称号及び氏名

博士（獣医学）Xiaomin Cai

学位授与の日付

平成 1８年２月２０日

論「Cryptosporidium parvum: From Distinct Molecular Features of文

名the Parasite Metabolism to an Improved Anti-protozoan Drug
Testing Assay」(Cryptosporidium parvum に関する研究：寄生虫代謝の分子生化学的解析を利用した抗原虫薬評価法の改良)

論文審査委員

主査

馬場栄一郎

副査

津山 伸吾

副査

竹内 正吉

副査

笹井 和美

論文要旨
Introduction
Cryptosporidium parvum is a unicellular parasite that can cause severe diarrhea in humans
and animals. Based on host specificity, C. parvum used to be separated into two types: Type I
isolates were primarily transmitted among humans, while Type II isolates were zoonotic. In 2002,
Type I C. parvum was renamed to C. hominis. In addition to C. parvum and C. hominis, at least 12
other Cryptosporidium species have been generally accepted as valid species that infect a wide rangeof animals, including mammals, birds, fishes, reptiles and amphibians. In domestic animals,
cryptosporidiosis may cause death or severely affect the growth and development in calves and foals.
In humans, this parasite can cause prolonged, life threatening infection in immunocompromizedindividuals. Therefore, C. parvum and C. hominis are also a significant opportunistic pathogen in
AIDS patients. Also because the environmental oocysts are highly resistant to almost all
disinfectants used in community and recreational waters, Cryptosporidium is a significant water- andfood-borne pathogen.
Cryptosporidium parvum belongs to the Phylum Apicomplexa, in which all the members areparasites and many of them are of medical or veterinary importance (e.g., Plasmodium [malaria],

Toxoplasma [toxoplasmosis], Babesia [Babesiosis] and Eimeria [coccidiosis]). Like other coccidia,
the life cycle of C. parvum consists of at least three distinct developmental stages: sporogony toproduce infectious sporozoites within oocysts; two generations of intracellular merogony to form
eight and four merozoites; and sexual gametogony to form micro- and macro-gametes that fuse to
become zygotes. Zygotes will develop into immature oocysts, which in turn undergo a next
generation of sporogony that produces infectious oocysts before releasing into the host digestive guts.
All apicomplexan life stages except for the short-lasting zygotes are haploid. Both sporogony andmerogony are types of cell multiplication differing from host somatic cell duplication.
Although globally important, effective treatments are limited in both human and animal
cryptosporidiosis. Currently, only a single drug (i.e., nitazoxanide [NTZ]) has been approved fortreating cryptosporidiosis in the United States, Central and South America. The slow drug
development in treating cryptosporidiosis is mainly caused by the author’s limited understanding ofbasic metabolism in C. parvum, which in turn is largely due to the technical difficulties in obtaining
a large quantity of pure parasite material for molecular and biochemical studies, and to the lack ofgenetic transfection systems. Current knowledge on the metabolism of Cryptosporidium is largely
acquired by gene discovery and more recently, the analysis of gene compositions based on complete
genome sequences. This parasite has a compact genome (~ 10.5 megabases) that constitutes a highly
streamlined metabolism. Many important pathways present in other apicomplexans are missing in
Cryptosporidium. For example, C. parvum lacks an apicoplast and associated metabolic pathways,including type II fatty acid synthase (FAS) and iospropynoid synthesis. This parasite may contain ahighly degenerated mitochondrion based on the presence of a number of mitochondrial-specificgenes (e.g., heat shock protein 70 [hsp70], ferredoxin [fdx], alternative oxidase [AOX], and theheadpiece of the F-ATPase alpha-subunit. However, it lacks Krebs cycle and cytochrome-based
respiration. It has lost almost all de novo biosynthetic capacity, including these for amino acid,nucleosides, and fatty acids. Instead, it apparently scavenges nutrients from the host using a highly
expanded set of transporters, such as those for sugars and amino acids.
All these molecular and biological features make Cryptosporidium unique amongapicomplexans. Indeed, recent molecular phylogenetic reconstructions have consistently placed theCryptosporidium genus at the base of the Apicomplexa, or even as a sister to the gregarines (a Classof parasites of invertebrates), which is in contrast to the conventional taxonomy that considers
Cryptosporidium as a sister to the Coccidia. As mentioned above, C. parvum is also divergent fromother apicomplexans at both molecular and biochemical levels. Therefore, one cannot simply learn
or deduce the fundamental biology of Cryptosporidium species from the knowledge generated onother model apicomplexans (e.g., Toxoplasma and Plasmodium). It is necessary to thoroughly
investigate its own molecular biology and biochemistry of Cryptosporidium in order to gain insight
into the fundamental biology of this parasite.

