Ebersole J L, Kraig E, Bauman G, Spitznagel J K, Kolodrubetz D
Department of Periodontics, University of Texas Health Science Center, San Antonio 78284.
Arch Oral Biol. 1990;35 Suppl:69S-78S. doi: 10.1016/0003-9969(90)90133-u.
A strategy has been developed to examine the hypothesis that leucotoxin is a critical virulence factor of Actinobacillus actinomycetemcomitans in a non-human primate (Macaca fascicularis). Firstly the leucotoxin gene from A. actinomycetemcomitans was cloned and sequenced. This DNA contained a functional leucotoxin gene, as protein extracts of Escherichia coli with the cloned sequences lysed appropriate human cell lines. The protein encoded by lktA shared at least 42% identity with P. haemolytica leucotoxin and with the alpha-haemolysins from E. coli and A. pleuropneumoniae. The lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be related to the LktC proteins from these other bacteria and with which it shared at least 49% amino acid identity. Despite the overall homology to the other leucotoxins/haemolysins, the LktA from A. actinomycetemcomitans has several unique properties including a very basic pI of 9.7, as compared to pIs approx. 6.2 for lktA proteins in other bacteria. Using the cloned genes as probes produced evidence that a TOX- strain contains the leucotoxin gene but fails to transcribe it at high levels. The second avenue of investigation was to develop methods for examining the humoral immune responses in the monkey to bacterial toxins such as lktA. A. actinomycetemcomitans was detected in subgingival plaque samples from approx. 40% of the animals. A. actinomycetemcomitans comprised less than 1% to 9% of the flora. Most A. actinomycetemcomitans isolates were serotype b and each of the monkeys had serum IgG antibody to A. actinomycetemcomitans serotype b (generally considered to be lktA-producing strains). An ELISA was developed to examine the isotype/subclass distribution, level and avidity of serum antibody in the monkey following parenteral immunization with a prototype bacterial exotoxin (tetanus toxoid). IgG1 and IgG3 antibody predominated over IgG2 and IgG4 after primary immunization. Secondary immunization elicited enriched IgG1 and IgG4 responses. Primary immunization increased avidity indices of IgG to tetanus toxoid from approx. 0.9 (baseline) to a mean of 1.72 and secondary immunization significantly increased the avidity index to 2.56.
已制定一项策略，以检验白细胞毒素是伴放线放线杆菌在非人灵长类动物（食蟹猴）中的关键毒力因子这一假说。首先，克隆并测序了伴放线放线杆菌的白细胞毒素基因。该DNA包含一个功能性白细胞毒素基因，因为带有克隆序列的大肠杆菌蛋白提取物可裂解合适的人类细胞系。lktA编码的蛋白质与溶血巴氏杆菌白细胞毒素以及大肠杆菌和胸膜肺炎放线杆菌的α-溶血素至少有42%的同源性。伴放线放线杆菌的lktA基因与另一个基因lktC相连，lktC被认为与这些其他细菌的LktC蛋白有关，且与其至少有49%的氨基酸同源性。尽管与其他白细胞毒素/溶血素总体上有同源性，但伴放线放线杆菌的LktA有几个独特特性，包括其非常碱性的9.7的pI，而其他细菌中lktA蛋白的pI约为6.2。用克隆基因作为探针的研究表明，一个TOX-菌株含有白细胞毒素基因，但未能高水平转录。第二条研究途径是开发方法来检测猴子对诸如lktA等细菌毒素的体液免疫反应。在约40%的动物的龈下菌斑样本中检测到了伴放线放线杆菌。伴放线放线杆菌占菌群的比例不到1%至9%。大多数伴放线放线杆菌分离株为b血清型，并且每只猴子都有针对伴放线放线杆菌b血清型的血清IgG抗体（通常认为是产生lktA的菌株）。开发了一种ELISA来检测猴子经注射原型细菌外毒素（破伤风类毒素）免疫后血清抗体的同种型/亚类分布、水平和亲和力。初次免疫后，IgG1和IgG3抗体占主导地位，超过IgG2和IgG4。二次免疫引发了富集的IgG1和IgG4反应。初次免疫使IgG对破伤风类毒素的亲和力指数从约0.9（基线）增加到平均1.72，二次免疫显著将亲和力指数提高到2.56。
