Siderophore-Mediated Iron-Uptake System in Vibrio parahaemolyticus and Its Relevance to Pathogenesis

1.在缺铁的副溶血性弧菌中，铁弧菌蛋白（VE）作为铁载体的功能被证明是通过摄取[55Fe]铁VF来显示典型的蛋白质受体介导过程的动力学。由VF介导的铁摄取被解偶联剂和atp酶抑制剂阻断。edda平板检测结果显示，VF对除溶藻弧菌外的其他弧菌均无活性，而溶藻弧菌后来被发现能合成VF.2。采用高效液相色谱法测定了32株不同来源的副溶血性弧菌培养上清液中VF的分泌量。临床分离株（n=8,33.6muM）产生的VF是环境分离株（n=22,3.9muM）的10倍，表明VF的产生有利于在人类宿主中存活和增殖。通过比较菌株AQ 3354与VF合成缺陷突变体的生长速度，证实了VF从饱和铁含量为30%的人转铁蛋白中吸收铁的能力。这表明生物在体内生存和增殖过程中可能通过VF的作用来利用宿主铁的来源。在铁限制条件下，副溶血性弧菌表达了两种主要的外膜蛋白，分别为78和83 kDa。先前用[55Fe] -VF孵育的外膜制剂的放射自显影分析显示单个放射性标记带，其中主要检测到78-kDa蛋白。用蛋白酶K处理整个细胞后，78-kDa蛋白被切割，表明其在细胞表面暴露的位置。这些结果表明78 kDa的Fe-VF结合蛋白可能是Fe-VF的受体。对纯化的78-kDa蛋白进行抗血清免疫印迹分析表明，该蛋白的分子质量和抗原特性在该物种中高度保守。一些临床分离物被检查了它们利用血红蛋白或血红蛋白作为铁的唯一来源的能力。这两种化合物似乎都是很好的铁源。血红蛋白-琼脂糖批量亲和法鉴定出83-kDa蛋白为血红蛋白结合蛋白。在感染的不同阶段或不同部位，拥有两个铁获取系统可能很重要，或者作为防止其中一个突变丢失的保险。制备这些受体蛋白中的一种或两种缺陷的突变体正在进行中，以评估这些铁摄取系统对副溶血性弧菌生理和感染过程的贡献。少

1.The function of vibrioferrin (VE) as a siderophore in iron-starved Vibrio parahaemolyticus was demonstrated by uptake of [55Fe] ferric VF displaying kinetics typical of a protein receptor-mediated process. The iron uptake mediated by VF was blocked by uncouplers and ATPase inhibitors. The EDDA-plate assays showed that VF was inactive to other Vibrio Species except for V.alginolyticus, which was found later to synthesize VF.2.The amounts of VF secreted to the culture supernatants were quantified by HPLC for 32 strains of V.parahaemolyticus isolated from different sources. The clinical isolates (n=8,33.6muM) produced VF 10-fold over the environmental isolates (n=22,3.9muM), indicating that the producibility of VF is advantageous of surviving and proliferating in human hosts.3.The ability of VF to assimilate iron for growth from 30% iron-saturated human transferrin was demonstrated by comparison of growth rate between strain AQ 3354 and its spontaneous mutant defective in VF synthesis. … More This suggests that the organism may utilize such a source of host iron through the action of VF during in vivo survival and proliferation.4.Under iron-restricted conditions, V.parahaemolyticus expressed two major outer membrane proteins of 78 and 83 kDa. Autoradiographic analysis of outer membrane preparations previously incubated with [55Fe] -VF revealed a single radiolabeled band, in which the 78-kDa protein was detected predominantly. The 78-kDa protein was cleaved by the treatment of whole cells with proteinase K,indicating its cell surface-exposed location. These results suggest that the Fe-VF binding protein of 78 kDa may function as the receptor for Fe-VF.Immunoblot analysis using the antiserum raised against the purified 78-kDa protein indicated that the molecular mass and antigenic properties of the protein were highly conserved among this species.5.Several clinical isolates were examined for their ability to utilize either hemin or hemoglobin as a sole source of iron. Both compounds appeared to be equally good iron sources. The hemin-agarose batch affinity method allowed us to identify the 83-kDa protein as the hemin-binding protein.6.Possession of two iron accquisition systems may be important at different stages or sites of infection, or as insurance against a mutational loss of one of them. The preparation of mutants defective of either or both of these receptor proteins is under progress to evaluate the contribution of these iron uptake systems to the physiology and infectious process of V.parahaemolyticus. Less