Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands

English version České info

In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

Vyšlo v časopise: Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands. PLoS Genet 9(4): e32767. doi:10.1371/journal.pgen.1003454
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003454

Souhrn

Zdroje

1. PettyNK, EvansTJ, FineranPC, SalmondGP (2007) Biotechnological exploitation of bacteriophage research. Trends Biotechnol 25: 7–15.

2. HendrixRW (2003) Bacteriophage genomics. Curr Opin Microbiol 6: 506–511.

3. WeinbauerMG (2004) Ecology of prokaryotic viruses. FEMS Microbiol Rev 28: 127–181.

4. LabrieSJ, SamsonJE, MoineauS (2010) Bacteriophage resistance mechanisms. Nat Rev Microbiol 8: 317–327.

5. FineranPC, BlowerTR, FouldsIJ, HumphreysDP, LilleyKS, et al. (2009) The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Proc Natl Acad Sci U S A 106: 849–899.

6. HorvathP, BarrangouR (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327: 167–170.

7. SorekR, KuninV, HugenholtzP (2008) CRISPR–a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 6: 181–186.

8. WestraER, SwartsDC, StaalsRH, JoreMM, BrounsSJ, et al. (2012) The CRISPRs, They Are A-Changin': How Prokaryotes Generate Adaptive Immunity. Annu Rev Genet 46: 311–339.

9. MarraffiniLA, SontheimerEJ (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11: 181–190.

10. RichterC, ChangJT, FineranPC (2012) The function and regulation of CRISPR/Cas systems. Viruses 4: 2291–2311.

11. FineranPC, CharpentierE (2012) Memory of viral infections by CRISPR-Cas adaptive immunue systems: Acquisition of new information. Virology 434: 202–209.

12. GrissaI, VergnaudG, PourcelC (2007) The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8: 172.

13. BarrangouR, FremauxC, DeveauH, RichardsM, BoyavalP, et al. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315: 1709–1712.

14. HaftDH, SelengutJ, MongodinEF, NelsonKE (2005) A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol 1: e60 doi:10.1371/journal.pcbi.0010060.

15. MakarovaKS, GrishinNV, ShabalinaSA, WolfYI, KooninEV (2006) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1: 7.

16. JansenR, EmbdenJD, GaastraW, SchoulsLM (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 43: 1565–1575.

17. MakarovaKS, AravindL, WolfYI, KooninEV (2011) Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct 6: 38.

18. MakarovaKS, HaftDH, BarrangouR, BrounsSJ, CharpentierE, et al. (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9: 467–477.

19. BrounsSJ, JoreMM, LundgrenM, WestraER, SlijkhuisRJ, et al. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321: 960–964.

20. CarteJ, WangR, LiH, TernsRM, TernsMP (2008) Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev 22: 3489–3496.

21. DeltchevaE, ChylinskiK, SharmaCM, GonzalesK, ChaoY, et al. (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471: 602–607.

22. HaurwitzRE, JinekM, WiedenheftB, ZhouK, DoudnaJA (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329: 1355–1358.

23. PrzybilskiR, RichterC, GristwoodT, ClulowJS, VercoeRB, et al. (2011) Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum. RNA Biol 8: 517–528.

24. HaleC, KleppeK, TernsRM, TernsMP (2008) Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14: 2572–2579.

25. HaleCR, MajumdarS, ElmoreJ, PfisterN, ComptonM, et al. (2012) Essential Features and Rational Design of CRISPR RNAs that Function with the Cas RAMP Module Complex to Cleave RNAs. Mol Cell 45: 292–302.

26. ZhangJ, RouillonC, KerouM, ReeksJ, BruggerK, et al. (2012) Structure and Mechanism of the CMR Complex for CRISPR-Mediated Antiviral Immunity. Mol Cell 45: 303–313.

27. GarneauJE, DupuisME, VillionM, RomeroDA, BarrangouR, et al. (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468: 67–71.

28. MarraffiniLA, SontheimerEJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322: 1843–1845.

29. WestraER, van ErpPB, KunneT, WongSP, StaalsRH, et al. (2012) CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3. Mol Cell 46: 595–605.

30. YosefI, GorenMG, QimronU (2012) Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res 40: 5569–5576.

31. DatsenkoKA, PougachK, TikhonovA, WannerBL, SeverinovK, et al. (2012) Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 3: 945.

32. SwartsDC, MosterdC, van PasselMW, BrounsSJ (2012) CRISPR Interference Directs Strand Specific Spacer Acquisition. PLoS ONE 7: e35888 doi:10.1371/journal.pone.0035888.

33. MojicaFJ, Diez-VillasenorC, Garcia-MartinezJ, AlmendrosC (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155: 733–740.

