知花 博治

The size of the genome in the opportunistic fungus Candida albicans is 15.6 Mb. Whole-genome shotgun sequencing was carried out at Stanford University where the sequences were assembled into 412 contigs. C. albicans is a diploid basically, and analysis of the sequence is complicated due to repeated sequences and to sequence polymorphism between homologous chromosomes. Chromosome 7 is 1 Mb in size and the best characterized of the 8 chromosomes in C. albicans. We assigned 16 of the contigs, ranging in length from 7309 to 267,590 bp, to chromosome 7 and determined sequences of 16 regions. These regions included four gaps, a misassembled sequence, and two major repeat sequences (MRS) of >16 kb. The length of the continuous sequence attained was 949,626 bp and provided complete coverage of chromosome 7 except for telomeric regions. Sequence analysis was carried out and predicted 404 genes, 11 of which included at least one intron. A 7-kb indel, which might be caused by a retrotransposon, was identified as the largest difference between the homologous chromosomes. Synteny analysis revealed that the degree of synteny between C. albicans and Saccharomyces cerevisiae is too weak to use for completion of the genomic sequence in C. albicans. Copyright © 2005 by the Genetics Society of America.

The major repeat sequence (MRS) is found at least once on all but one chromosome in Candida albicans, but as yet it has no known relation to the phenotype. The MRS affects karyotypic variation by serving as a hot spot for chromosome translocation and by expanding and contracting internal repeats, thereby changing chromosome length. Thus, MRSs on different chromosomes and those on chromosome homologues can differ in size. We proposed that the MRS's unique repeat structure and, more specifically, the size of the MRS could also affect karyotypic variation by altering the frequency of mitotic nondisjunction. Subsequent analysis shows that both natural and artificially induced differences in the size of the chromosome 5 MRS can affect chromosome segregation. Strains with chromosome 5 homologues that differ in the size of the naturally occurring MRSs show a preferential loss of the homologue with the larger MRS on sorbose, indicating that a larger MRS leads to a higher risk of mitotic nondisjunction for that homologue. While deletion of an MRS has no deleterious effect on the deletion chromosome under normal growth conditions and leads to no obvious phenotype, strains that have the MRS deleted from one chromosome 5 homologue preferentially lose the homologue with the MRS remaining. This effect on chromosome segregation is the first demonstration of a phenotype associated with the MRS. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The major repeat sequence (MRS) is found at least once on all but one chromosome in Candida albicans, but as yet it has no known relation to the phenotype. The MRS affects karyotypic variation by serving as a hot spot for chromosome translocation and by expanding and contracting internal repeats, thereby changing chromosome length. Thus, MRSs on different chromosomes and those on chromosome homologues can differ in size. We proposed that the MRS's unique repeat structure and, more specifically, the size of the MRS could also affect karyotypic variation by altering the frequency of mitotic nondisjunction. Subsequent analysis shows that both natural and artificially induced differences in the size of the chromosome 5 MRS can affect chromosome segregation. Strains with chromosome 5 homologues that differ in the size of the naturally occurring MRSs show a preferential loss of the homologue with the larger MRS on sorbose, indicating that a larger MRS leads to a higher risk of mitotic nondisjunction for that homologue. While deletion of an MRS has no deleterious effect on the deletion chromosome under normal growth conditions and leads to no obvious phenotype, strains that have the MRS deleted from one chromosome 5 homologue preferentially lose the homologue with the MRS remaining. This effect on chromosome segregation is the first demonstration of a phenotype associated with the MRS.

細胞間コミュニケーションであり、病原性発現の引き金として注目されているクオラムセンシング (QS) 機構は、細菌においてはかなり解明されてきている。一方 C. albicans における QS の研究は、近年その QS 分子の一つである farnesol が発見されたことにより解明が急速に進みだした。<BR> C. albicans は、QS により菌糸形への形態変換を起こすため、我々は酵母形から菌糸形への形態変換を指標とした C. albicans の QS システムの機構解明を目指して研究を行い、第 48 回本学会総会、第 78 回日本細菌学会総会でその成果の一部を発表した。<BR> 今回は DNA マイクロアレイの手法を用いた網羅的遺伝子解析を行い、QS 関連遺伝子を想定した。本大会では、この解析によって得られた遺伝子について、real-time RT-PCR による確認作業を報告するとともに、菌糸形成初期に関わる遺伝子 CGR1 の組換え体を利用した QS 関連想定遺伝子の検討についてもあわせて報告する。<BR>（会員外共同研究者：青山俊弘、鈴鹿高専・電子情報工学）

