Specific antibodies to Porphyromonas gingivalis Lys-gingipain by DNA vaccination inhibit bacterial binding to hemoglobin and protect mice from infection
Specific antibodies to Porphyromonas gingivalis Lys-gingipain by DNA vaccination inhibit bacterial binding to hemoglobin and protect mice from infection
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DOI：
10.1128/iai.69.5.2972-2979.2001
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发表时间：
2001-05-01
影响因子：
3.1
通讯作者：
Hamada, S
中科院分区：
文献类型：
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作者：
Kuboniwa, M;Amano, A;Hamada, S
Lys-gigiain(KGP)是一种赖氨酸特异的半胱氨酸蛋白酶，是牙龈卟啉单胞菌的主要毒力因子之一。在这里，我们研究了KGP的催化结构域(KGP(CD))在牙龈假单胞菌与血红蛋白结合中的作用，使用了编码KGP(CD)的质粒DNA或Arg-牙周酶催化结构域(RGP(CD))所诱导的特异性免疫球蛋白G(IgG)。通过连接Kgp(CD)和RGP(Cd)DNA片段，分别构建了pSeq2A/Kgp(Cd)和pSeq2B/Rgp(Cd)。用pSeq2A/KGP(CD)分别免疫雌性BALB/c尘螨，pSeq2A/KGP(CD)对重组KGP(CD)(rKGP(CD))产生较强的反应，并产生相应的RRGP(CD)反应性抗体。PSecTag2B/RGP(CD)诱导的血清抗体与RKGP(CD)和RRGP(CD)也发生交叉反应。抗KgP(CD)Ig G可显著抑制P牙周炎与Hb的结合，且抗Kgp(CD)与抗菌毛联合使用可明显增强对Hb结合的抑制。抗RGP(CD)免疫球蛋白的抑制作用不明显，而抗KGP(CD)和抗RGP(CD)免疫球蛋白均对Lys和Arg特异的蛋白水解酶活性有显著抑制作用，并且在补充血红蛋白为唯一铁源的铁限制条件下对牙周炎P菌的生长有明显的抑制作用。免疫小鼠经腹腔接种牙龈假单胞菌攻击。未免疫的小鼠均在72 h内死亡，而pSeq2A/Kgp(CD)和pSeq2B/RGP(CD)免疫小鼠可预防炎症反应，并使免疫小鼠的存活率分别提高43%和27%。这些结果表明，KGP(CD)既可以作为一种血红蛋白结合蛋白，也可以作为一种免疫原，诱导对牙龈假单胞菌感染的保护性反应。
Lys-gingipain (KGP), a lysine-specific cysteine proteinase, is one of the major virulence factors of Porphyromonas gingivalis. Here we examined the involvement of the catalytic domain of KGP (KGP(cd)) in hemoglobin binding by P. gingivalis, using a specific immunoglobulin G (IgG) elicited by the administration of plasmid DNA encoding KGP(cd) or the catalytic domain of Arg-gingipain (RGP(cd)). The pSeq2A/kgp(cd) and pSeq2B/rgp(cd) plasmids were constructed by the ligation of kgp(cd) and rgp(cd) DNA fragments, respectively. Female BALB/c mite were immunized with each of these plasmids, pSeq2A/kgp(cd) elicited a strong response to recombinant KGP(cd) (rKGP(cd)), as well as to comparably produced rRGP(cd)-reactive antibodies. The serum antibodies elicited by pSecTag2B/rgp(cd) also cross-reacted with rKGP(cd) as well as rRGP(cd). Anti-KGP(cd) IgG significantly inhibited hemoglobin binding by P, gingivalis, Furthermore, the inhibition of hemoglobin binding was markedly enhanced by a combination of anti-KGP(cd) and anti-fimbriae. Anti RGP(cd) IgG showed a negligible inhibitory effect, while both anti-KGP(cd) and anti-RGP(cd) IgGs showed significant inhibitory effects on Lys- and Arg- specific proteolytic activities and on the growth of P, gingivalis under iron-restricted conditions where supplemented hemoglobin was the sole iron source. Immunized mice were challenged by intraperitoneal inoculation with P. gingivalis. All nonimmunized mice died within 72 h; however, vaccination with pSeq2A/kgp(cd) and pSeq2B/rgp(cd) prevented inflammatory responses and prolonged the survival rate of immunized mice by 43 and 27%, respectively. These results suggest that KGP(cd) acts as a hemoglobin-binding protein and can also be useful as an immunogen inducing a protective response to P. gingivalis infection.