称号及び氏名

博士（獣医学） ISLAM AKM AZHARUL

学位授与の日付

2021 年 3 月 31 日

論The cytotoxicity and mechanism of Clostridium

文

名perfringens NetB toxin(ウエルシュ菌が産生する NetB 毒素の病原性発現に関する研究)
論文審査委員

主査
副査
副査

向本 雅郁
笹井 和美
山崎 伸二

論文要旨

Introduction
Avian necrotic enteritis by Clostridium perfringens is such an emergent disease that causes
significant economic loss to the poultry industry worldwide. Necrotic enteritis in chickens
caused by C. perfringens is a contagious disease in which sudden and sporadic deaths occur
due to coccidial infection, disturbance of the intestinal microflora by antibiotics, and highprotein feeding. Clinical signs include loss of energy, feather reversal, and diarrhea, and small
intestinal lesions are observed on gross examination. In the subclinical form of the disease, theintestinal mucosa is damaged, resulting in decreased digestion and absorption of nutrients,decreased weight gain, and increased feed requirements. This disease causes a loss ofapproximately 200 billion yen annually to the poultry industry worldwide, especially to thebroiler industry, and there is an urgent need for measures to reduce the incidence of this disease.
C. perfringens is a Gram-positive, anaerobic, large bacillus that is classified into types Ato G according to the production pattern of its major toxins (alpha, beta, epsilon, iota,enterotoxin and Necrotic Enteritis Toxin B-like (NetB toxin). It causes gas gangrene andnecrotic enteritis in livestock and poultry. For more than 30 years, the pathogenic factor ofchicken necrotic enteritis has been considered to be the alpha toxin (phospholipase C) produced
by C. perfringens. However, there has been a remarkable breakthrough in both our theoreticalunderstanding and practical application in 2006 when a mutant of C. perfringens in which thealpha-toxin gene was knocked out was found to cause necrotic enteritis, indicating that alphatoxin is not an essential pathogenic factor for necrotic enteritis. In 2008, a novel protein toxin
with 38% amino acid sequence homology to beta toxin produced by C. perfringens wasidentified in chicken isolates with necrotic enteritis and named NetB toxin. Its netB knockout
mutant does not cause necrotic enteritis in chickens but reintroduction of netB into the knockout
strain does, indicating that NetB toxin is an essential virulence factor for the development ofnecrotic enteritis
The author therefore isolated netB-carrying bacteria from the feces of necrotic enteritisprone chickens and healthy chickens in Japan analyzed the characteristics of the bacteria inChapter 1. The NetB toxin was then purified from the isolates and examined the species
specificity of the toxin activity using various strains of cells and erythrocytes from various
animal species and its pathogenicity to chickens in Chapter 2. In Chapter 3, the author analyzed
the molecular dynamics of NetB toxin on the target cell membrane leading to pore formation.
This thesis has provided several intriguing results, which will improve the understanding of themolecular mechanism of NetB toxin binding, oligomerization and cytotoxicity in avian cells.

Chapter 1. Molecular epidemiological analysis of netB-carrying C.
perfringens isolated necrotic enteritis affected chickens
The author isolated C. perfringens from specimens obtained in Japan and examined thepresence or absence of the major toxin gene and netB and compared the chicken erythrocyte
hemolytic activity of each strain. Pulsed-field gel electrophoresis (PFGE) was also used tocompare the genes and drug susceptibility of the isolates. In this study, NetB toxin-carrying C.
perfringens was isolated from feces of chickens with necrotic enteritis and healthy chickens atseveral farms, indicating that NetB toxin-carrying C. perfringens is distributed in Japan andinfects healthy chickens. However, NetB toxin-negative bacteria were also isolated fromchickens with necrotic enteritis, suggesting that there may be other factors associated with thedevelopment of necrotic enteritis. In DNA sequencing of isolated C. perfringens, NetB toxin isa very well conserved gene and that the NetB toxin-carrying strains in Japan are almost identicalto those distributed worldwide. In PFGE analysis, three strains which were isolated fromchickens with necrotic enteritis on different farms and harbored NetB toxin, were all classified
as different types. These results suggest that conjugative plasmids containing NetB toxin are
not easily transmitted, and that there may be a single or small number of NetB toxin-carryingstrains spreading in the same area. In addition, each type of NetB toxin-carrying strains isolated
in this study indicates that there are different NetB toxin-carrying strains in different regions.