出芽酵母 Saccharomyces cerevisiae における PHO システムは、環境中におけるリン酸濃度に応じて細胞内のリン酸代謝系を調節する巧みな制御機構である。このシステムにおいて中心的役割を担っている因子 PHO85 は、PHO システムの負の制御因子として分離され、非必須遺伝子であることが知られている。われわれは 先に病原性酵母 Candida albicans よりそのホモログを分離しその性状を調べ、酸性フォスファターゼに対する負の制御等々、出芽酵母との共通性がきわめて高いことを明らかにした。今回、C. albicans および C. glabrata のホモログ CaPHO85 , CgPHO85 の必須性について中山らの Tetracycline (Tet) 応答性遺伝子発現制御系 (TETシステム；Microbiology, 1998, Infect. Immun., 2000) を用いて検討を試みたところ、CaPHO85 で Tet 存在下-12 時間以上で菌の増殖が顕著に抑制され、本遺伝子は出芽酵母 PHO85 とは異なり必須遺伝子であることが示唆された。CgPHO85 では CaPHO85 ほどではないが同様の傾向が認められた。さらに現在、Tet＋/- 条件下での in vivo の実験が進行中である。

真菌細胞の表層にあるマンノプロテインは、宿主への付着因子ならびに病原因子として注目されている。GDP-マンノースは，タンパク質のマンノース糖鎖付加に用いられ，マンノプロテインの生合成に必須な基質であり，GDP-マンノースの合成酵素（PSA1）は生育に必須な遺伝子であると多くの真菌で報告されてきた。我々は，カンジダ症の原因菌で，分子生物学的解析が容易な一倍体真菌，Candida glabrata を用いて PSA1 遺伝子のプロモーターをテトラサイクリン応答性プロモーターに置き換えた変異株を作製し（この変異株では，テトラサイクリンの添加により容易に PSA1 遺伝子の発現を抑えられる），種々の培養環境での生育に対する必須性を調べた。その結果，炭素源がグルコースの場合は必須であったが，他の炭素源を用いた場合には PSA1 遺伝子が生育に必須ではないことが示された。パン酵母(Sacchromyces cerevisiae)のゲノム中には PSA1 は単独で存在しているが，最近公開されたC. glabrataのゲノム配列より，C. glabrataにはPSA1遺伝子のパラログ（類似遺伝子）PSA2 の存在が示された。本大会では、PSA1，PSA2 遺伝子の相補性とそれぞれの遺伝子の，種々の培養環境での発現変化について報告し, PSA1 と PSA2 の GDP-マンノース合成における役割について考察したい。

昨年、Candida albicans (16 Mb) と C. glabrata (13 Mb) の全ゲノムシークエンスが完全公開された。我々はこれらの情報を用いてカンジダの病原性解明と抗真菌薬開発をめざしている。異数体である C. albicans に比べ C. glabrata は一倍体であり遺伝子操作が簡便であるためゲノムワイドな機能解析において優れている。C. glabrata においては、in vitro ならびに in vivo での遺伝子解析が可能なテトラサイクリン応答型プロモーター (Tet-p) を用いたシステムが Nakayama et al. (1998) によって構築されている。我々はこのシステムを基本に Tet-p 導入のハイスループット化を実現させた独自のシステムを開発し、現在、5,300 全遺伝子に対し Tet-p 導入菌株を構築する計画 CGRP (Candida glabrata Genome Regulation Project) を推進中である。本計画ではまず、バイオインフォーマティックスにより抗真菌剤標的候補遺伝子を約 200 遺伝子抽出し、これら遺伝子に対して Tet-p を導入しているので、本総会において報告する。<br>会員外共同研究者： 笹本 要、伊藤桂子(千葉大)、青山俊弘 (鈴鹿高専)、釣谷克樹、中井謙太（東大）

Gene deletion in the pathogenic fungus Candida albicans has relied heavily on a URA3 cassette and a recipient Δura3 strain CA14. The IRO1 gene adjacent to URA3 was inadvertently deleted during construction of CAI4. We report here that a mutation in IRO1 reduces virulence of C. albicans.

By using real-time RT-PCR, we profiled the expression of CGR1, CaMSI3, EFG1, NRG1, and TUP1 in Candida albicans strains JCM9061 and CAI4 under several conditions, including induction of morphological transition, heat shock, and treatment with calcium inhibitors. Expression of CaMSI3 changed under these growth conditions except during heat shock. CGR1 expression increased during the early stages of hyphal growth in JCM9061, while expression was strain-dependent during heat shock. Both EFG1 and NRG1 were similarly expressed under hypha-inducing conditions and heat shock. Expression of TUP1 was slightly different from the expression of EFG1 or NRG1.

By using real-time RT-PCR, we profiled the expression of CGR1, CaMSI3, EFG1, NRG1, and TUP1 in Candida albicans strains JCM9061 and CAI4 under several conditions, including induction of morphological transition, heat shock, and treatment with calcium inhibitors. Expression of CaMSI3 changed under these growth conditions except during heat shock. CGR1 expression increased during the early stages of hyphal growth in JCM9061, while expression was strain-dependent during heat shock. Both EFG1 and NRG1 were similarly expressed under hypha-inducing conditions and heat shock. Expression of TUP1 was slightly different from the expression of EFG1 or NRG1. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

We present the diploid genome sequence of the fungal pathogen Candida albicans. Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis.

近年のDNA塩基配列読み取り技術の進歩に伴い全ゲノムシークエンスが次々に報告されており,病原性真菌においても多数のゲノムシークエンスプロジェクトが進行中である.その中でスタンフォード大学において進められて来たCandida albicansのゲノムシークエンスプロジェクトは終了に近付いている.ここではC.albicansのゲノム解析の歴史と合わせてシークエンスプロジェクトの現状について紹介する.

We used RNA fingerprinting of arbitrarily primed PCR to isolate genes upregulated during the yeast-hyphal transition in Candida albicans. The sequence and expression of one of these genes (CGR1, Candida growth regulation) are presented. Our results suggest that CGR1 expression is associated with a growth cessation of yeast cells, a prerequisite for germination in this organism. © 2001 Elsevier Science B.V.

We used RNA fingerprinting of arbitrarily primed PCR to isolate genes upregulated during the yeast-hyphal transition in Candida albicans. The sequence and expression of one of these genes (CGR1, Candida growth regulation) are presented. Our results suggest that CGR1 expression is associated with a growth cessation of yeast cells, a prerequisite for germination in this organism.

Parasexual genetic analysis of Candida albicans utilized the dominant selectable marker that conferred resistance to mycophenolic acid. We cloned and sequenced the IMH3r gene from C. albicans strain 1006, which was previously identified as resistant to mycophenolic acid (MPA) (A. K. Goshorn and S. Scherer, Genetics 123:213-218, 1989). MPA is an inhibitor of IMP dehydrogenase, an enzyme necessary for the de novo biosynthesis of GMP. G. A. Kohler et al. (J. Bacteriol. 179:2331-2338, 1997) have shown that the wild-type IMH3 gene, when expressed in high copy number, will confer resistance to this antibiotic. We demonstrate that the IMH3r gene from strain 1006 has three amino acid changes, two of which are nonconservative, and demonstrate that at least two of the three mutations are required to confer resistance to MPA. We used this gene as a dominant selectable marker in clinical isolates of C. albicans and Candida tropicalis. We also identified the presence of autonously replicating sequence elements that permit autonomous replication in the promoter region of this gene. Finally, we found the excision of a φ-type long terminal repeat element outside the IMH3 open reading frame of the gene in some strains. We used the IMH3r allele to disrupt one allele of ARG4 in two clinical isolates, WO-1 and FC18, thus demonstrating that a single ectopic integration of this dominant selectable marker is sufficient to confer resistance to MPA.

The pBAC 108L and pFos 1 vectors were developed as stable propagation vectors which, due to their extremely low copy number, facilitate the cloning of a large-sized insert containing repeated DNA. However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome walking. Here we describe efficient methods for end-DNA isolation. The entire process, including small-scale DNA preparation, restriction digestion, self-ligation, and PCR with vector-based primers, is carried out in 96-well formats. Using a Fosmid library of genomic DNA of Candida albicans, PCR products ranging in size from 0.1 to 8 kbp were generated from 118 end sequences in 140 reactions from 70 Fosmid clones. A single or a prominent band was found in 101 of these reactions. Twenty-six of these bands were tested for walking and all of them proved to be specific. Thus, the system overcomes the disadvantage caused by low copy number. This system allows rapid physical mapping of genomes, and is adaptable for several other vectors including BAC (bacterial artificial chromosome), PAC (P1-derived artificial chromosome) and YAC (yeast artificial chromosome).

