Andreishcheva Ekaterina N, Vann Willie F
Laboratory of Bacterial Toxins, Center for Biologics Evaluation and Research, Building 29, Room 103, US FDA, 8800 Rockville Pike, Bethesda, MD 20892, USA.
J Bacteriol. 2006 Mar;188(5):1786-97. doi: 10.1128/JB.188.5.1786-1797.2006.
Escherichia coli K1 is responsible for 80% of E. coli neonatal meningitis and is a common pathogen in urinary tract infections. Bacteria of this serotype are encapsulated with the alpha(2-8)-polysialic acid NeuNAc(alpha2-8), common to several bacterial pathogens. The gene cluster encoding the pathway for synthesis of this polymer is organized into three regions: (i) kpsSCUDEF, (ii) neuDBACES, and (iii) kpsMT. The K1 polysialyltransferase, NeuS, cannot synthesize polysialic acid de novo without other products of the gene cluster. Membranes isolated from strains having the entire K1 gene cluster can synthesize polysialic acid de novo. We designed a series of plasmid constructs containing fragments of regions 1 and 2 in two compatible vectors to determine the minimum number of gene products required for de novo synthesis of the polysialic acid from CMP-NeuNAc in K1 E. coli. We measured the ability of the various combinations of region 1 and 2 fragments to restore polysialyltransferase activity in vitro in the absence of exogenously added polysaccharide acceptor. The products of region 2 genes neuDBACES alone were not sufficient to support de novo synthesis of polysialic acid in vitro. Only membrane fractions harboring NeuES and KpsCS could form sialic polymer in the absence of exogenous acceptor at the concentrations formed by wild-type E. coli K1 membranes. Membrane fractions harboring NeuES and KpsC together could form small quantities of the sialic polymer de novo.
大肠杆菌K1导致了80%的大肠杆菌新生儿脑膜炎，并且是尿路感染中的常见病原体。这种血清型的细菌被α(2 - 8)-聚唾液酸NeuNAc(α2 - 8)包裹，这在几种细菌病原体中很常见。编码这种聚合物合成途径的基因簇被组织成三个区域：(i) kpsSCUDEF，(ii) neuDBACES，以及(iii) kpsMT。K1聚唾液酸转移酶NeuS在没有基因簇的其他产物时不能从头合成聚唾液酸。从具有完整K1基因簇的菌株中分离出的膜能够从头合成聚唾液酸。我们设计了一系列质粒构建体，它们在两个相容载体中包含区域1和2的片段，以确定在K1大肠杆菌中从CMP-NeuNAc从头合成聚唾液酸所需的最少基因产物数量。我们测量了区域1和2片段的各种组合在没有外源添加多糖受体的情况下在体外恢复聚唾液酸转移酶活性的能力。仅区域2基因neuDBACES的产物不足以支持体外聚唾液酸的从头合成。只有含有NeuES和KpsCS的膜部分在没有外源受体的情况下能够以野生型大肠杆菌K1膜形成的浓度形成唾液酸聚合物。含有NeuES和KpsC的膜部分一起能够从头形成少量的唾液酸聚合物。