Chapter 2. Purification and toxicity of Clostridium perfringens NetB toxin
The NetB toxin was purified from the supernatants of C. perfringens type G (CNEOP004)
isolated from chickens with necrotic enteritis in Japan. To select a suitable medium forproducing large amount of toxin a few media along with the culture time were examined.
Haemolytic activity showed the highest rate in culture supernatants in tryptose peptone glucose(TPG) medium for 36 hours. The toxin was purified by fractionating the culture supernatantwith ammonium sulphate precipitation, anion chromatography and cation exchange
chromatography. Finally, a band of NetB toxin (33kDa) was observed by these purification
procedures. EC50 of this purified NetB toxin toxin toward LMH cells was 0.63 μg/ml.
Haemolytic assay was done in erythrocytes of various animals and birds by measuring theamount of toxin inducing 50% hemolysis (HU50). The results of erythrocyte sensitivity tests
using the purified NetB toxin showed that the species specificity of NetB toxin is very narrow,
limited to the pheasant order to which chickens belong and some birds, and it is thought to havelittle toxicity to mammals. Cytotoxicity tests were conducted using various cell lines, and the50% cell lethal dose (EC50) was calculated. Chick liver-derived LMH cells showed remarkably
high sensitivity (EC50: 0.63 µg/ml). chick embryo fibroblast (CEF) cells were about 1/14 of thesensitivity of LMH cells (EC50: 8.7 µg / ml). In mammalian cell lines, IEC-6 cells (EC50: 18µg/ml), 3Y1 cells (EC50: 33 µg/ml), and MDBK cells (EC50: 31 µg/ml) were even less sensitive,and CRFK, MDCK, and Vero cells (EC50: 8.7 µg/ml) were less sensitive. CRFK cells, MDCKcells, Vero cells, P3U1 cells, C2C12 cells, L929 cells, and Caco-2 cells were not lethal even ata NetB toxin concentration of 50 µg/ml. Chicks died within 3 hr of intraperitoneal
administration of NetB toxin (50 µg). Necropsy showed extensive bleeding from the duodenum
to the jejunum and thinning of the caecum, but no lesions were found in other organs. The
intestinal mucosae in the duodenum, jejunum, and caecum were found to be desquamative withbleeding, but no abnormal findings were observed in the colon. Characteristic microscopic
lesions in moderate cases included focal, multifocal to coalescing necrosis of enterocytes,
whereas in severe cases, coagulative necrosis of the entire superficial mucosa separating
underlying viable lamina propria with infiltration of mild to moderate inflammatory cells wasobserved. Therefor it could be concluded that the NetB toxin was biologically active and thetoxicity was more specific to birds. From these results, NetB toxin has shown the possibility of
specifically binding to cell surface molecules expressed only on birds.

