称号及び氏名

博士（獣医学）Bernard Mudenda Hang' ombe

学位授与の日付

平成 17 年 3 月 31 日論

「Molecular mechanism of the toxic action of Clostridium

文

名septicum alpha-toxin on mammalian cells」（クロストリジウム・セプチカムα毒素の動物細胞に対する毒性発現機構に関する研究）

論文要旨
Introduction

Clostridium septicum is a ubiquitous, spore-forming, gram-positive anaerobic
bacterium that causes traumatic （ wound derived ） and non-traumatic（endogenous）myonecrosis, which are rapidly fatal diseases in man and animals.
Although the pathogen secretes several extracellular factors such asa alpha-toxin,
deoxyribonuclease, hyaluronidase and neuraminidase, alpha-toxin has been
implicated as a major virulent factor such that it possesses hemolytic, lethal andnecrotizing properties. However, the exact role of the toxin in disease remains
unclear. C. septicum alpha-toxin belongs to a family of toxin termed pore-forming
toxins. These toxins disrupt cellular membrane permeability by forming
unregulated channels. Previous studies have shown that the toxin is secreted as awater-soluble

protoxin

whichbinds

tocellsurfaces

via

glycosylphosphatidylinositol（GPI）-anchored proteins. Once the toxin binds to thecell, it is activated by proteolytic cleavage at the carboxyl-terminal propeptide
with cell surface proteases. Activation lets the activated monomers facilitate
oligomerization on the membrane followed by formation of functionaltransmembrane pores. The oligomerization on cell surface is considered to be a
critical step in the channel formation process. However, the precise mechanism on
oligomerization of the toxin on cells has not been fully understood. The present
study has been made to investigate the mechanism of toxic action on mammaliancells, in particular focusing upon toxin binding and subsequent oligomerization
on cell membranes. During the present investigation, a new toxin-binding proteinwas discovered and characterizad.
Chapter 1: Hemolytic activity and binding of C. septicum alpha-toxin on
erythrocyte membranes of various animals
In the preliminary experiments, the activity of C. septicum alpha-toxin wasdetermined in erythrocytes of various animals. The sensitivity of erythrocytes tothe toxin was observed in the order of mouse, rat, canine, equine, rabbit, chicken,
bovine, swine and ovine. Temperature and protease treatment affected thesensitivity of erythocytes to the toxin. Proteinase K tretment decreased thesensitivity of murine, canine, equine and bovine erythocytes, while ovine
erythrocytes did not change the sensitivity to the toxin. To the contrary, theactivity of the toxin on swine erythrocytes increased after treatment withproteinase K, trypsin, chymotrypsin or lysyl endopeptidase. In toxin overlay assay,
the toxin was shown to bind to erythrocyte membrane proteins with a molecularmass of 30 to 45-kDa in mouse, equine, bovine, swine and chicken, while the toxin
reacted with a 100-kDa protein in rat erythrocyte membranes. The treatment ofmurine and swine erythrocyte membranes with phosphatidylinositol-specificphospholipase C resulted in liberation of the toxin-binding protein fromindividual membranes in a native state. These results show that toxin associateswith specific erythocyte membrane proteins in any animal species, and aresubsets of GPI-anchored proteins in various animal species.
Chapter 2: Cytotoxicity and oligomerization of C. septicum alpha-toxin on
mammalian cellsThe cytotoxic activities were also examined with various mammalian cells. Incontrast with the activity to erythrocytes, mammalian cells were more sensitive
to protoxin than trypsinized toxin interacted with various molecular sizes ofcellular proteins. The size and SDS-PAGE pattern of the proteins were different
among cell lines, but they were liberated from the cells by the treatment withphosphatidylinositol-specific phospholipase C. The toxin appeared to target andutilize detergent resistant membranes（DRMs）for binding and subsequentoligomerization. In discontinuous sucrose density gradient followed byimmunoblotting, the toxin bound to DRMs contained in L929（fibroblast）cells andcaused oligomer formation. Furthermore, cholesterol depletion with cholesterolinteracting agents reduced toxin oligomerization and lowered cytotoxicity of thetoxin toward cells. These results suggest that the toxin preferentially exploits
DRMs for oligomerization.
Chapter 3: Interaction of C. septicum alpha-toxin with alpha-actin
3.1. Identification of alpha-actin as a C. septicum alpha-toxin binding protein
Alpha-toxin was found to interact with a43-kDa protein in L929 cells,

which was not associated with DRMs. In order to characterize the toxin-binding
protein, the protein was digested with lysyl endopeptidase and the liberated
peptides were separated by high performance liquid chromatography. When thepeptides obtained were applied to amino acid sequencing, the toxin-binding
protein was identified as a mouse cardiac alpha-actin. The cardiac alpha-actin
gene was then cloned, and the fusion protein was expressed as a histidine-tagged
protein in Escherichia coli. After purification by affinity chromatography, thetoxin binding activity of the recombinant protein was examined. The recombinantalpha-actin retained the toxin-binding ability, whereas no toxin binding wasobserved with bera-actin. The recombinant alpha-actin also appeared toaccelerate oligomer formation of the toxin in vitro.
3.2. Analysis of alpha-actin interacting with alpha-toxin in murine cells andtissues
A rat polyclonal anti-alpha-actin IgG was prepared and used to screen thepresence of alpha-actin in various murine cell lines and tissues. Alpha-actin waspresent in C2C12（myoblast）cells, L929 cells, heart and skeletal muscles as amonomer and polymer. After alpha-actin containing tissue lysates was entrappedwith beads coupled with anti-alpha-actin antibody, the immunoprecitates weresubjected to SDS-PAGE followed by toxin overlay assay. The results indicatedthat the toxin seemed to bind preferentially to alpha-actin polymer rather thanmonomer. To confirm further the interaction of the toxin with alpha-actin,
toxin-treated cell lysates were applied to immunoprecipitation with beads coupledwith anti-alpha-toxin. It was found that alpha-actin was entrapped together withthe toxin, suggesting that the toxin really associates with alpha-actin in cell

