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Diversity of CRISPR/Cas system in Clostridium perfringens

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Abstract

Clostridium perfringens is an important pathogen of human and livestock infections, posing a threat to health. The horizontal gene transfer (HGT) of plasmids that carry toxin-related genes is involved in C. perfringens pathogenicity. The CRISPR/Cas system, which has been identified in a wide range of prokaryotes, provides acquired immunity against HGT. However, information about the CRISPR/Cas system in Clostridium perfringens is still limited. In this study, 111 C. perfringens strains with publicly available genomes were used to analyze the occurrence and diversity of CRISPR/Cas system and evaluate the potential of CRISPR-based genotyping in this multi-host pathogen. A total of 59 out of the 111 genomes harbored at least one confirmed CRISPR array. Four CRISPR/Cas system subtypes, including subtypes IB, IIA, IIC, and IIID systems, were identified in 32 strains. Subtype IB system was the most prevalent in this species, which was subdivided into four subgroups displaying subgroup specificity in terms of cas gene content, repeat sequence content, and PAM. We showed that the CRISPR spacer polymorphism can be used for evolutionary studies, and that it can provide discriminatory power for typing strains. Nevertheless, the application of this approach was largely limited to strains that contain the CRISPR/Cas system. Spacer origin analysis revealed that approximately one-fifth of spacers showed significant matches to plasmids and phages, thereby suggesting the implication of CRISPR/Cas systems in controlling HGT. Collectively, our results provide new insights into the diversity and evolution of CRISPR/Cas system in C. perfringens.

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References

  • Andersen JM, Shoup M, Robinson C, Britton R, Olsen KE, Barrangou R (2016) CRISPR diversity and microevolution in Clostridium difficile. Genome Biol Evol 8:2841–2855

    Article  PubMed  PubMed Central  Google Scholar 

  • Barrangou R (2015) The roles of CRISPR-Cas systems in adaptive immunity and beyond. Curr Opin Immunol 32:36–41

    Article  CAS  PubMed  Google Scholar 

  • Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712

    Article  CAS  PubMed  Google Scholar 

  • Biswas A, Gagnon JN, Brouns SJ, Fineran PC, Brown CM (2013) CRISPRTarget: bioinformatic prediction and analysis of crRNA targets. RNA Biol 10:817–827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Boudry P, Semenova E, Monot M, Datsenko KA, Lopatina A, Sekulovic O, Ospina-Bedoya M, Fortier LC, Severinov K, Dupuy B, Soutourina O (2015) Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile. MBio 6:e01112–e01115

    CAS  PubMed  PubMed Central  Google Scholar 

  • Brussow H, Hendrix RW (2002) Phage genomics: small is beautiful. Cell 108:13–16

    Article  CAS  PubMed  Google Scholar 

  • Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, Neron B, Rocha EPC, Vergnaud G, Gautheret D, Pourcel C (2018) CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res 46:W246–W251

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Deveau H, Garneau JE, Moineau S (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 64:475–493

    Article  CAS  PubMed  Google Scholar 

  • Didovyk A, Borek B, Tsimring L, Hasty J (2016) Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications. Curr Opin Biotechnol 40:177–184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Friedland AE, Baral R, Singhal P, Loveluck K, Shen S, Sanchez M, Marco E, Gotta GM, Maeder ML, Kennedy EM, Kornepati AV, Sousa A, Collins MA, Jayaram H, Cullen BR, Bumcrot D (2015) Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol 16:257

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71

    Article  CAS  PubMed  Google Scholar 

  • Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F (2017) Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356:438–442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grissa I, Vergnaud G, Pourcel C (2007a) The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8:172

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Grissa I, Vergnaud G, Pourcel C (2007b) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–W57

    Article  PubMed  PubMed Central  Google Scholar 

  • Hargreaves KR, Flores CO, Lawley TD, Clokie MR (2014) Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen. MBio 5:e01045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hassan KA, Elbourne LD, Tetu SG, Melville SB, Rood JI, Paulsen IT (2015) Genomic analyses of Clostridium perfringens isolates from five toxinotypes. Res Microbiol 166:255–263

    Article  CAS  PubMed  Google Scholar 

  • Hirano H, Gootenberg JS, Horii T, Abudayyeh OO, Kimura M, Hsu PD, Nakane T, Ishitani R, Hatada I, Zhang F, Nishimasu H, Nureki O (2016) Structure and engineering of Francisella novicida Cas9. Cell 164:950–961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Horvath P, Romero DA, Coute-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R (2008) Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol 190:1401–1412

    Article  CAS  PubMed  Google Scholar 

  • Irikura D, Monma C, Suzuki Y, Nakama A, Kai A, Fukui-Miyazaki A, Horiguchi Y, Yoshinari T, Sugita-Konishi Y, Kamata Y (2015) Identification and characterization of a new enterotoxin produced by Clostridium perfringens isolated from food poisoning outbreaks. PLoS One 10:e0138183

