CpsK of Streptococcus agalactiae exhibits α2,3-sialyltransferase activity in Haemophilus ducreyi
CpsK of Streptococcus agalactiae exhibits α2,3-sialyltransferase activity in Haemophilus ducreyi
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DOI：
10.1046/j.1365-2958.2002.02988.x
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发表时间：
2002-07-01
影响因子：
3.6
通讯作者：
Rubens, CE
中科院分区：
文献类型：
--
作者：
Chaffin, DO;McKinnon, K;Rubens, CE
无乳链球菌（GBS）是新生儿严重细菌感染的主要原因。GBS逃避宿主免疫的关键是唾液酸修饰的荚膜多糖（CPS）的产生，其灭活了替代补体途径。已经描述了血清型Ia和III型GBS的CPS操纵子，但尚未鉴定出CPS唾液转移酶基因。我们在大多数血清型的CPS操纵子中发现了一个开放阅读框cpsK，它与杜氏嗜血杆菌的低脂寡糖（LOS）唾液转移酶基因lst同源。为了确定cpsK是否可能编码唾液基转移酶，我们用cpsK补充了H. ducreyi 1突变体。CpsK在H. ducreyi中表达，通过SDS-PAGE和高效液相色谱（HPLC）分离得到了LOS，并对其唾液酸含量进行了分析。Sialo-LOS在野生型，cpsK或最后补充突变株中可见，但在没有cpsK的突变株中未见。质谱分析证实了在LOS中添加了Neu5Ac。凝集素结合研究检测到野生型、cpsK或最后互补突变株LOS产生的LOS上的末端Neu5Ac(α 2—> 3)Gal (β 1-)，与单独突变株相比。我们的数据表征了来自革兰氏阳性细菌的第一个唾液转移酶基因，并提供了令人信服的证据，证明其产物催化了H. ducreyi LOS中Neu5Ac的α 2,3添加，从而催化了GBS CPS的末端侧链。系统发育研究进一步表明，lst和cpsK是相关的，但与大多数其他细菌的唾液基转移酶不同，并且它们具有相似的密码子使用偏好和G + C含量，表明它们是从祖先的低G + C生物体侧转移获得的。
Streptococcus agalactiae (GBS) is a major cause of serious newborn bacterial infections. Crucial to GBS evasion of host immunity is the production of a capsular polysaccharide (CPS) decorated with sialic acid, which inactivates the alternative complement pathway. The CPS operons of serotypes Ia and III GBS have been described, but the CPS sialyltransferase gene was not identified. We identified cpsK, an open reading frame in the CPS operon of most serotypes, which was homologous to the lipooligosaccharide (LOS) sialyltransferase gene, lst, of Haemophilus ducreyi. To determine if cpsK might encode a sialyltransferase, we complemented a H. ducreyi lst mutant with cpsK. CpsK was expressed in H. ducreyi and LOS was isolated and analysed for sialic acid content by SDS-PAGE and high-performance liquid chromatography (HPLC). Sialo-LOS was seen in the wild-type, cpsK- or lst-complemented mutant strains, but not in the mutant without cpsK. Addition of Neu5Ac to the LOS was confirmed by mass spectro-scopy. Lectin binding studies detected terminal Neu5Ac(alpha 2 --> 3)Gal (beta1- on LOS produced by the wild-type, cpsK or lst-complemented mutant strain LOS, compared with the mutant alone. Our data charac-terize the first sialyltransferase gene from a Gram- positive bacterium and provide compelling evidence that its product catalyses the alpha 2,3 addition of Neu5Ac to H. ducreyi LOS and therefore the terminal side-chain of GBS CPS. Phylogenetic studies further indicated that lst and cpsK are related but distinct from sialyltransferases of most other bacteria and, along with their similar codon usage bias and G + C content, suggests acquisition by lateral transfer from an ancestral low G + C organism.