Chaffin Donald O, McKinnon Katherine, Rubens Craig E
Department of Pediatrics, Division of Infectious Diseases, Children's Hospital and Regional Medical Center/University of Washington, Seattle, 98105, USA.
Mol Microbiol. 2002 Jul;45(1):109-22. doi: 10.1046/j.1365-2958.2002.02988.x.
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 spectroscopy. Lectin binding studies detected terminal Neu5Ac(alpha 2-->3)Gal(beta 1- on LOS produced by the wild-type, cpsK or lst-complemented mutant strain LOS, compared with the mutant alone. Our data characterize the first sialyltransferase gene from a Gram- positive bacterium and provide compelling evidence that its product catalyses the alpha2,3 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.
无乳链球菌（GBS）是新生儿严重细菌感染的主要病因。GBS逃避宿主免疫的关键在于产生一种带有唾液酸的荚膜多糖（CPS），该多糖可使替代补体途径失活。已对血清型Ia和III型GBS的CPS操纵子进行了描述，但未鉴定出CPS唾液酸转移酶基因。我们鉴定出cpsK，它是大多数血清型CPS操纵子中的一个开放阅读框，与杜氏嗜血杆菌的脂寡糖（LOS）唾液酸转移酶基因lst同源。为了确定cpsK是否可能编码一种唾液酸转移酶，我们用cpsK对杜氏嗜血杆菌lst突变体进行了互补。CpsK在杜氏嗜血杆菌中表达，分离出LOS，并通过SDS-PAGE和高效液相色谱（HPLC）分析其唾液酸含量。在野生型、cpsK或lst互补突变菌株中可见唾液酸化LOS，但在没有cpsK的突变体中未见。通过质谱证实了Neu5Ac添加到了LOS上。凝集素结合研究检测到，与单独的突变体相比，野生型、cpsK或lst互补突变菌株产生的LOS上存在末端Neu5Ac（α2→3）Gal（β1-）。我们的数据表征了来自革兰氏阳性细菌的首个唾液酸转移酶基因，并提供了令人信服的证据，证明其产物催化Neu5Ac向杜氏嗜血杆菌LOS的α2,3添加，从而催化GBS CPS的末端侧链形成。系统发育研究进一步表明，lst和cpsK相关，但与大多数其他细菌的唾液酸转移酶不同，并且它们具有相似的密码子使用偏好和G + C含量，这表明它们是通过横向转移从一个祖先的低G + C生物体获得的。