Recently, several important metabolic pathways in C. parvum have been more extensively
investigated in the authors’ laboratory, such as the fatty acid biosynthesis, glycolysis, DNA replicationand transcription. The ultimate goal for studying the metabolism in C. parvum is to truly understandthe fundamental biology of this parasite and build a solid ground for the development of drugs and/or
means to control or treat cryptosporidiosis in humans and animals. Therefore, this dissertation will
cover the studies ranging from the molecular and biochemical analysis of distinct parasite genes andproteins to the development of improved molecular assays for the drug testing against C. parvum invitro.

Chapter 1:

Functional Characterization of an Evolutionarily Distinct Phosphopantetheinyl
Transferase in the Apicomplexan, Cryptosporidium parvum

Recently, two types of fatty acid synthases (FASs) have been discovered fromapicomplexan parasites. Although significant progress has been made in characterizingthese apicomplexan FASs, virtually nothing was previously known about the activation
and regulation of these enzymes. In this study, we report the discovery andcharacterization of two distinct types of phosphopantetheinyl transferase (PPTase) thatare responsible for synthesizing holo-acyl carrier protein (ACP) from threeapicomplexan parasites: surfactin production element (SFP)-type in Cryptosporidium

parvum (CpSFP-PPT), holo-ACP synthase (ACPS)-type in Plasmodium falciparum(PfACPS-PPT), and both SFP and ACPS types in Toxoplasma gondii (TgSFP-PPT andTgACPS-PPT). CpSFP-PPT and TgSFP-PPT are monofunctional, cytosolic, andphylogenetically related to animal PPTases. However, PfACPS-PPT and TgACPS-PPT
are bifunctional (fused with a metal-dependent hydrolase), likely targeted to theapicoplast, and more closely related to proteobacterial PPTases.
The function of apicomplexan PPTases has been confirmed by detailed
functional analysis using recombinant CpSFP-PPT expressed from an artificiallysynthesized gene with codon usage optimized for Escherichia coli. The recombinant
CpSFP-PPT was able to activate the ACP domains from the C. parvum Type I FAS in

vitro using either CoA or acetyl-CoA as a substrate, or in vivo when co-expressed inbacteria, with kinetic characteristics typical of PPTases. These observations suggestthat the two types of fatty acid synthases in the Apicomplexa are activated andregulated by two evolutionarily distinct PPTases.

Chapter 2:

Intron-containing β-Tubulin Transcripts in Cryptosporidium parvum Cultured invitro

The genome of Cryptosporidium parvum contains a relatively small number of introns,which includes the β-tubulin gene with only a single intron. Recently, we observed that the intron
was not removed from some of the β−tubulin transcripts in the late life cycle stages cultured in vitro.
Although normally spliced β-tubulin mRNA were detected in all parasite intracellular stages byRT-PCR (e.g., HCT-8 or Caco-2 cells infected with C. parvum for 12 to 72 hr), at 48 – 72 hr post
infection unprocessed β-tubulin transcripts containing intact introns started to appear in parasite
mRNA within infected host cells. The intron-containing transcripts could be detected byfluorescence in situ hybridization using an intron-specific probe.
The intron-containing β-tubulin transcripts appeared unique to the in vitro cultured C.
parvum, since they were not detected in parasite-infected calves at 72 hr. It is yet unclear whether thelate life cycle stages of C. parvum are partially deficient in intron-splicing, or the intron-splicing
processes have merely slowed, both of which would allow the detection of intron-containing
transcripts. Another possible explanation is that the decay in transcript processing might be simply
due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts
coincides with the arrest of C. parvum development in vitro. This unusual observation prompts us to
speculate that the abnormal intron-splicing of β-tubulin transcripts may be one of the factors
preventing complete development of this parasite in vitro. Furthermore, the presence of bothprocessed and unprocessed introns in β−tubulin transcripts in vitro may provide a venue for studying
overall mechanisms for intron-splicing in this parasite.