Chapter 3. Binding and oligomerization of NetB toxin on avian cells
As the author has seen in Chapter 2 that significant hemolytic activity and cytotoxicity
happen only in an avian cell line so we attempted to investigate the oligomerization and pore
formation capabilities of NetB toxin only on chicken liver derived LMH cells. LMH cells werereacted with freshly purified NetB toxin solution (10 μg/ml) followed by lysis withmorpholinoethanesulfonic acid-buffered saline containing 1% Triton X-114. Subsequently, thecell lysates were fractionated by sucrose density gradient ultracentrifugation and run on SDS–
PAGE followed by immunoblotting. Results showed that both monomer and oligomer
molecule of NetB toxin were localized in non-lipid raft region. Furthermore, to analyze theinvolvement of cholesterol in the cell membrane in NetB toxin binding and oligomer formation,
methyl-β-cyclodex trin (MCD) was applied to LMH cells to remove cholesterol. After
cholesterol removal, NetB toxin was reacted, dissolved using a surfactant. Then the cell lysate
was centrifuged into soluble fraction and an insoluble pellet and then these both wereimmunoblotted using an anti-NetB toxin antibody. It was also immunoblotted using anti-actin
antibody to differentiate the band intensity between oligomers found in MCD treated cells andMCD untreated cells. The result showed that oligomer of NetB toxin can be detected in thecells even after removal of cholesterol. These data suggested that this NetB toxin may
recognize different membrane receptors or use a different mechanism for membrane-protein
interactions than usual pore forming toxins do. In order to analyze the localization of the NetB
toxin binding molecules on LMH cells, a toxin overlay assay was done followed byimmunoblotting. In this assay, the LMH cells were separated into detergent-soluble anddetergent-insoluble fractions and the soluble parts were fractionated by sucrose densitygradient ultracentrifugation. Results showed that molecules of approximately 60, 53, 47, 40
and 25 kDa that were bound to NetB toxin were detected in the non-lipid raft supernatantfractions.

Conclusions
The author found that netB-carrying C. perfringens is widespread in Japan and infects
healthy chickens. NetB toxin is on a zygotic plasmid, but it is not easily transmitted, and onlya single or a small number of netB-carrying strains are thought to spread in the same area. NetB
toxin was also a pore-forming toxin with highly specific toxin activity restricted to the pheasantorder, to which chickens belong, and some birds.
It was found that NetB toxin binds to the target cell in the non-raft region without theinvolvement of cholesterol in the cell membrane, and that the nearby monomers polymerize toform oligomers without being affected by the fluidity of the membrane. Thus, the molecular
dynamics of NetB toxin, from binding to the cell to pore formation, was partially elucidated.
Since netB-carrying bacteria are widely detected even in healthy chickens, prevention of
the onset of necrotizing enterocolitis, such as vaccination, is considered to be important. In thefuture, the identification of binding molecules to NetB toxin and the determination of the region
on the NetB toxin molecule involved in the dynamics from binding on cell membranes to pore
formation will elucidate the pathogenic mechanism of necrotic enteritis in chickens andcontribute to the development of vaccines.

