Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b.
Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b.
复制标题
b 型流感嗜血杆菌中铁调节血红素受体的鉴定和表征。
DOI：
10.1128/iai.62.1.48-59.1994
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发表时间：
1994
影响因子：
3.1
通讯作者：
Williams,P
中科院分区：
文献类型：
--
作者：
Wong,JC;Holland,J;Parsons,T;Smith,A;Williams,P
血红素可以作为流感嗜血杆菌的必需卟啉和铁的来源。在哺乳动物细胞外体液中，既没有游离血红素也没有游离铁，因为它们分别与血红素结合蛋白和转铁蛋白紧密结合。自H.流感病毒在铁-转铁蛋白和血红素-血红素结合蛋白存在下生长，并且已知表达转铁蛋白的饱和受体，我们研究了该病原体从血红素结合蛋白获得血红素用作铁源的过程。人和兔的血红素结合蛋白能够将血红素作为铁源提供给H。通过平板生物测定证实了流感B型菌株。以生物素化血红素结合蛋白为配体，用斑点酶法检测H。流感病毒在铁限制培养基中生长后结合血红素-血红素结合蛋白和脱辅基-血红素结合蛋白，但在铁充足培养基中不结合。与血红素血红素结合蛋白和脱辅基血红素结合蛋白的竞争性结合研究证明了结合的饱和性。无论是血红素，原卟啉IX，血红蛋白，也不转铁蛋白阻止血红素结合蛋白结合到整个细胞，证明了结合的特异性。治疗全H。流感病毒细胞与胰蛋白酶的结合消失。综上所述，这些观察结果表明，H。流感病毒B型表达外膜蛋白，该外膜蛋白作为血红素结合蛋白的受体，并受生长培养基中铁的可利用性调节。在铁限制培养基中，H.流感病毒706705和DL 42不表达先前报道的100-kDa血红素结合蛋白（M.S. Hanson，S.E. Pelzel，J. Latimer，U. Muller-Eberhard和E. J.汉森，美国国家科学院院刊。Acad. Sci. USA 89：1973-1977，1992）。假定的铁调节血红素结合蛋白受体溶解从H。流感病毒706705、DL 42和埃根聚糖与去污剂CHAPS（3-[（3-胆酰胺丙基）-二甲基-铵基]-1-丙磺酸盐）接触，并通过血红素-血红素结合蛋白-琼脂糖4 B亲和层析分离。结合到亲和树脂的蛋白质的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示29、38和57 kDa的三种蛋白质，其中57-和29-kDa的蛋白质在Western印迹（免疫印迹）后结合血红素结合蛋白。706705的57-kDa血红素结合蛋白的单克隆抗体识别706705、DL 42和埃根的细胞包膜蛋白的Western印迹上的57-kDa蛋白;未观察到与DL 42的100-kDa血红素结合蛋白的反应。这些数据表明，一些H。流感病毒株具有至少两种血红素结合蛋白受体，其表达由主要生长环境决定。
Heme can serve Haemophilus influenzae as a source of both essential porphyrin and iron. In extracellular mammalian body fluids neither free heme nor free iron is available, since they are tightly bound to hemopexin and transferrin, respectively. Since H. influenzae grows in the presence of iron-transferrin and heme-hemopexin and is known to express a saturable receptor for transferrin, we investigated the process by which this pathogen acquired heme from hemopexin for use as an iron source. The ability of human and rabbit hemopexin to donate heme as a source of iron to H. influenzae type b strains was demonstrated by plate bioassays. With a dot enzyme assay with biotinylated hemopexin as ligand, H. influenzae bound heme-hemopexin and apo-hemopexin following growth in iron-restricted, but not in iron-sufficient, medium. Competitive binding studies with heme-hemopexin and apo-hemopexin demonstrated saturability of binding. Neither heme, protoporphyrin IX, hemoglobin, nor transferrin blocked the binding of hemopexin to whole cells, demonstrating the specificity of binding. Treatment of whole H. influenzae cells with trypsin abolished binding. Taken together, these observations suggest that H. influenzae type b expresses an outer membrane protein(s) which acts as a receptor for hemopexin and which is regulated by the availability of iron in the growth medium. In iron-restricted media, H. influenzae 706705 and DL42 did not express the 100-kDa hemopexin-binding protein previously reported (M.S. Hanson, S.E. Pelzel, J. Latimer, U. Muller-Eberhard, and E.J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). The putative iron-regulated hemopexin receptor was solubilized from cell envelopes of H. influenzae 706705, DL42, and Eagan with the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and isolated by affinity chromatography on heme-hemopexin-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins bound to the affinity resin revealed three proteins of 29, 38, and 57 kDa, of which the 57- and 29-kDa proteins bound hemopexin after Western blotting (immunoblotting). A monoclonal antibody to the 57-kDa hemopexin-binding protein of 706705 recognized a 57-kDa protein on Western blots of the cell envelope proteins of 706705, DL42, and Eagan; no reaction was observed with the 100-kDa hemopexin-binding protein of DL42. These data suggest that some H. influenzae strains possess at least two hemopexin receptors, the expression of which is determined by the prevailing growth environment.