Chapter 3:

Application of Quantitative Real-Time Reverse Transcriptase-Polymerase Chain
Reaction (qRT-PCR) in Assessing Drug Efficacy Against the Intracellular Pathogen
Cryptosporidium parvum in vitro

We report here a quantitative real-time RT-PCR (qRT-PCR) assay for assessing drug
efficacy against the intracellular pathogen, Cryptosporidium parvum. The qRT-PCR assay detects
18S rRNA transcripts from both parasites (CT[P18S]) and host cells (CT[H18S]), and evaluates therelative expression between parasite and host rRNA levels (i.e., ΔCT = CT[P18S] - CT[H18S]) to minimize
experimental and operational errors. The choice of qRT-PCR over qPCR in this study is based on theobservations that: 1) the relationship between the logarithm of infected parasites (log[P]) and thenormalized relative level of rRNA (ΔΔCT) is linear with a 4-fold dynamic range using qRT-PCR, but
sigmoidal (non-linear) using qPCR; and 2) the level of RNA represents that of live parasites better
than that of DNA, because the decay of RNA (99% in ~3 hr) in dead parasites is faster than that ofDNA (99% in ~24 - 48 hr) under in vitro conditions.
The reliability of the qRT-PCR method was validated by testing the efficacy of nitazoxanide
and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8
cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values fornitazoxanide and paromomycin were 0.30 - 0.45 μg/ml and 89.7 - 119.0 μg/ml, respectively,

comparable to the previously reported values. Using the qRT-PCR assay, we have also observed thatpyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that therecently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets.

Conclusions
Although Cryptosporidium genus has been traditionally considered as a sister to the Class
Coccidia, the present molecular and metabolic data indicate that this genus is highly divergent fromother apicomplexans. This notion is also supported by a number of recent molecular phylogenetic
reconstructions that have consistently placed Cryptosporidium at the base of the Apicomplexa.
Cryptosporidium possesses an SFP-type PPTase (CpSFP-PPT) that is responsible foractivating the ACP domains in the multifunctional Type I CpFAS1 and CpPKS1. However, thisparasite lacks an ACPS-type PPTase and Type II FAS that are present in Toxoplasma and Plasmodium.
Recombinant CpSFP-PPT is able to activate recombinant ACP domains from CpFAS1 in vitro or invivo when co-expressed in bacteria, with enzymatic kinetics characteristic to other characterizedPPTases.
In vitro cultivated C. parvum may have a deficiency in intron-splicing (at least for theβ-tubulin gene), which may be partially associated with the difficulties to complete the entire parasite
life cycle under in vitro conditions. It may also provide a potential tool for dissecting the mechanism of
intron-splicing in this parasite.
The present qRT-PCR is an improved method for evaluating the efficacies of drugs against C.
parvum in vitro. It is a good alternative to the conventional methods and may be developed into
high-throughput screening of compounds against C. parvum in vitro.