審査結果の要旨
Clostridium perfringens は嫌気性、芽胞形成のグラム陽性大桿菌であり、産生する毒素（α,β,ε,ι,CPE,NetB）の産生性の違いから A〜G 型に分類される。本菌は土壌や下水など環境中および動物の腸内細菌叢から分離されており、動物に様々な疾患を引き起こす。その一つが
鶏壊死性腸炎であり、A および G 型菌まれに C 型菌によって引き起こされる。鶏壊死性腸炎は、世界の養鶏業に大きな経済的損失をもたらす、致死性の高い急性の伝染性疾病であり、発生軽減への対策が急務となっている。鶏壊死性腸炎の病原因子は、本菌が産生する α 毒素（ホスホリパーゼ C）であると考えられてきた。近年、本菌が産生する β 毒素とアミノ酸配列の相同性が38％の新規蛋白毒素（NetB 毒素）が、壊死性腸炎の発症鶏から発見され、現在では、NetB 毒素が鶏壊死性腸炎の発症に関わる必須の病原性因子であると考えられている。NetB 毒素は細胞膜に結合後、7 量体となり孔を形成することで細胞の溶解を引き起こす孔形成毒素であり、G 型菌のみが産生する。本研究では NetB 毒素の病原性発現機構を解明する目的で、日本国内の壊死性腸炎発症鶏より分離した株から精製した NetB 毒素を用いて、種特異性および標的細胞膜上における分子動態の解析を行い、以下の成果を得た。
第１章では、わが国の壊死性腸炎発症鶏および健常鶏から C. perfringens を分離し，netBの有無を調べ，株間の鶏赤血球溶血活性を比較した。また、パルスフィールドゲル電気泳動（PFGE）を用いて、遺伝子の系統樹解析を行いさらに薬剤感受性を比較した。本研究により，複数の農場において壊死性腸炎発症鶏と健常鶏の糞便から netB を保有する C. perfringens が分離されたことから、netB を保有する C. perfringens が日本国内に分布し，健常鶏にも感染していることを証明した。しかし，壊死性腸炎を発症した鶏からは netB 陰性菌も分離されており，壊死性腸炎の発症には他の病原因子も関与している可能性が示唆された．分離された C. perfringens において、netB の塩基配列は非常によく保存されていた。PFGE 解析では，異なる農場の壊死性腸炎発症鶏から分離された netB 保有株は，いずれも異なるタイプに分類された。これらの結果から、日本において、地域ごとに異なる netB 保有株が存在することが明らかとなった。
第 2 章では、壊死性腸炎発症鶏から分離された C. perfringens 培養上清より 2 種類のイオン交換クロマトグラフィーを用いて NetB 毒素（33kDa）を精製した。様々な哺乳類・鳥類の赤血球および株化細胞に対する NetB 毒素の溶血・細胞致死活性を測定した。NetB 毒素の種特異性は非常に高く、溶血および細胞致死活性はニワトリが属するキジ目や一部の鳥類に限定されており、哺乳類の赤血球や株化細胞に対してはほとんど毒性を示さなかった。ニワトリ肝癌由来株化細胞である LMH 細胞に対する 50％細胞致死量（EC50）は 0.63μg/ml と非常に高値を示した。NetB 毒素（50 μg）を 2 日齢ニワトリの腹腔内に投与したとき、 3 時間以内にすべての個体が死亡した。剖検では十二指腸から空腸への大量出血と盲腸の菲薄化が認められた。組織学的には腸管粘膜の剥離・脱落および出血が確認され、壊死性腸炎発症鶏と同様の病態を示した。以上より，NetB毒素は生物学的に活性を有しており，その毒性は鳥類に特異的であると結論づけた。
第 3 章 では NetB 毒素の活性は鳥類に対してのみ有していることから LMH 細胞を用いて、細胞膜上での NetB 毒素のオリゴマー化と孔形成能を調べた。LMH 細胞と NetB 毒素を反応させた後、細胞溶解物をショ糖密度勾配超遠心分離法で分画し、SDS-PAGE で分析した後、免疫ブロッティングを行った。その結果、NetB 毒素のモノマー分子とオリゴマー分子の両方が細胞膜上の非ラフト画分に存在していた。さらに、コレステロールを除去した LMH 細胞に対しても NetB 毒素のオリゴマーが検出されたことから、NetB 毒素の細胞膜への結合やオリゴマー化にはコレステロールが関与しないことが明らかとなった。また、LMH 細胞上の NetB 毒素と結合する分子が toxin
overlay assay により非ラフト画分に検出された。
以上、本研究により netB を保有する C. perfringens は日本に広く蔓延しており、健常鶏にも感染していることが明らかとなった。NetB 毒素は、ニワトリが属するキジ目や一部の鳥類に限定された種特異性の非常に高い活性を有する孔形成性毒素であった。NetB 毒素は、細胞膜上の非ラフト領域で標的細胞に結合し、近傍のモノマー同士がコレステロールの関与なく重合してオリゴマーを形成することを明らかにし、NetB 毒素の細胞への結合から孔形成までの分子動態の一部を解明した。これらの成果は、今後の細胞膜上の NetB 毒素結合分子の同定や、細胞膜への結合から孔形成に至る動態に関与する NetB 毒素分子上の領域決定の一助となり、鶏壊死性腸炎の発症メカニズムの解明さらには、ワクチン開発に繋がり、獣医学および畜産学の発展に貢献するものである。本論文の審査ならびに最終試験の結果と併せて博士（獣医学）の学位を授与することを適当と認める。