The focus of this symposium was to present new information on the morphogenesis of Candida albicans, particularly how it relates to signal transduction pathways and other genes involved in the regulation of morphogenesis. In addition, we discuss the role of adherence and colonization of the oral cavity by the organism and discuss the role of mannan as an adhesin that recognizes the human red blood cell. C. albicans utilizes at least two signal pathways to regulate its conversion from a yeast form to filamentous growth (hyphae). One of these two pathways is similar to the Saccharomyces cerevisiae pseudohyphal/mating pathway, which utilizes the regulatory protein, Cph1p. The other pathway is not totally defined but requires a second regulatory protein, referred to as Efg1p. Other signal pathways may exist, which include a two-component histidine kinase and response regulator proteins. The latter pathway(s) may include proteins such as Chk1p, Ssk1p, Shi1p and Cos1p/Nik1p. Mutations in strains, which specifically target these proteins, result in morphogenesis defects and avirulence or attenuation of strains. A growth regulatory gene has also been recently defined whose expression is associated with growth cessation and which appears to be a necessary prerequisite in conversion of the organism to a filamentous growth form. Starvation of yeast cells induces exponentially grown cells (and usually non-germinative) to germinate. This phenomenon is also observed in cells that are transiently treated with metabolic inhibitors. During each of these treatments (starvation, metabolic inhibition), expression of a growth regulatory gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is complex; several proteins, which appear to have host recognition functions, have been defined. In the oral cavity, C. albicans selectively adheres to salivary proteins, which are absorbed to many oral surfaces. This mechanism enables the cells to colonize surfaces of the oral cavity. An understanding of these interactions may lead to strategies to prevent oral disease. Mannan from C. albicans may provide a host recognition function for C. albicans. Recent experiments indicate that mannan binds to human red blood cells and causes hemolysis. Binding of mannan to the band 3 protein of human red blood cells has been established. This activity may be associated with the ability of the organism to utilize hemoglobin (and iron).

It has been suggested that Candida albicans, a diploid asexual fungus, achieves genetic diversity by genomic rearrangement. This important human pathogen may provide a system in which to analyze alternate routes to genomic diversity. C. albicans has a highly variable karyotype; its chromosomes contain a middle repeated DNA sequence called the Major Repeat Sequence (MRS), composed of subrepeats HOK, RPS, and RB2. RPS is tandemly repeated while the other subrepeats occur once in each MRS. Chromosome 7, the smallest of the eight chromosomes, has been previously mapped. The complete physical map of this chromosome was used to analyze chromosome 7 diversity in six strains, including two well-characterized laboratory strains (1006 and WO-I) and four clinical ones. We found four types of events to explain the genomic diversity: 1) Chromosome length polymorphism (CLP) results from expansion and contraction of the RPS; 2) reciprocal translocation occurs at the MRS loci; 3) chromosomal deletion; and (4) trisomy of individual chromosomes. These four phenomena play an important role in generating genomic diversity in C. albicans.

The focus of this symposium was to present new information on the morphogenesis of Candida albicans, particularly how it relates to signal transduction pathways and other genes involved in the regulation of morphogenesis. In addition, we discuss the role of adherence and colonization of the oral cavity by the organism and discuss the role of mannan as an adhesin that recognizes the human red blood cell. C. albicans utilizes at least two signal pathways to regulate its conversion from a yeast form to filamentous growth (hyphae). One of these two pathways is similar to the Saccharomyces cerevisiae pseudohyphal/mating pathway, which utilizes the regulatory protein, Cphlp. The other pathway is not totally defined but requires a second regulatory protein, referred to as Efg1p. Other signal pathways may exist, which include a two-component histidine kinase and response regulator proteins. The latter pathway(s) may include proteins such as Chk1p, Ssk1p, Shi1p and Cos1p/Nik1p. Mutations in strains, which specifically target these proteins, result in morphogenesis defects and avirulence or attenuation of strains. A growth regulatory gene has also been recently defined whose expression is associated with growth cessation and which appears to be a necessary prerequisite in conversion of the organism to a filamentous growth form. Starvation of yeast cells induces exponentially grown cells (and usually non-germinative) to germinate. This phenomenon is also observed in cells that are transiently treated with metabolic inhibitors. During each of these treatments (starvation, metabolic inhibition), expression of a growth regulatory gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is complex; several proteins, which appear to have host recognition functions, have been defined. In the oral cavity, C. albicans selectively adheres to salivary proteins, which are absorbed to many oral surfaces. This mechanism enables the cells to colonize surfaces of the oral cavity. An understanding of these interactions may lead to strategies to prevent oral disease. Mannan from C. albicans may provide a host recognition function for C. albicans. Recent experiments indicate that mannan binds to human red blood cells and causes hemolysis. Binding of mannan to the band 3 protein of human red blood cells has been established. This activity may be associated with the ability of the organism to utilize hemoglobin (and iron).

