Strong immunostimulatory activity of AT-oligodeoxynucleotide requires a six-base loop with a self-stabilized 5'-C...G-3' stem structure.
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.
Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
Takeshi
T
Shimosato
Toshiro
T
Kimura
Masanori
M
Tohno
Iliyan Dimitrov
ID
Iliev
Shinichiro
S
Katoh
Yoshiyuki
Y
Ito
Yasushi
Y
Kawai
Takashi
T
Sasaki
Tadao
T
Saito
Haruki
H
Kitazawa
D020856:AT Rich Sequence / Q000276:immunology*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007422:Intestines / Q000166:cytology / Q000276:immunology; D007778:Lactobacillus / Q000737:chemistry / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000276:immunology*; D010581:Peyer's Patches / Q000166:cytology / Q000276:immunology; D019936:Probiotics; D013552:Swine; D051217:Toll-Like Receptor 9 / Q000235:genetics; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D051217:Toll-Like Receptor 9