Hemoglobin hydrolysis and heme acquisition by Porphyromonas gingivalis
Hemoglobin hydrolysis and heme acquisition by Porphyromonas gingivalis
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DOI：
10.1046/j.0902-0055.2003.00113.x
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发表时间：
2004-02-01
影响因子：
--
通讯作者：
Reynolds, EC
中科院分区：
文献类型：
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作者：
Dashper, SG;Cross, KJ;Reynolds, EC
牙龈卟啉单胞菌与慢性牙周炎的进展有关，慢性牙周炎是牙齿支持组织的炎性疾病。这种细菌是一种革兰氏阴性、黑色色素、溶砷厌氧菌，其依赖于氨基酸的发酵来产生代谢能量。牙龈卟啉单胞菌的精氨酸和赖氨酸特异性胞外半胱氨酸蛋白酶RgpA、RgpB和Kgp被认为是主要的毒力因子。在本研究中，我们研究了牙龈卟啉单胞菌W50和突变体W501（RgpA（-）），W50 AB（RgpA（-）RgpB（-））和W50 ABK（RgpA（-）RgpB（-）Kgp（-））在严格厌氧条件下在生理缓冲液（pH 7.5）中水解人血红蛋白的质谱分析。将牙龈卟啉单胞菌W50与血红蛋白一起孵育30分钟，检测到20种血红蛋白肽，所有肽均具有C-末端Arg或Lys残基。除了每条链的C-末端一半的两个相似区域α（92-127）和β（83-120）外，大多数血红蛋白α-和β-链序列以肽的形式回收。未回收序列的残基形成α链和β链之间的界面的一部分，以及血红蛋白四聚体的暴露表面积，其可能参与与牙龈卟啉单胞菌的结合。牙龈卟啉单胞菌W501（RgpA（-））产生的肽与野生型中所见的肽相似。由牙龈卟啉单胞菌W50 AB（RgpA（-）RgpB（-））突变体水解血红蛋白而鉴定的所有肽都是在Lys处切割的结果。三重突变体W50 ABK在所使用的测定条件下不能水解血红蛋白，这表明在全细胞上负责水解血红蛋白的主要细胞表面活性来自RgpA/B和Kgp蛋白酶。然而，三重蛋白酶突变体W50 ABK在含有血红蛋白作为唯一铁源的培养基中生长良好，表明RgpA/B和Kgp蛋白酶对于牙龈卟啉单胞菌从血红蛋白同化铁不是必需的。
Porphyromonas gingivalis has been implicated in the progression of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. This bacterium is a gram-negative, black-pigmented, asaccharolytic anaerobe that relies on the fermentation of amino acids for the production of metabolic energy. The Arg- and Lys-specific extracellular cysteine proteinases of P. gingivalis, RgpA, RgpB and Kgp have been implicated as major virulence factors. In this study we investigated the hydrolysis of human hemoglobin by whole cells of P. gingivalis W50 and the mutants W501 (RgpA(-)), W50AB (RgpA(-)RgpB(-)) and W50ABK (RgpA(-)RgpB(-)Kgp(-)) under strictly anaerobic conditions in a physiological buffer (pH 7.5) using mass spectrometric analysis. Incubation of P. gingivalis W50 with hemoglobin over a period of 30 min resulted in the detection of 20 hemoglobin peptides, all with C-terminal Arg or Lys residues. The majority of the hemoglobin alpha- and beta-chain sequences were recovered as peptides except for two similar regions of the C-terminal half of each chain, alpha(92-127) and beta(83-120). The residues of the unrecovered sequences form part of the interface between the alpha- and beta-chains and an exposed surface area of the hemoglobin tetramer that may be involved in binding to P. gingivalis. P. gingivalis W501 (RgpA(-)) produced similar peptides to those seen in the wild-type. All identified peptides from the hydrolysis of hemoglobin by the P. gingivalis W50AB (RgpA(-)RgpB(-)) mutant were the result of cleavage at Lys. The triple mutant W50ABK was unable to hydrolyze hemoglobin under the assay conditions used, suggesting that on whole cells the major cell surface activity responsible for hydrolysis of hemoglobin is from the RgpA/B and Kgp proteinases. However, the triple proteinase mutant W50ABK grew as well as the wild-type in a medium containing hemoglobin as the only iron source, indicating that the RgpA/B and Kgp proteinases are not essential for iron assimilation from hemoglobin by P. gingivalis.