The Candida albicans CHS4 gene encoding chitin synthase 4 has been isolated using the Saccharomyces cerevisiae CHS4/SKT5 gene as a probe. The gene contains a 2061 bp open reading frame capable of encoding a protein of 687 amino acids (76053 Da). No intron was observed in the gene. Disruption of CHS4 in C. albicans yielded a Calcofluor-resistant phenotype, indicating that Chs4p contributes to chitin biosynthesis. Consistent with this, overexpression of Chs4p under the regulation of the ScGAL1 promoter enhanced chitin synthase 3 activity in S. cerevisiae 7- to 38-fold. In addition, chs3 and chs4 null mutants were significantly defective in Calcofluor white staining and their chitin content was 10% of that of the parental strain. Chs4p of C. albicans and S. cerevisiae showed 61% identity in the C-terminal half of the proteins and that region of C albicans Chs4p complemented the Chs4p function of a mutant of S. cerevisiae resistant to Calcofluor white. Therefore, it appears that Chs4p is involved in chitin synthase 3 activity by combining with Chs3p to interact synergistically in chitin biosynthesis.

The Candida albicans CHS4 gene encoding chitin synthase 4 has been isolated using the Saccharomyces cerevisiae CHS4/SKT5 gene as a probe. The gene contains a 2061 bp open reading frame capable of encoding a protein of 687 amino acids (76053 Da). No intron was observed in the gene. Disruption of CHS4 in C. albicans yielded a Calcofluor-resistant phenotype, indicating that Chs4p contributes to chitin biosynthesis. Consistent with this, overexpression of Chs4p under the regulation of the ScGAL1 promoter enhanced chitin synthase 3 activity in S. cerevisiae 7- to 38-fold. In addition, chs3 and chs4 null mutants were significantly defective in Calcofluor white staining and their chitin content was 10% of that of the parental strain. Chs4p of C. albicans and S. cerevisiae showed 61% identity in the C-terminal half of the proteins and that region of C. albicans Chs4p complemented the Chs4p function of a mutant of S. cerevisiae resistant to Calcofluor white. Therefore, it appears that Chs4p is involved in chitin synthase 3 activity by combining with Chs3p to interact synergistically in chitin biosynthesis.

As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the Sill restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Re12 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.

As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the Sill restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Re12 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.

A novel sequence designated HOK, which is next to the RPS, a repetitive sequence specific to Candida albicans, was cloned and sequenced. HOK hybridized with all of the chromosomes on which the RPSs were located, but did not hybridize with chromosome 3, which does not harbour any RPSs. Sequence determination revealed that a portion of HOK has significant homology with the B and C1 fragments of Ca3, which is used as a molecular epidemiological probe. A homology search of the deduced amino acids of HOK against the protein database showed partial homology with an isocitrate dehydrogenase of Saccharomyces cerevisiae, although an ORF large enough to encode the enzyme was not detected. To verify the existence of other sequences homologous with HOK, a portion of the HOK sequence was amplified using PCR. Sequence determination of the 41 clones from the PCR products resulted in at least six HOK-homologous clones. Another RPS-containing clone, RB2, was isolated from the Pst1-digested chromosome R or 1. It was determined that RB2a, one of the subclones from RB2, hybridized with all of the chromosomes, including chromosome 3, with which neither HOK nor RPS hybridized. The hybridization profile also showed that RPS is located between HOK and RB2a on chromosomes other than chromosome 3.

A primer pair, PB and BSH, which amplified alts, a portion of Candida albicans-specific repetitive sequence, RPS, gave stable and reproducible fingerprint patterns of the strains by polymerase chain reaction (PCR). We applied this method to clinical isolates of C. albicans for strain discrimination. Using PCR fingerprint patterns, we could analyze the relatedness of C. albicans strains including those isolated from children with leukemia and their bedside parents. The results indicated that PCR analysis targeting an alt region gives rise to the same conclusion as the previous study obtained by SmaI RFLP analysis.