審査結果の要旨
クリプトスポリジウムは、ヒトと動物に重篤な下痢を引き起こすアピコンプレックス門の原虫であり、現在、十数種に分類されている。幼齢動物はクリプトスポリジウム症により死亡することがあり、免疫力が低下したヒトでは本症によって生命の危機にさらされる。一方でクリプトスポリジウムは、環境中のオーシストが一般的な浄水過程で使用される消毒薬に対して強い耐性を示すので、重要な飲水あるいは食物媒介性病原体である。このように非常に重要な感染性原虫であるにもかかわらず、ヒトや動物のクリプトスポリジウム症に対する効果的な治療法は限られており、現在、米国、中南米では主に病原性の強い Cryptosporidium parvum 治療薬として一種類の薬剤（nitazoxanide）が承認されているに過ぎない。
申請者は、クリプトスポリジウム属の代謝経路、例えば脂肪酸生合成、解糖系、DNA の複製と転写などについて広範囲な研究を行い、本原虫がアピコンプレックス門のなかで特異な性質を持つことを見いだした。この論文では、C. parvum の遺伝子とタンパク質を分子生化学的に解析した結果を利用して in vitro で抗原虫薬を試験する改良型定量的リアルタイム RT-PCR（qRT-PCR）法を開発し、以下の内容を得た。
第１章では、脂肪酸生合成過程において重要な役割を演じる脂肪酸シンターゼ(FAS)とその活性化を担うphosphophanotetheinyl transferase (PPTase)について解析し、以下の結果を得た。FASには、脂肪酸生合成に係わる複数の酵素が、アシルキャリアータンパク質(ACP)と結合した状態で複合体を形成して統合的に機能するType I FASと、複数の酵素がばらばらに存在して個々にACPと結合した状態のType II FASが存在する。アピコンプレックス門におけるFASに関する過去の報告では、C. parvumはType I を、 Plasmodium falciparumはTypeを、Toxoplasma gondii はType I とType IIの双方を持つと報告されている。また、FASの活性化には、PPTaseが関与しており、Type IFASにはsurfactin産生因子 (SFP)-PPTが、Type II FASにはACPシンターゼ-PPT (ACPS-PPT)が作用すると考えられているが、アピコンプレックス門のPPTaseに関する詳細は不明であった。そこで、本章においては分子生物学的手法を用いて、上述の３種のアピコンプレックス門原虫のPPTaseを解析した。その結果、C. parvumはSFP-PPTを、P. falciparumはACPS-PPTを、T. gondiiはSFP-PPTとACPS-PPTを保有していた。C. parvumにおいては、SFP-PPTのmRNAが全てのライフサイクルにおいて発現し、宿主細胞感染12時間後、次いで、感染前のスポロゾイトのステージにおいてその発現が高かった。さらに、C. parvumのSFP-PPTとACPの組換えタンパク質を作製し、組換えSFP-PPTが組換えACPのSer残基にphosphophanotetheinyl motifが付加されることをMALDI-TOF-mass
spectrometry法により検証した。また、組換えSFP-PPTはMg2+の存在下で酵素活性を示すことを確認した。最後に組換えSFP-PPTを用いてウサギ免疫血清を作製し、C. parvumのスポロゾイトおよびメロントでの局在を間接免疫蛍光法で観察し、SFP-PPTが細胞質全体に分布している可能性を示唆した。
第２章では、C. parvum の発育・増殖における β-tubulin 遺伝子に含まれる intron の役割について述べており、以下の結果を得た。C. parvum は他の寄生虫と比較してゲノム中の intron が少ないが、β-tubulin 遺伝子の 5’末端付近には１つの intron が存在することが報告されている。β-tubulinは、細胞内の微小管の主要構成タンパク質で、C. parvum の発育・増殖に深く関わっている。C.
parvum は、宿主体内で感染 72 時間後に無数のオーシストを産生し、発育・増殖を繰り返すが、invitro の培養細胞での発育・増殖は非常に悪いことが知られている。そこで、ヒト大腸腺癌由来培養細胞(HCT-8)とヒト結腸癌由来細胞（Caco-2）、および宿主である牛を用いて感染実験を行い、RT-PCR 法により intron の有無を検討した。培養細胞においては、HCT-8 では感染 48-72 時間後、Caco-2 では感染 30-60 時間後に採取したサンプルからは intron を含む配列が増幅されたが、感染後 72 時間後の牛の小腸上皮細胞から採取したサンプルからは intron を含む配列は増幅されなかった。ゲノムからのコンタミネーションを除外するために培養細胞から抽出した RNA サンプルをDNase で繰り返し処置したところ、同様の結果を得た。さらに RNase で処理した群では intron を含んだ配列は認められなかった。培養細胞における intron の存在をさらに確認するために、
anti-intron および anti-exon プローブを用いた in situ ハイブリダイゼーション法を試みた結果、C.

parvum の虫体にのみ anti-intron による蛍光が観察された。以上の結果から C. parvum が培養細胞で発育・増殖が悪い原因の一つに β-tubulin の intron が関与している可能性を示唆している。
第３章では、C. parvumに対する抗原虫薬のin vitro評価法としてのqRT-PCR法の応用について以下の結果を得た。C. parvumに対する薬剤の評価法は、従来、培養細胞を用いたin vitro法において、形態学的計測法や抗体を用いた測定法が用いられてきたが、感度と精度に大きな問題があった。近年、PCR法によりDNA量を測定することによる評価法が提唱されているが、死滅した寄生虫由来のDNAによって測定結果が影響を受けることが判明した。そこで、qRT-PCR法によりC.
parvumの 18S rRNAと宿主細胞の 18S rRNAを比較検討することによる測定法を開発した。qRTPCRの信頼性は、in vitroでのHCT-8 細胞内C. parvum（IOWA株とKSU-1 株）の発育に関して、nitazoxanideとparomomycinの効果を試験することによって確認され、両薬剤は、用量依存的な抑制 を 示 し た 。 Nitazoxanide と paromomycin の MIC50 値 は 、 そ れ ぞ れ 0.30-0.45μg/ml と89.7-119.0μg/mlであり、以前の報告と同様であった。さらに、pyrazoleがin vitroでC. parvumの発育を阻害する結果を得た。
本研究は、今後大きな社会問題になると予想されるクリプトスポリジウム感染症の治療と予防のために不可欠な薬剤を開発するための画期的な方法を提供するものである。一方で、感染症の中でも対応が遅れがちである原虫疾患への対処方法について分子生化学的手法を実践的に用いる点は、獣医学に大きく貢献するものと評価できる。したがって、本論文の審査および学力確認の結果とあわせて博士（獣医学）の学位を授与することを適当と認める。