Wessels M R, Haft R F, Heggen L M, Rubens C E
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Infect Immun. 1992 Feb;60(2):392-400. doi: 10.1128/iai.60.2.392-400.1992.
The type III capsular polysaccharide of group B streptococci (GBS) consists of a linear backbone with short side chains ending in residues of N-acetylneuraminic acid, or sialic acid. The presence of sialic acid on the surface of the organism inhibits activation of the alternative pathway of complement and is thought to be an important element in the virulence function of the capsule. We showed previously that a mutant strain of GBS that expressed a sialic acid-deficient, or asialo, form of the type III polysaccharide was avirulent, supporting a virulence function for capsular sialic acid. We now report the derivation of an asialo capsule mutant from a highly encapsulated wild-type strain of type III GBS, strain COH1, by insertional mutagenesis with transposon Tn916 delta E. In contrast to the wild-type strain, the asialo mutant strain COH1-11 was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat model of GBS infection. The asialo mutant accumulated free intracellular sialic acid, suggesting a defect subsequent to sialic acid synthesis in the biosynthetic pathway leading to capsule sialylation. The specific biosynthetic defect in mutant strain COH1-11 was found to be in the activation of free sialic acid to CMP-sialic acid: CMP-sialic acid synthetase activity was present in the wild-type strain COH1 but was not detected in the asialo mutant strain COH1-11. One of the two transposon insertions in the asialo mutant COH1-11 mapped to the same chromosomal location as one of the two Tn916 insertions in the previously reported asialo mutant COH31-21, identifying this site as a genetic locus necessary for expression of CMP-sialic acid synthetase activity. These studies demonstrate that the enzymatic synthesis of CMP-sialic acid by GBS is an essential step in sialylation of the type III capsular polysaccharide.
B族链球菌（GBS）的III型荚膜多糖由一条线性主链和短侧链组成，侧链末端为N - 乙酰神经氨酸或唾液酸残基。该生物体表面唾液酸的存在会抑制补体替代途径的激活，并且被认为是荚膜毒力功能的一个重要因素。我们之前表明，一株表达III型多糖唾液酸缺陷型或脱唾液酸形式的GBS突变菌株是无毒的，这支持了荚膜唾液酸的毒力功能。我们现在报告通过用转座子Tn916 delta E进行插入诱变，从高度荚膜化的III型GBS野生型菌株COH1中获得脱唾液酸荚膜突变体。与野生型菌株相比，脱唾液酸突变体菌株COH1 - 11在体外对人白细胞的吞噬杀伤敏感，并且在GBS感染的新生大鼠模型中相对无毒。脱唾液酸突变体积累了游离的细胞内唾液酸，这表明在导致荚膜唾液酸化的生物合成途径中，唾液酸合成之后存在缺陷。发现突变体菌株COH1 - 11中的特定生物合成缺陷在于游离唾液酸激活为CMP - 唾液酸：野生型菌株COH1中存在CMP - 唾液酸合成酶活性，但在脱唾液酸突变体菌株COH1 - 11中未检测到。脱唾液酸突变体COH1 - 11中的两个转座子插入之一与先前报道的脱唾液酸突变体COH31 - 21中的两个Tn916插入之一映射到相同的染色体位置，确定该位点为表达CMP - 唾液酸合成酶活性所必需的基因座。这些研究表明，GBS对CMP - 唾液酸的酶促合成是III型荚膜多糖唾液酸化的关键步骤。
