Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core
Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core
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DOI：
10.1046/j.1462-2920.2002.00288.x
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发表时间：
2002-12-01
影响因子：
5.1
通讯作者：
Lam, JS
中科院分区：
文献类型：
--
作者：
Daniels, C;Griffiths, C;Lam, JS
铜绿假单胞菌是一种革兰氏阴性机会致病菌，感染免疫功能低下的患者和创伤受害者，并导致囊性纤维化患者致命的肺部感染。这种微生物产生许多毒力因子，其中之一是脂多糖（LPS），其已被证明介导许多生物学效应，包括对血清杀伤和吞噬作用的抗性。这些生物活性与O-多糖的长度及其在外膜上的分布相关。Wzz负责调节O-抗原的大小分布。已经发现Wzz仅参与LPS生物合成的Wzy依赖性途径，其产生杂聚0-多糖，例如铜绿假单胞菌的B带LPS。我们的实验室以前曾报道过PAO 1的B带O-抗原生物合成簇中编码的Wzz蛋白的表征。铜绿假单胞菌PAO 1基因组序列的可用性使得鉴定第二种功能性Wzz蛋白（PA 0938，Wzz（2））成为可能。基因置换用于产生未标记的wzz（2）Delta敲除和wzz（2）Delta/wzz（1）：：Gm双敲除。如预期的，wzz（2）Delta菌株产生具有由Wzz（1）赋予的模态长度的LPS，并且wzz（2）Delta/wzz（1）：：Gm菌株产生具有非模态（随机）长度的LPS O-抗原。来自铜绿假单胞菌PAO 1的wzz（1）和wzz（2）均被克隆并表达，具有N-末端His（6）标签。His（6）-Wzz（1）和His（6）-Wzz（2）通过固定化金属亲和层析（IMAC）纯化至接近均一。这些制备物用于开发针对每种蛋白质的特异性多克隆抗体。体内蛋白质交联后的Western免疫印迹表明，Wzz（1）形成二聚体，而Wzz（2）形成八聚体。通过wzz（2）Delta/rmlC双突变体的产生和LPS的分析，我们已经做出了新颖的观察，即在连接到脂质A核心之前发生众数链长分布的O-抗原的聚合。我们已经显示了Wzz蛋白和O-抗原聚合物链之间的关联，使用免疫沉淀与抗O 5 O-抗原单克隆抗体MF 15 -4。Wzz（1）和Wzz（2）都可以与O 5聚合物共沉淀。
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that infects immunocompromised patients and trauma victims and causes fatal lung infections in people with cystic fibrosis. This microorganism produces a number of virulence factors, one of which is lipopolysaccharide (LPS), which has been shown to mediate many biological effects including resistance to serum killing and phagocytosis. These biological activities have been correlated to the length of the O-polysaccharide and its distribution on the outer membrane. Wzz is responsible for regulation of the size distribution of the O-antigen. Wzz has been found to participate solely in the Wzy-dependent pathway for LPS biosynthesis, which produces heteropolymeric O-polysaccharide such as the B-band LPS of P. aeruginosa . Our laboratory has previously reported characterization of a Wzz protein encoded in the B-band O-antigen biosynthesis cluster of PAO1. The availability of the genome sequence of P. aeruginosa PAO1 has made it possible to identify a second functional Wzz protein (PA0938, Wzz(2)). Gene replacement was used to generate an unmarked wzz(2)Delta knock-out and a wzz(2)Delta/wzz(1)::Gm double knock-out. As expected, the wzz(2)Delta strain produced LPS with modal length imparted by Wzz(1), and the wzz(2)Delta/wzz(1)::Gm strain produced LPS O-antigen with a non-modal (random) length. Both wzz(1) and wzz(2) from P. aeruginosa PAO1 were cloned and expressed with an N-terminal His(6) tag. His(6)-Wzz(1) and His(6)-Wzz(2) were purified to near homogeneity by immobilized metal affinity chromatography (IMAC). These preparations were used to develop specific polyclonal antibodies against each of the proteins. In vivo protein cross-linking followed by Western immunoblotting indicated that Wzz(1) forms dimers whereas Wzz(2) forms octamers. By generation of a wzz(2)Delta/rmlC double mutant and analysis of the LPS, we have made the novel observation that polymerization of modal chain length-distributed O-antigen occurred before ligation to the lipid A core. We have shown an association between the Wzz proteins and O-antigen polymer chains using immunoprecipitation with anti-O5 O-antigen monoclonal antibody MF15-4. Both Wzz(1) and Wzz(2) could be co-precipitated with O5 polymer.