Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model
Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model
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DOI：
10.1128/iai.69.12.7527-7534.2001
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发表时间：
2001-12-01
影响因子：
3.1
通讯作者：
Reynolds, EC
中科院分区：
文献类型：
--
作者：
O'Brien-Simpson, NM;Paolini, RA;Reynolds, EC
细胞外Arg-x和Lys-x特异性半胱氨酸蛋白酶被认为是牙龈卟啉单胞菌的重要毒力因子和致病标志物，是慢性牙周炎的主要病原体。三个基因。RgpA、rgpB和Kgp分别编码Arg-x特异的蛋白酶和粘附素(RgpA)、Arg-x特异的蛋白酶(RgpB)和Lys-x特异的蛋白酶和粘附素(Kgp)。使用缺乏RgpA、RgpB和Kgp的同源突变株，在小鼠病变模型中研究了牙龈假单胞菌W50的每一个蛋白水解酶基因对致病性的贡献。与野生型W50相比，RgpA(-)和RgpB(-)等基因突变体的全细胞Arg-x特异性蛋白分解活性显著降低(3-4倍)。而Kgp(-)等基因突变株的全细胞Arg-x活性与野生型相似。RgpA和RgpB突变体的全细胞Lys-x蛋白水解酶活性与野生型W50无显著差异，而Kgp突变体缺乏Lys-x全细胞蛋白水解酶活性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和蛋白质印迹分析表明，突变株和野生型菌株的胞浆蛋白水解酶催化结构域均不表达。分析进一步表明，RgpB以72 kDa和80 kDa的条带出现，RgpA和KGP的催化结构域分别以加工的45 kDa和48 kDa的条带出现。在小鼠病变模型中，小鼠被用三种剂量的突变株和野生型菌株攻击。在较低剂量(3.0×10(9)活细胞)下，每个突变体都没有记录到病变，而野生型W50引起了较大的溃烂病变。在6.0×10(9)活细胞剂量下，用野生型菌株攻击的小鼠全部死亡，而用RgpA和RgpB等基因突变体攻击的小鼠没有死亡，但出现了病变。在这个剂量下，用KGP-等基因突变体挑战的小鼠没有出现病变。在1.2×10(10)活细胞剂量下，受到kgp突变株攻击的小鼠中只有40%出现损伤，而且这些损伤明显小于野生型菌株在3.0×109活细胞剂量下引起的损伤。所有被RgpA突变体攻击的小鼠在1.2×10(10)活细胞剂量下死亡，而当被RgpB突变体攻击时只有20%的小鼠在这个剂量下死亡。通过与含有rgpB基因的质粒pNJR12：：rgpB互补，将野生型恢复为rgpB-突变体。在小鼠病变模型中，pNJR12：：rgpB互补的RgpB突变株与野生型W50株在全细胞Arg-x活性、蛋白谱和毒力方面没有差异。这些结果表明，在小鼠病变模型中，RgpA、RgpB和Kgp这三种蛋白酶都对牙龈假单胞菌W50的毒力有贡献，并且它们的贡献顺序是Kgp远大于RgpB大于或等于RgpA。
Extracellular Arg-x- and Lys-x-specific cysteine proteinases are considered important virulence factors and pathogenic markers for Porphyromonas gingivalis, a bacterium implicated as a major etiological agent of chronic periodontitis. Three genes. rgpA, rgpB, and kgp, encode an Arg-x-specific proteinase and adhesins (RgpA), an Arg-x-specific proteinase (RgpB), and a Lys-x-specific proteinase and adhesins (Kgp), respectively. The contribution to pathogenicity of each of the proteinase genes of P. gingivalis W50 was investigated in a murine lesion model using isogenic mutants lacking RgpA, RgpB, and Kgp. Whole-cell Arg-x-specific proteolytic activity of both the RgpA(-) and RgpB(-) isogenic mutants was significantly reduced (3- to 4-fold) relative to that of the wild-type W50. However, for the Kgp(-) isogenic mutant, whole-cell Arg-x activity was similar to that of the wild-type strain. Whole-cell Lys-x proteolytic activity of the RgpA- and RgpB- mutants was not significantly different from that of wild-type W50, whereas the Kgp- mutant was devoid of Lys-x whole-cell proteolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using proteinase-specific antibodies of cell sonicates of the wild-type and mutant strains showed that the proteinase catalytic domain of each of the mutants was not expressed. This analysis further showed that RgpB appeared as 72- and 80-kDa bands, and the catalytic domains of RgpA and Kgp appeared as processed 45-kDa and 48-kDa bands, respectively. In the murine lesion model, mice were challenged with three doses of each mutant and wild-type strain. At the lower dose (3.0 x 10(9) viable-cells), no lesions were recorded for each of the mutants, whereas wild-type W50 induced large ulcerative lesions. At a dose of 6.0 x 10(9) viable-cells, all the mice challenged with the wild-type strain died, whereas mice challenged with the RgpA- and RgpB- isogenic mutants did not die but developed lesions. Mice challenged with the Kgp- isogenic mutant at this dose did not develop lesions. At a 1.2 x 10(10) viable-cell dose, only 40% of mice challenged with the Kgp- mutant developed lesions, and these lesions were significantly smaller than lesions induced by the wild-type strain at the 3.0 x 109 viable-cell dose. All the mice challenged with the RgpA- mutant died at the 1.2 x 10(10) viable-cell dose, whereas only 20% died when challenged with the RgpB- mutant at this dose. Wild-type phenotype was restored to the RgpB- mutant by complementation with plasmid pNJR12::rgpB containing the rgpB gene. There was no difference between the pNJR12::rgpB-complemented RgpB- mutant and the wild-type W50 strain in whole-cell Arg-x activity, protein profile, or virulence in the murine lesion model. These results show that the three proteinases, RgpA, RgpB, and Kgp, all contributed to virulence of P. gingivalis W50 in the murine lesion model and that the order in which they contributed was Kgp much greater than RgpB greater than or equal to RgpA.