Title
Studies on the Virulence and Genome Structure of Atypical
Toxoplasma gondii Strains Isolated in Japan( 要約版(Digest) )
Author(s)
谷口, 裕二
Report No.(Doctoral
Degree)
博士(獣医学) 甲第587号
Issue Date
2021-03-15
Type
博士論文
Version
none
URL
http://hdl.handle.net/20.500.12099/81631
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。Ꮫ ㄽ ᩥ せ ⣙
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㢟 ┠ Studies on the Virulence and Genome Structure of Atypical
Toxoplasma gondii
Strains Isolated in Japan ᪥ᮏ࡛ศ㞳ࡉࢀࡓࢺ࢟ࢯࣉࣛࢬ࣐㠀ᆺᰴࡢཎᛶ ࢤࣀ࣒ᵓ㐀㛵ࡍࡿ◊✲Toxoplasma gondii㸦ࢺ࢟ࢯࣉࣛࢬ࣐㸧ࡢศᏊᏛ◊✲ࡣḢᕞࡸ࣓ࣜ࢝࡞ࡢᆅᇦ࡛ศ㞳ࡉࢀࡓᰴࢆ୰ᚰ㐍ࡵ ࡽࢀࡓࠋ1990 ᖺ௦,ࡇࢀࡽࡢᆅᇦ࡛ศ㞳ࡉࢀࡓࢺ࢟ࢯࣉࣛࢬ࣐ࡘ࠸࡚ 6 ࡘࡢ㑇ఏᏊ࣐࣮࣮࢝ࢆ⏝࠸ࡓ PCR/RFLP 㸦polymerase chain reaction/restriction fragment length polymorphism㸧ἲࡼࡗ࡚㑇ఏᏊᆺศ㢮ࡀ⾜ࢃࢀࡓࠋ100 ᰴ௨ୖࡢࢺ࢟ࢯࣉࣛࢬ࣐ࢆㄪࡓ⤖ᯝ,ぢࡘࡗࡓ㑇ఏᏊᆺࡣ 15 ࡛,ከᩘࡢᰴࡣ 3 ࡘࡢ㑇ఏᏊᆺ㸦TypeI,II,III㸧ศ 㢮ࡉࢀࡓࠋࡉࡽTypeI,II,III ศ㢮ࡉࢀࡿࢺ࢟ࢯࣉࣛࢬ࣐ࡣ࣐࢘ࢫ࠾࠸࡚㑇ఏᏊᆺࡈỴࡲࡗࡓẘᛶࢆ♧ࡍࠋࡇ ࡢࡼ࠺ࢺ࢟ࢯࣉࣛࢬ࣐ࡢࢇࡀTypeI,II,III ࡢ 3 ࡘࡢ㑇ఏᏊᆺศ㢮࡛ࡁࡿ࠸࠺ࠕ3 ࢡ࣮ࣟࣥ௬ㄝࠖࡣ,༡ ࣓ࣜ࢝ࡸࣇࣜ࢝,ࢪᆅᇦ࡞࡛᪂ࡓ࡞㑇ఏᏊᆺࢆᣢࡘᰴࡀศ㞳ࡉࢀࡿࡲ࡛ୡ⏺ⓗᗈࡃᨭᣢࡉࢀ࡚࠸ࡓࠋᆺⓗ ࡞3 ࡘࡢ㑇ఏᏊᆺᙜ࡚ࡣࡲࡽ࡞࠸ᰴࡣࠕatypicalࠖࡸࠕexoticࠖࡤࢀ,ࡇࢀࡽࡢ㠀ᆺᰴࡣᆺⓗ࡞ TypeI,II,III ᰴẚ࡚࣐࢘ࢫ࡛ᙉ࠸ẘᛶࢆ♧ࡍഴྥࡀ࠶ࡿࠋࡇࢀࡽࡢ⤖ᯝࡣࢺ࢟ࢯࣉࣛࢬ࣐ࡢ㑇ఏⓗከᵝᛶࡀணࡼࡾࡶ㧗࠸ࡇ ࢆ♧ࡋ࡚࠸ࡿࠋࡇࢀࡲ࡛ࢺ࢟ࢯࣉࣛࢬ࣐ࡢẘᛶⓎ⌧࣓࢝ࢽࢬ࣒ࡣ࣐࢘ࢫࢆ⏝࠸࡚◊✲ࡉࢀ࡚ࡁࡓࡀ,࣐࢘ࢫ࠾ ࡅࡿࢺ࢟ࢯࣉࣛࢬ࣐ឤᰁᑐࡍࡿචᛂࡣࣄࢺࡁࡃ␗࡞ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࢺ࢟ࢯࣉࣛࢬ࣐ࡢẘᛶ㛵 ࡋ࡚ᗈࡃ⌮ゎࡍࡿࡓࡵࡣ,࣐࢘ࢫ௨እࡢᐟື≀ࢆ⏝࠸ࡓ◊✲ࡀᚲせ࡛࠶ࡿࠋ㏆ᖺ,ࣈࢱ⣽⬊࡛ぢࡽࢀࡿࢺ࢟ࢯࣉࣛࢬ ࣐ឤᰁᑐࡍࡿࣥࢱ࣮ࣇ࢙ࣟࣥȚㄏᑟᛶࡢචᛂࡀࣄࢺࡢ⣽⬊㢮ఝࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ,ࣈࢱࡣࣄࢺ ࢺ࢟ࢯࣉࣛࢬ࣐ࡢࣔࢹࣝື≀࡞ࡾ࠺ࡿࠋࡲࡓ㠀ᆺⓗ࡞ࢺ࢟ࢯࣉࣛࢬ࣐ࡘ࠸࡚ヲ⣽ㄪࡿࡇ࡛᪂ࡓ࡞ẘᛶ Ⓨ⌧࣓࢝ࢽࢬ࣒ࡢⓎぢࡘ࡞ࡀࡿྍ⬟ᛶࡀ࠶ࡿࠋࢪᆅᇦ,≉୰ᅜ࡛ࡣ㠀ᆺᰴࡀᩘከࡃศ㞳ࡉࢀ࡚࠾ࡾ,ᴟᮾ ⨨ࡍࡿ᪥ᮏ࠾࠸࡚ࡶ㠀ᆺᰴࡀศᕸࡋ࡚࠸ࡿྍ⬟ᛶࡀ㧗࠸ࠋࡑࡢࡓࡵ᪥ᮏศᕸࡍࡿࢺ࢟ࢯࣉࣛࢬ࣐ࡢ㑇ఏⓗከᵝ ᛶ࠾ࡼࡧẘᛶࢆ᫂ࡽࡍࡿࡇࡣࢺ࢟ࢯࣉࣛࢬ࣐ࡢẘᛶ◊✲࠾࠸࡚㔜せࡔゝ࠼ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣ,᪥ᮏศ ᕸࡍࡿࢺ࢟ࢯࣉࣛࢬ࣐㠀ᆺᰴࢆศ㞳ࡋ,࣐࢘ࢫ࠾ࡼࡧ࣐ࢡ࣑ࣟࢽࣈࢱࢆ⏝࠸ࡓཎᛶホ౯ヲ⣽࡞ࢤࣀ࣒ᵓ㐀ゎᯒ ࢆᐇࡋࡓࠋ Ἀ⦖┴࡛ศ㞳ࡉࢀࡓࢺ࢟ࢯࣉࣛࢬ࣐ᑐࡋ࡚1 ࡘࡢ㑇ఏᏊ࣐࣮࣮࢝㸦gra6㸧ࢆ⏝࠸ࡓ PCR/RFLP ἲࡼࡿ㑇ఏᏊᆺ ศ㢮ࢆ⾜ࡗࡓ◊✲࡛ࡣ,࣐࢘ࢫ࡛ᙉ࠸ẘᛶࢆ♧ࡍࡇࡀ▱ࡽࢀ࡚࠸ࡿ TypeI ᰴྠࡌ㑇ఏᏊᆺࢆᣢࡘࢺ࢟ࢯࣉࣛࢬ࣐ࡀ ከࡃぢࡘࡗ࡚࠸ࡿࠋࡲࡓ,᪥ᮏࡢ 47 㒔㐨ᗓ┴ࡢ୰࡛ࣈࢱࢺ࢟ࢯࣉࣛࢬ࣐ࡢሗ࿌ࡀ࠶ࡿࡢࡣ 2010 ᖺ௨㝆Ἀ⦖┴ࡢࡳ ࡛࠶ࡿࠋࡇࢀࡽࡢᐇࡣᙉẘᆺࡢࢺ࢟ࢯࣉࣛࢬ࣐ࡀἈ⦖┴ᒁᅾࡍࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋࡋࡋ,ࡍ࡛㏙ࡓࡼ࠺ ࣐࢘ࢫࣈࢱ࡛ࡣ␗࡞ࡿẘᛶࢆ♧ࡍྍ⬟ᛶࡀ࠶ࡾ,ࣈࢱ࠾ࡅࡿẘᛶࢆホ౯ࡍࡿࡓࡵࡣࣈࢱࢆ⏝࠸ࡓឤᰁᐇ㦂ࡀᚲ せ࡛࠶ࡿࠋࡑࡇ࡛➨1 ❶࡛ࡣ,Ἀ⦖┴࡛ศ㞳ࡉࢀ,ᙅẘ࡛▱ࡽࢀࡿ TypeII ㏆࠸㑇ఏᏊᆺࢆᣢࡘࡶ㛵ࢃࡽࡎ࣐࢘ࢫ࡛ ᙉ࠸ẘᛶࢆ♧ࡍࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࢺ࢟ࢯࣉࣛࢬ࣐TgCatJpOk4 ᰴࡘ࠸࡚࣐ࢡ࣑ࣟࢽࣈࢱࢆ⏝࠸ࡓཎᛶホ౯ ࢆᐇࡋࡓࠋ5 㢌ࡢ࣐ࢡ࣑ࣟࢽࣈࢱ㸦5 ᭶㱋㸧 TgCatJpOk4 ᰴࡢࢱ࢟ࢰࢺ㸦107㸧ࢆ⭡⭍᥋✀ࡋࡓࠋ࠺ࡕ4 㢌 ࡣ᥋✀4-7 ᪥ᚋࡽ㣗ḧᗫ⤯,Ⓨ⇕,ඖẼᾘኻ,㢖྾࡞ࡢ⮫ᗋ≧ࢆ♧ࡋࡓࠋ⮫ᗋ≧ࢆ♧ࡋࡓ 4 㢌ࡢ࠺ࡕ 3 㢌ࡣ᥋ ✀ᚋ9-11 ᪥ࡢ㛫ṚஸࡋࡓࠋṚஸಶయࡢ⫵ࡣయ㉥ࡳࡀࡗ࡚࠾ࡾ⮋⒆㒊ࡀᩓぢࡉࢀࡓࠋ⌮᳨ᰝࡢ⤖ᯝ,Ẽ⟶ᨭቨ ࠾ࡼࡧ⫵⬊ቨከ㔞ࡢ⅖⣽⬊ࡀᾐ₶ࡋ㛫㉁ᛶ⫵⅖ࢆ࿊ࡋ࡚࠸ࡓࠋᮏ❶ࡼࡾ,Ἀ⦖┴࡛ศ㞳ࡉࢀࡓࢺ࢟ࢯࣉࣛࢬ࣐
TgCatJpOk4 ᰴࡀ࣐࢘ࢫࡔࡅ࡛࡞ࡃࣈࢱ࡛ࡶᙉ࠸ẘᛶࢆ♧ࡍࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