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Karvelis T, Gasiunas G, Young J, Bigelyte G, Silanskas A, Cigan M, Siksnys V (2015) Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements. Genome Biol 16:253

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Kitterer D, Braun N, Jehs MC, Schulte B, Alscher MD, Latus J (2014) Gas gangrene caused by Clostridium perfringens involving the liver, spleen, and heart in a man 20 years after an orthotopic liver transplant: a case report. Exp Clin Transplant 12:165–168

    PubMed  Google Scholar 

  • Kiu R, Caim S, Alexander S, Pachori P, Hall LJ (2017) Probing genomic aspects of the multi-host pathogen Clostridium perfringens reveals significant pangenome diversity, and a diverse array of virulence factors. Front Microbiol 8:2485

    Article  PubMed  PubMed Central  Google Scholar 

  • La Sala LF, Redondo LM, Diaz Carrasco JM, Pereyra AM, Farber M, Jost H, Fernandez-Miyakawa ME (2015) Carriage of Clostridium perfringens by benthic crabs in a sewage-polluted estuary. Mar Pollut Bull 97:365–372

    Article  PubMed  CAS  Google Scholar 

  • Lacey JA, Johanesen PA, Lyras D, Moore RJ (2016) Genomic diversity of necrotic enteritis-associated strains of Clostridium perfringens: a review. Avian Pathol 45:302–307

    Article  PubMed  Google Scholar 

  • Lacey JA, Keyburn AL, Ford ME, Portela RW, Johanesen PA, Lyras D, Moore RJ (2017) Conjugation-mediated horizontal gene transfer of Clostridium perfringens plasmids in the chicken gastrointestinal tract results in the formation of new virulent strains. Appl Environ Microbiol 83:e01814–e01817

    Article  PubMed  PubMed Central  Google Scholar 

  • Lee CM, Cradick TJ, Bao G (2016) The Neisseria meningitidis CRISPR-Cas9 System enables specific genome editing in mammalian cells. Mol Ther 24:645–654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Leenay RT, Beisel CL (2017) Deciphering, Communicating, and Engineering the CRISPR PAM. J Mol Biol 429:177–191

    Article  CAS  PubMed  Google Scholar 

  • Li J, Adams V, Bannam TL, Miyamoto K, Garcia JP, Uzal FA, Rood JI, McClane BA (2013) Toxin plasmids of Clostridium perfringens. Microbiol Mol Biol Rev 77:208–233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li C, Lillehoj HS, Gadde UD, Ritter D, Oh S (2017) Characterization of Clostridium perfringens strains isolated from healthy and necrotic enteritis-afflicted broiler chickens. Avian Dis 61:178–185

    Article  PubMed  Google Scholar 

  • Lin L, Petersen TS, Jensen KT, Bolund L, Kuhn R, Luo Y (2017) Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells. J Biotechnol 247:42–49

    Article  CAS  PubMed  Google Scholar 

  • Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467–477

    Article  CAS  PubMed  Google Scholar 

  • Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV (2015) An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 13:722–736

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marais BJ, Mlambo CK, Rastogi N, Zozio T, Duse AG, Victor TC, Marais E, Warren RM (2013) Epidemic spread of multidrug-resistant tuberculosis in Johannesburg, South Africa. J Clin Microbiol 51:1818–1825

    Article  PubMed  PubMed Central  Google Scholar 

  • McGhee GC, Sundin GW (2012) Erwinia amylovora CRISPR elements provide new tools for evaluating strain diversity and for microbial source tracking. PLoS One 7:e41706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Minkenberg B, Wheatley M, Yang Y (2017) CRISPR/Cas9-enabled multiplex genome editing and its application. Prog Mol Biol Transl Sci 149:111–132

    Article  PubMed  CAS  Google Scholar 

  • Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J (2016) Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science 353:5147

    Article  CAS  Google Scholar 

  • Molhuizen HO, Bunschoten AE, Schouls LM, van Embden JD (1998) Rapid detection and simultaneous strain differentiation of Mycobacterium tuberculosis complex bacteria by spoligotyping. Methods Mol Biol 101:381–394

    CAS  PubMed  Google Scholar 

  • Nakade S, Yamamoto T, Sakuma T (2017) Cas9, Cpf1 and C2c1/2/3-What’s next? Bioengineered 8:265–273

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nakano V, Ignacio A, Llanco L, Bueris V, Sircili MP, Avila-Campos MJ (2017) Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes. Anaerobe 44:99–105

    Article  CAS  PubMed  Google Scholar 

  • Obeng N, Pratama AA, Elsas JDV (2016) The Significance of mutualistic phages for bacterial ecology and evolution. Trends Microbiol 24:440–449

    Article  CAS  PubMed  Google Scholar 

  • Ohtani K, Shimizu T (2016) Regulation of Toxin Production in Clostridium perfringens. Toxins (Basel) 8:207