membranes.
Conclusions
1．Alpha-toxin binds to specific GPI-anchored proteins in various mammalian cells.
2．The protoxin binds to GPI-anchored proteins in non-DRMs for activation bycell surface proteases before moving to DRMs and oligomerize leading topore-formation.3 ． Cholesterol

plays

asignificantroleinalpha-toxin

oligomerization and cytotoxicity.4 ． Alpha-actin was identified as a new
toxin-binding protein, which provides some information to elucidate peculiarity interms of symptoms such as malignant edema involving C. septicum infection.

審査結果の要旨

ヒトのガス壊疽や動物の悪性水腫の原因菌である Clostridium septicum は皮膚や腸管の創傷部位から侵入し、産生される数種類の毒素･酵素の相乗作用により壊死や毒血症を引き起こす。C. septicum の主要な病原因子であるα毒素は細胞膜上に pore を形成することにより細胞を傷害する細胞溶解毒素（溶血毒）である。α毒素は活性のない分子量 46kDa の前駆体(protoxin)として産生された後、蛋白分解酵素により限定分解を受け、41kDa の活性化毒素となる。α毒素は GPI アンカー蛋白を受容体として細胞膜上に結合後、6～7 量体の高分子複合体(oligomer)として孔を形成して膜を破壊する代表的な細胞溶解毒素である。
本研究では、α毒素の細胞障害機構における受容体への結合から孔形成に至るまでの細胞膜上でのα毒素の動態について分子レベルで詳細に解析することにより、次のような成果を得た。
１．種々の動物由来赤血球を用いて C. septicumα毒素の溶血活性と受容体との関係を比較検討した。各赤血球を proteinase K で処理したとき、マウス、イヌ、ウマ、ウシ赤血球の感受性が低下した。ヒツジ赤血球では変化がみられず、ブタ赤血球では proteinase K、トリプシン、キモトリプシン、リシルエンドペプチダーゼのいずれの蛋白分解酵素処理によってもα毒素の活性は増加した。Toxin overlay assay でα毒素はマウス、ブタ、ウシ、ウマ、ニワトリ赤血球膜の 35～45ｋDa 蛋白と結合し、ラットでは 100ｋDa の膜蛋白と反応する こ と が 分 か っ た 。 マ ウ ス お よ び ブ タ 赤 血 球 膜 を phosphatidylinositol-specificphospholipase C(PI-PLC)処理すると、α毒素結合蛋白は膜から遊離することを明らかにした。これらの結果は、α毒素が動物種によって異なる赤血球膜蛋白と結合し、これらの蛋白はいずれも GPI-アンカー蛋白であることを示しており、このことがα毒素の動物種による溶血活性の違いに影響を与えていると考えられた。
２．動物由来株化細胞に対するα毒素の細胞障害および受容体結合活性、オリゴマー形成に関係する細胞側分子について解析を行った。α毒素は様々な分子量の異なる蛋白と結合したが、これらの結合蛋白は細胞株間で異なっていた。また、PI-PLC で処理するとα毒素結合蛋白は細胞から遊離することから、赤血球同様、株化細胞においてもα毒素は種々のGPI-アンカー蛋白と結合することにより活性を発現する明らかにした。α毒素で処理した細胞を界面活性剤で可溶化後、不連続ショ糖密度勾配遠心により分画し抗α毒素抗体を用いた immunoblotting により局在を調べた。α毒素は細胞膜の detergent 不溶画分（DRM）、いわゆるラフトでオリゴマーを形成することが分かった。さらにコレステロール阻害剤で処理するとα毒素のオリゴマー形成が減少し、細胞障害活性も低下した。これらの結果か

ら、α毒素は受容体に結合した後、ラフトに集積しオリゴマーを形成すること、さらにラフト集積およびオリゴマー形成には膜に存在するコレステロールが重要な役割を担っていることを明らかにした。
３．α毒素に結合する細胞の新たな蛋白としてαアクチンを同定した。L929 細胞を界面活性剤で処理後、ショ糖密度勾配遠心により分画し、toxin overlay assay を行った。α毒素
は不溶画分の GPI-アンカー蛋白以外に、可溶画分に存在する分子とも結合した。この分子の部分アミノ酸配列からマウス心臓αアクチンであることが分かった。種々の臓器、組織の可溶分画を抗αアクチン抗体で免疫沈降し、その沈降物を用いて toxin overlay assayを行った。その結果、α毒素は心臓および骨格筋では単量体より重合体αアクチンと結合することが分かった。α毒素を結合させた筋肉由来株化細胞を可溶化した後、抗α毒素抗体で免疫沈降した結果、沈降物にはαアクチンが含まれていた。これらの結果は、α毒素が細胞に結合し孔を形成した後、αアクチンと特異的に結合すること示し、さらにαアクチンはα毒素の細胞障害活性を発揮するために重要な役割を担っていることを初めて明らかにした。
この成果は、細胞溶解毒素の持つ病原性発現機構における新たな見地を与え、感染制御学の分野に貢献するところが大きく、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。