᪥ᮏ࠾ࡅࡿࢺ࢟ࢯࣉࣛࢬ࣐㠀ᆺᰴࢆ᥈⣴ࡍࡿ㐣⛬࡛,ᒱ㜧┴࡛ࢺ࢟ࢯࣉࣛࢬ࣐ࢆศ㞳ࡋࡓ㸦TgCatJpGi1/TaJ ᰴ㸧ࠋ ➨2 ❶࡛ࡣ TgCatJpGi1/TaJ ᰴࢆ⏝࠸࡚,ẘᛶᅉᏊࡋ࡚▱ࡽࢀࡿ rhoptry(ROP)ࢱࣥࣃࢡࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢ㑇ఏ Ꮚᆺศ㢮࣐࢘ࢫ࠾ࡼࡧ࣐ࢡ࣑ࣟࢽࣈࢱ࠾ࡅࡿཎᛶホ౯ࢆᐇࡋࡓࠋROP ࢱࣥࣃࢡ㸦ROP5,16,17,18㸧ࡣࢺ࢟ ࢯࣉࣛࢬ࣐ࡢẘᛶᅉᏊࡋ࡚▱ࡽࢀ࡚࠾ࡾ,ࡇࢀࡽࡢ㑇ఏᏊᆺࡽ࣐࢘ࢫ࠾ࡅࡿẘᛶࢆண ࡛ࡁࡿࡇࡀሗ࿌ࡉࢀ࡚ ࠸ࡿࠋ㐣ཤࡢሗ࿌ᚑ࠸PCR/RFLP ἲࢆ⏝࠸࡚ TgCatJpGi1/TaJ ᰴࡢ ROP 㑇ఏᏊࢆㄪࡓࡇࢁ,ࡍ࡚ࡢ ROP 㑇 ఏᏊࡘ࠸࡚↓ẘᰴྠࡌ㑇ఏᏊᆺࢆ♧ࡋࡓࠋࡇࡢ⤖ᯝࡣ,TgCatJpGi1/TaJ ᰴࡀ࣐࢘ࢫ࠾࠸࡚↓ẘ࡛࠶ࡿࡇࢆ♧ ၀ࡋ࡚࠸ࡿࠋࡋࡋ,ᐇ㝿 TgCatJpGi1/TaJ ᰴࡢࢩࢫࢺ㸦102㸧ࢆ࣐࢘ࢫ⤒ཱྀ᥋✀ࡋࡓࡇࢁ,᥋✀ࡋࡓࡍ࡚ࡢ࣐ ࢘ࢫࡀṚஸࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࡽ,᪤▱ࡢẘᛶ㑇ఏᏊࡢ㑇ఏᏊᆺࡽࡣண࡛ࡁ࡞࠸ẘᛶࢆ࣐࢘ࢫ࡛♧ࡍࢺ࢟ࢯࣉࣛ ࢬ࣐ᰴࡀ᪥ᮏศᕸࡍࡿࡇࡀࢃࡗࡓࠋ TgCatJpGi1/TaJ ᰴࡣ ROP 㑇ఏᏊࡢ㑇ఏᏊᆺࡽࡢணࡣ␗࡞ࡿẘᛶࢆ࣐࢘ࢫ࡛♧ࡍࡇࢆ➨ 2 ❶࡛♧ࡋࡓࠋࡇ ࡢࡇࡣTgCatJpGi1/TaJ ᰴࡀᮍ▱ࡢẘᛶᅉᏊࢆᣢࡗ࡚࠸ࡿྍ⬟ᛶࢆ♧၀ࡋ࡚࠸ࡿࠋTgCatJpGi1/TaJ ᰴࡢࢤࣀ࣒ ࢩ࣮ࢣࣥࢫゎᯒࡢ⤖ᯝ,ࢩࣥࢢࣝࢥࣆ࣮㑇ఏᏊ࡛࠶ࡿ ROP16,17,18 ࢆࢥ࣮ࢻࡋ࡚࠸ࡿ㑇ఏᏊࡢሷᇶ㓄ิࡘ࠸࡚ࡣ↓ ẘ࡛▱ࡽࢀࡿTypeIII ᰴ୍⮴ࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡋࡋ,ROP5 ࡢ㑇ఏᏊࡘ࠸࡚ࡣࢱࣥࢹ࣒ࣜ ࣆ࣮ࢺ㓄ิࢆྵࢇ࡛࠸ࡿࡓࡵṇ☜࡞ࢥࣆ࣮ᩘࡀ᥎ᐃ࡛ࡁ࡞ࡗࡓࠋ➨3 ❶࡛ࡣ,ROP5 ࡢࡼ࠺࡞ከࢥࣆ࣮㑇ఏᏊࡢᵓ㐀 ࢆ᫂ࡽࡋTgCatJpGi1/TaJ ᰴࡢẘᛶᅉᏊࢆ᥈⣴ࡍࡿࡓࡵ,ࣟࣥࢢ࣮ࣜࢻࢩ࣮ࢣࣥࢧ࣮ࢆ⏝࠸ࡓࢤࣀ࣒ࢩ࣮ࢣࣥࢫ ゎᯒࢆᐇࡋࡓࠋຍ࠼࡚RNA-seq ゎᯒࢆ⾜࠸ TgCatJpGi1/TaJ ᰴ↓ẘ࡛▱ࡽࢀࡿ CTG ᰴࢆẚ㍑ࡋࡓሙྜࡢⓎ⌧ኚ ື㑇ఏᏊࡘ࠸࡚ࡶㄪࡓࠋࡲࡎ TgCatJpGi1/TaJ ᰴ CTG ᰴࡢ㧗⢭ᗘ࡞ࢻࣛࣇࢺࢤࣀ࣒ࢆᚓࡿࡓࡵ,ᮏ◊✲࡛ࡣ Illumina ࡼࡿࢩ࣮ࣙࢺ࣮ࣜࢻࢹ࣮ࢱ Nanopore ࡼࡿࣟࣥࢢ࣮ࣜࢻࢹ࣮ࢱࢆ⏝࠸ࡿࣁࣈࣜࢵࢻࢭࣥࣈࣜࢆ᥇ ⏝ࡋࡓࠋᅇᚓࡽࢀࡓTgCatJpGi1/TaJ ᰴ CTG ᰴࡢࢻࣛࣇࢺࢤࣀ࣒ࡣࢹ࣮ࢱ࣮࣋ࢫୖᏑᅾࡍࡿࣞࣇࣞࣥࢫᰴࡢ ࢤࣀ࣒ẚ࡚㠀ᖖᑡ࡞࠸ࢥࣥࢸࢢ࡛ᵓᡂࡉࢀ࡚࠾ࡾ,ࡉࡽࢥࣥࢸࢢ㛫ࡢࢠࣕࢵࣉࡣࡳࡽࢀ࡞ࡗࡓࠋࡇࢀࡽ ࡢ⤖ᯝࡣࣁࣈࣜࢵࢻࢭࣥࣈࣜࡀࢺ࢟ࢯࣉࣛࢬ࣐ࡢࢤࣀ࣒ᵓ㐀ࢆ㧗࠸⢭ᗘ࡛Ỵᐃ࡛ࡁࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋḟከ ࢥࣆ࣮ࡘࢺ࢟ࢯࣉࣛࢬ࣐ࡢẘᛶᙉࡃ㛵ࢃࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿROP5 ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢሷᇶ㓄ิ࠾ࡼࡧ㑇ఏ Ꮚᵓ㐀ࡘ࠸࡚ゎᯒࢆ⾜ࡗࡓࠋTgCatJpGI1/TaJ ᰴ CTG ᰴࡢ ROP5 ࡢࢥࣆ࣮ᩘࢆỴᐃࡍࡿࡓࡵࡑࢀࡒࢀࡢࢤࣀ࣒ ࢆඖBLAST ゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ,TgCatJPGi1/TaJ ᰴࡣ 4 ࢥࣆ࣮,CTG ᰴࡣ 3 ࢥࣆ࣮࡛ᵓᡂࡉࢀࡿ ROP5 ࢆᣢ ࡗ࡚࠸ࡿࡇࡀࢃࡗࡓࠋ⥆ࡅ࡚TgCatJpGi1/TaJ ᰴ,CTG ᰴ࠾ࡼࡧࣞࣇࣞࣥࢫᰴࡢ ROP5 ሷᇶ㓄ิࢆ⏝࠸࡚ࣁࣉ ࣟࢱࣉࢿࢵࢺ࣮࣡ࢡゎᯒࢆ⾜ࡗࡓࠋTgCatJpGi1/TaJ ᰴ࠾ࡼࡧ CTG ᰴࡢ ROP5 ࢯࣇ࢛࣮࣒ࡣ GT-1㸦TypeI㸧 ᰴ,VEG㸦TypeIII㸧ᰴࡢ ROP5 ࢡ࣮ࣞࢻࢆᙧᡂࡋࡓࠋࡇࡢ⤖ᯝࡣ TgCatJpGi1/TaJ ᰴࡢ ROP5 ࡀ᪤▱ࡢ TypeI ᰴ࠾ ࡼࡧ TypeIII ᰴࡀᣢࡘ ROP5 ࡼࡃఝ࡚࠸ࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋ࣑ࣀ㓟㓄ิࡢẚ㍑ࢆ⾜ࡗࡓࡇࢁ,᪤▱ࡢ↓ẘᆺ TypeII ᰴ㸦CTG ᰴ࠾ࡼࡧ VEG ᰴ㸧࡛ࡣぢࡽࢀ࡞࠸࣑ࣀ㓟⨨ࡀ TgCatJpGi1/TaJ ᰴ࠾࠸࡚」ᩘぢࡘࡗࡓࡀ, ࢀࡶ ROP5 ࡢᶵ⬟㛵ࢃࡿ㒊ศ࡛ࡣ࡞ࡗࡓࠋ௨ୖࡢ⤖ᯝࡽ TgCAtJpGi1/TaJ ᰴࡢ ROP5 ࡣ↓ẘᆺࡢ TypeIII ᰴࡀᣢࡘ ROP5 ࡁࡃኚࢃࡽ࡞࠸ࡇࡀࢃࡗࡓࠋࡉࡽ RNA-seq ࡢࢹ࣮ࢱࡽⓎ⌧ኚື㑇ఏᏊゎᯒࢆ⾜ࡗࡓ ࡇࢁ,TgCatJpGi1/TaJ ᰴ CTG ᰴ࡛ 2106 ಶࡢⓎ⌧ኚື㑇ఏᏊࡀぢࡘࡗࡓࠋࡇࢀࡽࡢ୰ࡽ TgCatJpGi1/TaJ ᰴ CTG ᰴࡢẘᛶࡢ㐪࠸㛵ࢃࡿ㑇ఏᏊࢆぢࡘࡅࡿࡓࡵ,ࢺ࢟ࢯࣉࣛࢬ࣐ࡢẘᛶࡶࡋࡃࡣᐟ⣽⬊ࡢධࡢ㛵ࡀ▱ ࡽࢀ࡚࠸ࡿࢱࣥࣃࢡᶆⓗࢆ⤠ࡗ࡚㑇ఏᏊⓎ⌧㔞ࡢẚ㍑ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ,ᐟ⣽⬊ࡢ᥋╔㛵ࢃࡿࢱࣥࣃࢡ యࡢ㐠ືᛶ㛵ࢃࡿࢱࣥࣃࢡࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ⌧㔞ࡀCTG ᰴẚ࡚ TgCatJpGi1/TaJ ᰴ࡛㧗࠸ࡇࡀࢃࡗ ࡓࠋ ᮏ◊✲࠾࠸࡚ᴟᮾ⨨ࡍࡿ᪥ᮏ࡛ศ㞳ࡉࢀࡓ ᰴࡢࢺ࢟ࢯࣉࣛࢬ࣐ࡀ㠀ᆺⓗ࡞ẘᛶࢆ♧ࡍࡇࡀ᫂ࡽ࡞ ࡗࡓࠋᮏ◊✲ࡣࢪᆅᇦ࠾ࡅࡿࢺ࢟ࢯࣉࣛࢬ࣐ࡢከᵝᛶࢆ♧ࡍࡔࡅ࡛࡞ࡃᮍ▱ࡢẘᛶᅉᏊࡢᏑᅾࡶ♧ࡋ࡚࠸ࡿࠋ
ࡲࡓࢺ࢟ࢯࣉࣛࢬ࣐ࡢ㑇ఏᏊゎᯒ࠾ࡅࡿ᪂ࡋ࠸ᡭἲࡢ᭷ຠᛶࢆ♧ࡍࡇ࡛ࢺ࢟ࢯࣉࣛࢬ࣐ࡢẘᛶ㛵ࡍࡿ◊✲ࢆ ࡁࡃ๓㐍ࡉࡏࡓࠋ
Ꮫ ㄽ ᩥ せ ⣙
Ặ ྡTANIGUCHI, Yuji
㢟 ┠ Studies on the Virulence and Genome Structure of Atypical
