Studies of the capsular-like antigen of F. tularensis

由于其极强的致病性和作为生物武器的潜在用途，图拉氏方济氏菌是一种A类细菌选择剂。卡莱尔和胡德的早期研究表明，图拉夫酵母产生一种胶囊状物质。许多细菌产生胶囊，生产胶囊所需的基因编码蛋白质，用于运输、生物合成和调节。与衣壳生物合成有关的基因的同源基因存在于弗朗西塞拉基因组数据库中。使用电子显微镜进行的初步研究表明，并证实了图拉夫酵母周围有一种胶囊状物质(CLM)。我们现在可以根据化学、层析和免疫化学分析来分离这种不含弗朗西塞氏菌内毒素的物质。这种物质与细菌细胞联系松散，很容易从细菌表面移除，并由重复的四糖重复组成。利用转座子诱变，我们已经鉴定了一些对我们的CLM特异性抗体XE8没有反应或有限反应的突变体。我们目前已经确定了7个改变CLM表达的转座子突变体的插入位置，这将作为本提案中研究的无囊化菌株。基于这些观察，我们将提出以下假设：1)土拉方杆菌表达一种在发病机制中至关重要的被囊样物质，我们认为它是第一组被膜；2)通过参与生物合成和CLM表达的基因突变来改变这种被囊样结构，将改变土拉方杆菌在细胞培养模型和呼吸道感染动物模型中的致病性；3)诱导针对CLM的免疫反应将提供宿主免疫。以下特定目标将被用于解决这些假说：1)有缺陷的土拉氏原虫Schu S4突变体的特征，以及2)土拉氏原虫S4囊样物质生物学作用的特征，3)研究被动免疫和主动免疫在小鼠弗朗西斯杆菌感染模型中的免疫原性和保护效果

Francisella tularensis is a class A bacterial select agent due to its extreme pathogenicity and potential use as a bioweapon. Early studies by Carlisle and Hood have indicated that F. tularensis produces a capsule-like material. Many bacteria produce capsules and the genes required to produce them encode proteins for transport, biosynthesis and regulation. Homologs of genes implicated in capsular biosynthesis are present in the Francisella genome database. Preliminary studies, using electron microscopy, indicate and confirm that a capsule-like material (CLM) surrounds F. tularensis. We can now isolate this material, free from Francisella LPS, based on chemical, chromatographic and immunochemical analysis. This material is loosely associated with the bacterial cell, is easily removed from the bacterial surface and is composed of a repeating tetrasaccharide repeat. Using transposon mutagenesis in F. tularensis Schu S4, we have identified a number of mutants with no or limited reactivity to our CLM specific antibody XE8. We have currently identified the site of insertion of 7 of these transposon mutants that have altered CLM expression that will serve as acapsular strains for the studies in this proposal. Based on these observations, we would pose the following hypotheses 1) Francisella tularensis expresses a capsular-like material that is important in pathogenesis and that we believe is a group 1 capsule; 2) Alteration of this capsule-like structure by mutations of the genes involved in biosynthesis and expression of CLM will alter pathogenicity of Francisella tularensis in cell culture models and in an animal model of respiratory infection; 3) Induction of an immune response targeted to the CLM will provide host immunity. The following specific aims will be used to resolve these hypotheses: 1) Characterization of the F. tularensis Schu S4 mutants that are defective in production of capsule-like material and 2) Characterization of the biological role of the P. tularensis Schu S4 capsule like material and 3) Study the immunogenicity and protective efficacy of passive and active immunization in murine models of Francisella infection