Ala'Aldeen D A, Powell N B, Wall R A, Borriello S P
Microbial Pathogenicity Research Group, Northwick Park Hospital, Harrow, Middlesex, United Kingdom.
Infect Immun. 1993 Feb;61(2):751-9. doi: 10.1128/iai.61.2.751-759.1993.
The interaction between gold-labelled human transferrin (Au-HTF) with live meningococci after growth in vivo or in different in vitro conditions was examined by electron microscopy to localize and quantify the numbers of HTF-binding sites on the cell surface. It was clearly demonstrated that HTF binds to the surface of live meningococci (of different serogroups and serotypes) after growth in either iron-sufficient or iron-restricted cultures, although the degree of labelling was always higher (2- to 35-fold) in the latter case. The commensal Neisseria polysaccharea behaved similarly. Ultrathin sections showed that Au-HTF was localized predominantly on the outer membrane of the cells and vesicles, with hardly any internalization. Au-HTF labelling on meningococci was significantly reduced after incubation with unlabelled HTF or with rabbit antiserum containing antibodies against transferrin-binding proteins (TBPs), demonstrating the specificity of the interaction. These sera also blocked binding between HTF and outer membrane proteins on Western immunoblots. Direct evidence of the expression of the TBPs (Western blots) and localization of the HTF receptor (electron microscopy) on in vivo-grown meningococci was obtained from organisms derived without laboratory culturing from the cerebrospinal fluid of a patient. There was considerable cell-to-cell variation in the amount of labelling present on cells of the same sample (in vitro- or in vivo-grown organisms) and between different strains. The degree of binding varied with time of incubation of the cells with Au-HTF. The gold particles frequently formed discrete circles on the cell surfaces of the in vitro-grown organisms; these circles appear to be associated with outer membrane vesicle formation. The results show that the TBPs, which form part of the active components of the HTF receptor(s), are expressed in vivo and are surface exposed and immunogenic and that antibodies against them can interfere with the HTF binding of the meningococcal cells, which may affect iron utilization. This study further supports the concept of regarding the TBPs as future vaccine candidates.
通过电子显微镜检查了体内生长或在不同体外条件下生长后，金标记的人转铁蛋白（Au-HTF）与活的脑膜炎球菌之间的相互作用，以定位和量化细胞表面HTF结合位点的数量。结果清楚地表明，在铁充足或铁限制培养物中生长后，HTF均能与活的脑膜炎球菌（不同血清群和血清型）表面结合，尽管在后一种情况下标记程度总是更高（2至35倍）。共生的多糖奈瑟菌表现类似。超薄切片显示，Au-HTF主要定位于细胞和囊泡的外膜上，几乎没有内化现象。用未标记的HTF或含有抗转铁蛋白结合蛋白（TBP）抗体的兔抗血清孵育后，脑膜炎球菌上的Au-HTF标记显著减少，证明了这种相互作用的特异性。这些血清在Western免疫印迹中也阻断了HTF与外膜蛋白之间的结合。从一名患者脑脊液中未经实验室培养获得的生物体中，获得了体内生长的脑膜炎球菌上TBP表达（Western印迹）和HTF受体定位（电子显微镜）的直接证据。同一样本（体外或体内生长的生物体）的细胞之间以及不同菌株之间，标记量存在相当大的细胞间差异。结合程度随细胞与Au-HTF孵育时间的变化而变化。金颗粒经常在体外生长的生物体的细胞表面形成离散的圆圈；这些圆圈似乎与外膜囊泡形成有关。结果表明，构成HTF受体活性成分一部分的TBP在体内表达，位于表面且具有免疫原性，针对它们的抗体可干扰脑膜炎球菌细胞的HTF结合，这可能会影响铁的利用。这项研究进一步支持了将TBP视为未来疫苗候选物的概念。