Toxoplasma gondii
Strains Isolated in Japan ᪥ᮏ࡛ศ㞳ࡉࢀࡓࢺ࢟ࢯࣉࣛࢬ࣐㠀ᆺᰴࡢཎᛶ ࢤࣀ࣒ᵓ㐀㛵ࡍࡿ◊✲Molecular epidemiological studies on Toxoplasma gondii (T. gondii) have mainly advanced in Europe and North America. In 1990s, over one-hundredT. gondii strains mainly isolated in Europe and North America could be classified into only fifteen different genotypes based on polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) using 6 genetic markers and almost all strains could be divided into major three groups, Type I, II, and III. The “three-clone hypothesis” which theorized that the majority of
T. gondii strains globally could be classified into Type I, II and III had been accepted until many new strains found in South America, Africa and Asia did not fit into these classifications. These strains are designated as atypical or exotic and tend to show higher virulence in mice than the typical three-clones. These results indicate that the genetic diversity of T. gondii was higher than previously thought. The virulence mechanism of T. gondii had been studied using a mouse model. However, it has been reported that the immune response against T. gondii in mouse is completely different from human. To further understand the molecular mechanism of T. gondii virulence, studies using experimental animals other than mice are also necessary. Especially, there is a possibility that pig can be a model animal of human toxoplasmosis because it has been reported that interferon-γ induced immune response against T. gondii infection in pig cell is similar with that in human cell. The study about atypical T. gondii strains has contributed to the discovery of new virulence mechanism of T. gondii. In Asia, especially China, it has been reported that the atypical T. gondii strains which have different genotype from “three-clone” strains were frequently isolated. So, there is a possibility that atypical strains distribute also in Japan which is located in far east. It is necessary to reveal the genetic property and virulence of Japanese isolates in detail. Therefore, the aim of this study is to isolate atypical T. gondii strains in Japan (Far East), compare their virulence in mice and Microminipigs, and then analyze their detailed genome structures.
There are some reports studying about the genotype of T. gondii strains isolated in Okinawa depending on PCR/RFLP analysis using gra6 gene. According to this genotyping, many strains isolated in Okinawa have same genotype with Type I strains, which are known as high virulence in mice. In fact, even though there are 47 prefectures in Japan, clinical toxoplasmosis in pigs has been reported only from Okinawa prefecture since 2010. This suggests that high virulent strains in pigs distribute in Okinawa locally. However, there is a possibility that T. gondii shows different virulence between in mice and pigs. Therefore, it is necessary to perform the infection experiment using pigs for the evaluation of the virulence of T. gondii in pigs. In CHAPTER 1, I examined the virulence of TgCatJpOk4 strain, which was isolated in Okinawa, using Microminipigs. Five Microminipigs (5-month-old) were injected with 107 TgCatJpOk4 tachyzoites
intraperitoneally. Four infected pigs showed inappetence, fever, hypoactivity and tachypnea from 4-7 days post infection. Among the four pigs that showed clinical symptoms, three pigs died during the observation period. Macroscopic analysis revealed that lungs of three pigs which died were diffusely reddened and had several whitish spots. Microscopic analyses also revealed that numerous leukocytes infiltration occurred in the bronchial septa and the alveolar septa, and those septa were severely thickened, representing the pathological characteristics of interstitial pneumonia. In CHAPTER 1, it was revealed that a T. gondii strain isolated in Okinawa showed high virulence in pigs.
