均显示有荚膜。这些菌株在对中性粒细胞吞噬作用的敏感性方面存在差异。酵母细胞在正常人血清中孵育进行调理。五株菌株对吞噬具有抗性，两株菌株对吞噬表现出中等水平的抗性，一株菌株对吞噬相当敏感。用热灭活血清（56℃ 30分钟）调理的酵母细胞既不与中性粒细胞附着也不被其吞噬。通过使用含有125I标记C3的正常人血清来定量测定与酵母细胞结合的C3量。结果显示，无论酵母细胞对吞噬敏感还是抗性，每个酵母细胞约有5×10⁶至10×10⁶个C3分子。通过用0.1 M NH₂OH（pH 10）处理从酵母细胞中洗脱结合的C3，并在还原条件下通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳检测洗脱的片段。该分析结果表明，很少有C3以C3b的形式存在，且大量降解为iC3b，即C3b的无活性降解产物。所有菌株的这种降解模式相似。免疫电子显微镜用于评估与酵母细胞结合的C3片段的超微结构定位。无论分离株对吞噬敏感还是抗性，C3片段均结合在荚膜周边。因此，就结合到隐球菌荚膜上的C3片段的量、分子形式和超微结构定位而言，对吞噬敏感和抗性的分离株是相似的。这些结果进一步表明，补体级联反应的激活对于酵母细胞的吞噬作用是必要的，但并不充分。