    Article  CAS  Google Scholar 

  • Park M, Deck J, Foley SL, Nayak R, Songer JG, Seibel JR, Khan SA, Rooney AP, Hecht DW, Rafii F (2016) Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids. Anaerobe 38:25–35

    Article  CAS  PubMed  Google Scholar 

  • Petit L, Gibert M, Popoff MR (1999) Clostridium perfringens: toxinotype and genotype. Trends Microbiol 7:104–110

    Article  CAS  PubMed  Google Scholar 

  • Schouls LM, Reulen S, Duim B, Wagenaar JA, Willems RJ, Dingle KE, Colles FM, Van Embden JD (2003) Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination. J Clin Microbiol 41:15–26

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shariat N, Sandt CH, DiMarzio MJ, Barrangou R, Dudley EG (2013) CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates. BMC Microbiol 13:254

    Article  PubMed  PubMed Central  Google Scholar 

  • Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F (2016) Rationally engineered Cas9 nucleases with improved specificity. Science 351:84–88

    Article  CAS  PubMed  Google Scholar 

  • Sontheimer EJ, Marraffini LA (2010) Microbiology. Nature 468:45–46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stern A, Mick E, Tirosh I, Sagy O, Sorek R (2012) CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome. Genome Res 22:1985–1994

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takei Y, Shah S, Harvey S, Qi LS, Cai L (2017) Multiplexed dynamic imaging of genomic loci by combined CRISPR imaging and DNA sequential FISH. Biophys J 112:1773–1776

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tillman GE, Simmons M, Garrish JK, Seal BS (2013) Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-l-alanine amidase as a potential antimicrobial to control the bacterium. Arch Microbiol 195:675–681

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Uzal FA, Freedman JC, Shrestha A, Theoret JR, Garcia J, Awad MM, Adams V, Moore RJ, Rood JI, McClane BA (2014) Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol 9:361–377

    Article  CAS  PubMed  Google Scholar 

  • van der Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJ (2009) CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci 34:401–407

    Article  PubMed  CAS  Google Scholar 

  • Westra ER, Buckling A, Fineran PC (2014) CRISPR-Cas systems: beyond adaptive immunity. Nat Rev Microbiol 12:317–326

    Article  CAS  PubMed  Google Scholar 

  • Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, Cai W, Yang G, Bronson R, Crowley DG, Zhang F, Anderson DG, Sharp PA, Jacks T (2014) CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514:380–384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yamashiro Y (2017) Gut Microbiota in Health and Disease. Ann Nutr Metab 71:242–246

    Article  CAS  PubMed  Google Scholar 

  • Zuo E, Cai YJ, Li K, Wei Y, Wang BA, Sun Y, Liu Z, Liu J, Hu X, Wei W, Huo X, Shi L, Tang C, Liang D, Wang Y, Nie YH, Zhang CC, Yao X, Wang X, Zhou C, Ying W, Wang Q, Chen RC, Shen Q, Xu GL, Li J, Sun Q, Xiong ZQ, Yang H (2017) One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Res 27:933–945

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This study was funded by the National Science and Technology Specific Projects (2018ZX10301407, 2013ZX10004607) and Henan Province University Science and Technology Innovation Talent Projects (17HASTIT045).

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Authors and Affiliations

  1. Department of Epidemiology, College of Public Health, Zhengzhou University, No. 100 Kexue Avenue, Zhengzhou, 450001, Henan, People’s Republic of China

    Jinzhao Long, Liuyang Ou, Haiyan Yang, Yuanlin Xi, Shuaiyin Chen & Guangcai Duan

  2. Henan Innovation Center of Molecular Diagnosis and Laboratory Medicine, Xinxiang Medical University, Xinxiang, Henan, People’s Republic of China

    Guangcai Duan

  3. School Hospital, Henan University of Science and Technology, Luoyang, Henan, People’s Republic of China

    Yake Xu

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  1. Jinzhao Long
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  2. Yake Xu
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  3. Liuyang Ou
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  4. Haiyan Yang
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  5. Yuanlin Xi
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  6. Shuaiyin Chen
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Contributions

Jinzhao Long, Guangcai Duan, and Shuaiyin Chen designed the study, Jinzhao Long and Yake Xu analyzed the data and wrote the paper. Haiyan Yang, Yuanlin Xi, and Liuyang Ou collected some data. All authors read and approved the final manuscript.

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Correspondence to Guangcai Duan.

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Jinzhao Long declares that he has no conflict of interest. Yake Xu declares that she has no conflict of interest. Liuyang Ou declares that he has no conflict of interest. Haiyan Yang declares that she has no conflict of interest. Yuanlin Xi declares that he has no conflict of interest. Shuaiyin Chen declares that he has no conflict of interest. Guangcai Duan declares that he has no conflict of interest.

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Long, J., Xu, Y., Ou, L. et al. Diversity of CRISPR/Cas system in Clostridium perfringens. Mol Genet Genomics 294, 1263–1275 (2019). https://doi.org/10.1007/s00438-019-01579-3

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