In the process of searching for strains with various pathogenicity, I isolated a T. gondii strain in Gifu prefecture, which was located in Honshu island, Japan. This strain was here as TgCatJpGi1/TaJ strain. In CHAPTER 2, I performed genotyping of several genes which had been known as virulence factors and examined the virulence of TgCatJpGi1/TaJ strain in mice and Microminipigs. It has been reported that the allele types of rop5, rop16, rop17, and rop18 genes, as determined by RFLP analysis of their polymorphic regions, are predictive of virulence in mice across the global distributed T. gondii strains. Therefore, I analyzed rop5, rop16, rop17, and rop18 genes from TgCatJpGi1/TaJ strain by RFLP using a previously reported method. According to the genotyping, TgCatJpGi1/TaJ strain has an identical RFLP pattern in its
rop genes as avirulent type III strains. This result suggests that TgCatJpGi1/TaJ strain show no virulence in mice. However, TgCatJpGi1/TaJ strain showed high virulence in mice. From these results, it was revealed that T. gondii strain which shows unexpected virulence in mice according to the genotyping of genes coding previously known virulence factors distributes in Japan.
As described in CHAPTER 2, TgCatJpGi1/TaJ strain showed higher virulence than expected from its genotype of rop genes. This result suggests that TgCatJpGi1/TaJ strain has an unknown virulence factor other than ROPs. As a result of the whole genome sequence analysis of TgCatJpGi1/TaJ strain using the short read sequencer, it had been revealed that the sequence of genes for ROP16, ROP17 and ROP18, these are single copy genes, are identical with that of avirulent Type III strain. However, the copy number of genes coding ROP5 was unable to be estimated accurately because the gene for ROP5 is composed of tandem repeat in T. gondii genome. In CHAPTER 3, to overcome the problem and reveal the structure of multi-copy genes of TgCatJpGi1/TaJ strain, I performed whole genome sequencing using long read sequencer. Additionally, I performed comparison analysis of differentially expressed genes (DEGs) identified by RNA-seq analysis between TgCatJpGi1/TaJ and CTG strains, which is a typical avirulent Type III strain. To obtain the draft genome of T. gondii TgCatJpGi1/TaJ and CTG strains with high quality, I applied the technique called the hybrid assembly using Illumina short-reads and Nanopore long-reads. After the sequencing, the whole genome sequence of two strains were assembled using both short and long read data. Draft genome of TgCatJpGi1/TaJ and CTG strains showed very few numbers of contigs and didn’t contain the gap of contigs. These results indicate that the structure of T. gondii genome was able to be determined high accuracy using the hybrid assembly. Next, I performed sequence and structural analysis for rop5 gene. To determine the copy number of ROP5 of TgCatJpGi1/TaJ and CTG strains, I searched ROP5 sequence in their genome using BLAST. It was revealed that TgCatJpGi1/TaJ strain and CTG strain had 4 copies and 3 copies of ROP5 in their genome. Subsequently, I performed haplotype network analysis using the sequence of rop5 gene of TgCatJpGi1/TaJ, CTG and reference strains. Each isoform of ROP5 of TgCatJpGi1/TaJ and CTG strains build a clade together with GT-1 strain (Type I) and VEG strain (Type III). This result indicates
that the sequence of rop5 gene of TgCatJpGi1/TaJ strain is similar to its of Type I or Type III strains. The comparison of amino acid sequence of ROP5 among TgCatJpGi1/TaJ and Type III strains (CTG and VEG) revealed that there was no mutation in the region which was associated with the function of ROP5. These results indicate that ROP5 of TgCatJpGi1/TaJ strain is not so different from that of avirulent Type III strains. Differentially expressed genes (DEGs) analysis revealed the existence of 2106 DEGs between TgCatJpGi1/TaJ and CTG strains. Among all DEGs, to find the genes which are associated with the difference of phenotype between TgCatJpGi1/TaJ and CTG strains, coding genes of a group of virulence- or invasion-associated effectors were subjected to the comparison of gene expression level. It was revealed that the expression level of rhoptry neck proteins and microneme proteins, which are associated with host cell invasion and gliding motility, were higher in TgCatJpGi1/TaJ straiin.
In this study, two T. gondii strains isolated in Japan, which is an island country in far east Asia, showed unexpected virulence from their genotypes. These findings indicate not only the genetic diversity of T. gondii
strains in Asia but also the existence of unknown virulence factors in T. gondii. In addition, this study advanced the research of virulence of T. gondii by indicating usefulness of the new effective method in genetic analysis of T. gondii.
