de la Fuente José, Garcia-Garcia Jose C, Blouin Edmour F, Kocan Katherine M
Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, 250 McElroy Hall, 74078-2007, Stillwater, OK, USA.
Vet Microbiol. 2003 Feb 2;91(2-3):265-83. doi: 10.1016/s0378-1135(02)00309-7.
The major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been shown to mediate adhesion, infection and transmission of the organism, as well as to contribute to protective immunity in cattle. MSP1a contains a variable number of tandemly repeated peptides in the amino-terminal region, while the remainder of the protein is highly conserved among isolates. The number of repeats varies among geographic isolates of A. marginale but is constant within an isolate and has been used as a stable genetic marker of isolate identity. Because the sequence of the tandem repeats is the most variable part of the protein among isolates, this region of the protein is most likely to be involved in adhesion to host cells, a prerequisite to infection. The purpose of this study was to characterize the organization and function of the MSP1a tandem repeats of A. marginale in adhesion to host cells. We demonstrated by use of recombinant mutant proteins that the tandemly repeated region of MSP1a was necessary and sufficient to mediate adhesion of MSP1a to tick cells and bovine erythrocytes. Synthetic peptides representing the predominant sequences of individual repeats were tested for their adhesive capacity for tick cell extract (TCE). Peptides containing acidic amino acids D or E at position 20 bound to TCE, while peptides with a G as the 20th amino acid were not adhesive to TCE. Antibodies produced in rabbits against a synthetic repeat peptide neutralized A. marginale infection of cultured tick cells, and the neutralization observed was similar to that effected by antibodies produced against the whole MSP1a recombinant protein. Analysis of tandemly repeated MSP1a peptides of several geographic isolates of A. marginale revealed a complex relationship between the msp1alpha genotype and the tick-transmissible phenotype of the isolate and suggested that both the sequence and conformation of the repeated peptides influenced the adhesive properties of MSP1a. These studies demonstrated that the tandemly repeated region of the protein mediates the adhesive function of MSP1a.
边缘无形体（立克次氏体目：无形体科）属模式种的主要表面蛋白（MSP）1a已被证明可介导该生物体的黏附、感染和传播，以及在牛体内产生保护性免疫。MSP1a在氨基末端区域含有数量可变的串联重复肽段，而该蛋白的其余部分在不同分离株中高度保守。重复序列的数量在边缘无形体的不同地理分离株中有所不同，但在一个分离株内是恒定的，并已被用作分离株身份的稳定遗传标记。由于串联重复序列是该蛋白在不同分离株中最具变异性的部分，因此该蛋白的这一区域最有可能参与与宿主细胞的黏附，这是感染的先决条件。本研究的目的是表征边缘无形体MSP1a串联重复序列在与宿主细胞黏附中的组织和功能。我们通过使用重组突变蛋白证明，MSP1a的串联重复区域对于介导MSP1a与蜱细胞和牛红细胞的黏附是必要且充分的。测试了代表各个重复序列主要序列的合成肽对蜱细胞提取物（TCE）的黏附能力。在第20位含有酸性氨基酸D或E的肽与TCE结合，而第20位氨基酸为G的肽则不与TCE黏附。用兔抗合成重复肽产生的抗体中和了培养蜱细胞中的边缘无形体感染，观察到的中和作用与抗整个MSP1a重组蛋白产生的抗体所产生的中和作用相似。对边缘无形体几个地理分离株的串联重复MSP1a肽段的分析揭示了msp1alpha基因型与分离株的蜱传播表型之间的复杂关系，并表明重复肽段的序列和构象均影响MSP1a的黏附特性。这些研究表明，该蛋白的串联重复区域介导了MSP1a的黏附功能。
