Shimosato Takeshi, Kimura Toshiro, Tohno Masanori, Iliev Iliyan Dimitrov, Katoh Shinichiro, Ito Yoshiyuki, Kawai Yasushi, Sasaki Takashi, Saito Tadao, Kitazawa Haruki
Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
Cell Microbiol. 2006 Mar;8(3):485-95. doi: 10.1111/j.1462-5822.2005.00640.x.
Lactobacillus gasseri OLL2716 has recently been discovered as a probiotic that suppresses the growth of Helicobacter pylori and reduces gastric mucosal inflammation in humans. This has resulted in the development of a new type of probiotic yoghurt 'LG21' in Japan. In our previous study, we found an immunostimulatory AT5ACL oligodeoxynucleotide (AT-ODN) containing a unique core sequence (5'-ATTTTTAC-3') in L. gasseri JCM1131(T). Interestingly, although the AT-ODN does not contain any CpG sequences, it exerts mitogenic activity in B cells and augments Th-1-type immune responses via Toll-like receptor 9. These findings prompted us to identify strong immunostimulatory non-CpG AT-ODNs that contain the 5'-ATTTTTAC-3' motif in the genomic sequence of L. gasseri OLL2716. We identified 280 kinds of AT-ODNs in the L. gasseri OLL2716 genome. Mitogenicity and NF-kappaB gene reporting assays showed that 13 of the 280 AT-ODNs were strongly immunostimulatory when in the TLR9 transfectant. Of these, AT-ODNs LGAT-145 and LGAT-243 were the most potent. With respect to the induction of Th-1-type cytokines, LGAT-243 had the greatest activity and was more potent than the swine prototype, ODN D25. We further found that a six-base secondary loop structure containing a self-stabilized 5'-C...G-3' stem sequence is important for potent immunostimulatory activity. These results show for the first time that AT-ODNs with a specific loop and stem structure are important factors for immunostimulatory activity. Finally, we found that novel strong immunostimulatory non-CpG AT-ODNs exist in the genome of probiotic lactic acid bacteria.
加氏乳杆菌OLL2716最近被发现是一种益生菌，它能抑制幽门螺杆菌的生长并减轻人类胃黏膜炎症。这促使日本研发出了一种新型益生菌酸奶“LG21”。在我们之前的研究中，我们在加氏乳杆菌JCM1131(T)中发现了一种含有独特核心序列（5'-ATTTTTAC-3'）的免疫刺激型AT5ACL寡脱氧核苷酸（AT-ODN）。有趣的是，尽管该AT-ODN不包含任何CpG序列，但它在B细胞中具有促有丝分裂活性，并通过Toll样受体9增强Th-1型免疫反应。这些发现促使我们在加氏乳杆菌OLL2716的基因组序列中鉴定出含有5'-ATTTTTAC-3'基序的强免疫刺激型非CpG AT-ODN。我们在加氏乳杆菌OLL2716基因组中鉴定出了280种AT-ODN。促有丝分裂活性和NF-κB基因报告分析表明，在TLR9转染细胞中，这280种AT-ODN中有13种具有强免疫刺激作用。其中，AT-ODN LGAT-145和LGAT-243的活性最强。就Th-1型细胞因子的诱导而言，LGAT-243的活性最大，比猪源原型ODN D25更有效。我们进一步发现，含有自我稳定的5'-C...G-3'茎序列的六碱基二级环结构对于有效的免疫刺激活性很重要。这些结果首次表明，具有特定环和茎结构的AT-ODN是免疫刺激活性的重要因素。最后，我们发现益生菌乳酸菌基因组中存在新型强免疫刺激型非CpG AT-ODN。
