基本情報
研究分野
1経歴
5-
2018年11月 - 現在
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2015年4月 - 2018年10月
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2011年4月 - 2015年3月
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2008年5月 - 2011年3月
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2008年4月 - 2008年5月
委員歴
3-
2018年4月 - 現在
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2018年4月 - 2024年3月
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2016年4月 - 2017年3月
論文
56-
Anaerobe 93 102968-102968 2025年6月
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PNAS Nexus pgaf176 2025年5月31日
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Internal Medicine 2025年4月
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Microbiology Resource Announcements 2024年10月2日 査読有り
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Communications biology 7(1) 1129-1129 2024年9月13日In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.
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Scientific reports 14(1) 16225-16225 2024年7月13日In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.
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mBio e00339-24 2024年6月12日 筆頭著者責任著者
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International Journal of Molecular Sciences 24(23) 17029-17029 2023年12月1日RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.
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Microbiology and Immunology 67(10) 438-446 2023年8月13日Abstract The skin and mucous membranes are the primary sites of Staphylococcus aureus colonization, particularly those of health care personnel and patients in long‐term care centers. We found that S. aureus colonized with a higher abundance ratio on skins which had recovered from pressure injury (PI) than on normal skins in our earlier research on the skin microbiota of bedridden patients. Multilocus sequence typing (MLST) is a useful tool for typing S. aureus isolated from clinical specimens. However, the MLST approach cannot be used in microbiota DNA owing to the contamination from other bacteria species. In this study, we developed a multiplex‐nested PCR method to determine S. aureus MLST in samples collected from human skins. The seven pairs of forward and reverse primers were designed in the upstream and downstream regions, which were conserved specifically in S. aureus. The first amplifications of the seven pairs were conducted in a multiplex assay. The samples were diluted and applied to conventional PCR for MLST. We confirmed that the method amplified the seven allele sequences of S. aureus specifically in the presence of untargeted DNAs from human and other skin commensal bacteria. Using this assay, we succeeded in typing sequence types (STs) of S. aureus in the DNA samples derived from the skins healed from PI. Peaks obtained by Sanger sequencing showed that each sample contained one ST, which were mainly categorized into clonal complex 1 (CC1) or CC5. We propose that this culture‐free approach may be used in detecting S. aureus in clinical specimens without isolation.
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Scientific Reports 12(1) 21297-21297 2022年12月9日
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Streptococcus pyogenes: Basic Biology to Clinical Manifestations [Internet]. 2nd edition. 2022年10月8日
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Journal of Cutaneous Immunology and Allergy 2022年9月8日 査読有り
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Respiratory Medicine Case Reports 38 101673-101673 2022年6月 査読有り
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Frontiers in Microbiology 13 765317-765317 2022年3月14日 査読有り筆頭著者
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Frontiers in Microbiology 13 818398-818398 2022年3月1日 査読有り
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International Journal of Molecular Sciences 23(1) 402-402 2021年12月30日 査読有り
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Antibiotics 11(1) 36-36 2021年12月29日 査読有り
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Journal of the Association for Rapid Method and Automation in Microbiology 31(1) 29-40 2021年12月 査読有り
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Microbiology Resource Announcements 10(27) 2021年7月8日 査読有り筆頭著者
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Annals of Clinical Microbiology and Antimicrobials 20(1) 2021年7月 査読有り
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Scientific Reports 10(1) 16907-16907 2020年10月 査読有り筆頭著者
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Scientific Reports 10(1) 16107-16107 2020年10月 査読有り
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Nature communications 11(1) 2934-2934 2020年6月10日 査読有りThe emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.
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Microbiology resource announcements 9(23) 2020年6月4日 査読有りThe association of Panton-Valentine leukocidin (PVL) toxin with necrotizing soft tissue infection (NSTI) caused by Staphylococcus aureus remains controversial. Here, we report the complete genome sequence of the PVL-negative S. aureus strain JMUB1273, isolated from a patient with pervasive NSTI.
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Microorganisms 7(10) 463 2019年10月 査読有り
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Frontiers in Microbiology 10 2838-2838 2019年 査読有り筆頭著者Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.
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Journal of Infection and Chemotherapy 25(1) 1-5 2019年1月 査読有り
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Microbiology Resource Announcements 8(4) e01652-18 2019年1月 査読有りSevere community-acquired pneumonia (CAP) caused by methicillin-resistant Staphylococcus aureus (MRSA) is relatively rare and is usually associated with rapid progression to death. Here, we report the complete genome sequence of the MRSA strain JMUB3031, which was isolated from a patient with fatal CAP.
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BMC Genomics 19(1) 810-810 2018年11月8日 査読有り筆頭著者BACKGROUND: Staphylococcus caprae is an animal-associated bacterium regarded as part of goats' microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. RESULTS: We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. CONCLUSION: The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections.
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Journal of Microbiological Methods 146 25-32 2018年3月 査読有りElectroporation is a common technique necessary for genomic manipulation of Staphylococci. However, because this technique has too low efficiency to be applied to some Staphylococcal species and strains, especially to coagulase-negative Staphylococcus (CNS) isolates, basic researches on these clinically important Staphylococci are limited. Here we report on the optimization of electroporation parameters and conditions as well as on the generation of a universal protocol that can be efficiently applicable to both CNS and Coagulase-positive Staphylococci (CPS). This protocol could generate transformants of clinical Staphylococcus epidermidis isolate, with an efficiency of up to 1400 CFU/μg of plasmid DNA. Transformants of 12 other clinically important Staphylococcal species, including CNS and CPS, were also generated with this protocol. To our knowledge, this is the first report on successful electroporation in nine these Staphylococcal species.
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Japanese Journal of Chemotherapy 65(5) 745-750 2017年 査読有り
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Infection and Immunology 84(8) 2264-2273 2016年8月 査読有り筆頭著者
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Genome Announcements 4(5) e01133-16 2016年 査読有り筆頭著者
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Microbiology and Immunology 60(1) 1-9 2016年1月 招待有り筆頭著者
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Molecular Microbiology 91(3) 538-547 2014年2月 査読有り
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Journal of Infectious Diseases 208(9) 1482-1493 2013年11月1日 査読有り筆頭著者
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Genome Biology and Evolution 5(9) 1644-1651 2013年 査読有り筆頭著者
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Journal of Bacteriology 194(19) 5466-5466 2012年10月 査読有り
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Journal of Infection and Chemotherapy 17(5) 609-621 2011年10月 査読有り
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Plos Pathogens 7(10) e1002287 2011年10月 査読有り筆頭著者
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PLOS ONE 4(5) e5714 2009年5月 査読有り筆頭著者
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Journal of Antimicrobial Chemotherapy 63(1) 32-41 2009年1月 査読有り
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Journal of Microbiological Methods 75(2) 312-317 2008年10月 査読有り
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Journal of Antimicrobial Chemotherapy 62(2) 324-328 2008年8月 査読有り
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Brazilian journal of infectious diseases 12(3) 213-216 2008年6月 査読有り
MISC
71-
日本細菌学雑誌 78(1) 86-86 2023年2月
担当経験のある科目(授業)
1-
細菌学 (自治医科大学)
共同研究・競争的資金等の研究課題
18-
日本学術振興会 科学研究費助成事業 2024年4月 - 2029年3月
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2024年4月 - 2026年3月
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日本医療研究開発機構 (AMED) 医薬品研究開発 2021年 - 2026年
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日本学術振興会 科学研究費助成事業 2022年4月 - 2025年3月
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日本学術振興会 科学研究補助金 2022年8月 - 2024年3月