34. DeveauH, BarrangouR, GarneauJE, LabonteJ, FremauxC, et al. (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190: 1390–1400.

35. SemenovaE, JoreMM, DatsenkoKA, SemenovaA, WestraER, et al. (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A 108: 10098–10103.

36. HaleCR, ZhaoP, OlsonS, DuffMO, GraveleyBR, et al. (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139: 945–956.

37. LintnerNG, KerouM, BrumfieldSK, GrahamS, LiuH, et al. (2011) Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE). J Biol Chem 286: 21643–21656.

38. WiedenheftB, van DuijnE, BultemaJ, WaghmareS, ZhouK, et al. (2011) RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci U S A 108: 10092–10097.

39. NamKH, HaitjemaC, LiuX, DingF, WangH, et al. (2012) Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System. Structure 20: 1574–1584.

40. HorvathP, Coute-MonvoisinAC, RomeroDA, BoyavalP, FremauxC, et al. (2009) Comparative analysis of CRISPR loci in lactic acid bacteria genomes. Int J Food Microbiol 131: 62–70.

41. LillestolRK, RedderP, GarrettRA, BruggerK (2006) A putative viral defence mechanism in archaeal cells. Archaea 2: 59–72.

42. AklujkarM, LovleyDR (2010) Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus. BMC Evol Biol 10: 230.

43. EdgarR, QimronU (2010) The Escherichia coli CRISPR System Protects from {lambda} Lysogenization, Lysogens, and Prophage Induction. J Bacteriol 192: 6291–6294.

44. ManicaA, ZebecZ, TeichmannD, SchleperC (2011) In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon. Mol Microbiol 80: 481–491.

45. SternA, KerenL, WurtzelO, AmitaiG, SorekR (2010) Self-targeting by CRISPR: gene regulation or autoimmunity? Trends Genet 26: 335–340.

46. KuninV, SorekR, HugenholtzP (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 8: R61.

47. RichterC, GristwoodT, ClulowJS, FineranPC (2012) In vivo protein interactions and complex formation in the Pectobacterium atrosepticum subtype I-F CRISPR/Cas system. PLoS ONE 7: e49549 doi:10.1371/journal.pone.0049549.

48. LiuH, CoulthurstSJ, PritchardL, HedleyPE, RavensdaleM, et al. (2008) Quorum sensing coordinates brute force and stealth modes of infection in the plant pathogen Pectobacterium atrosepticum. PLoS Pathog 4: e1000093 doi:10.1371/journal.ppat.1000093.

49. AlmendrosC, GuzmanNM, Diez-VillasenorC, Garcia-MartinezJ, MojicaFJ (2012) Target Motifs Affecting Natural Immunity by a Constitutive CRISPR-Cas System in Escherichia coli. PLoS ONE 7: e50797 doi:10.1371/journal.pone.0050797.

50. HuismanO, D'AriR (1981) An inducible DNA replication-cell division coupling mechanism in E. coli. Nature 290: 797–799.

51. CadyKC, Bondy-DenomyJ, HeusslerGE, DavidsonAR, O'TooleGA (2012) The CRISPR/Cas Adaptive Immune System of Pseudomonas aeruginosa Mediates Resistance to Naturally Occurring and Engineered Phages. J Bacteriol 194: 5728–5738.

52. SternbergSH, HaurwitzRE, DoudnaJA (2012) Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA 18: 661–672.

53. VangaBR, ButlerRC, TothIK, RonsonCW, PitmanAR (2012) Inactivation of PbTopo IIIbeta causes hyper-excision of the Pathogenicity Island HAI2 resulting in reduced virulence of Pectobacterium atrosepticum. Mol Microbiol 84: 648–663.

54. BellKS, SebaihiaM, PritchardL, HoldenMT, HymanLJ, et al. (2004) Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors. Proc Natl Acad Sci U S A 101: 11105–11110.

55. Mohd-ZainZ, TurnerSL, Cerdeno-TarragaAM, LilleyAK, InzanaTJ, et al. (2004) Transferable antibiotic resistance elements in Haemophilus influenzae share a common evolutionary origin with a diverse family of syntenic genomic islands. J Bacteriol 186: 8114–8122.

56. CarterMQ, ChenJ, LoryS (2010) The Pseudomonas aeruginosa pathogenicity island PAPI-1 is transferred via a novel type IV pilus. J Bacteriol 192: 3249–3258.

57. CadyKC, O'TooleGA (2011) Non-identity-mediated CRISPR-bacteriophage interaction mediated via the Csy and Cas3 proteins. J Bacteriol 193: 3433–3445.

58. Diez-VillasenorC, AlmendrosC, Garcia-MartinezJ, MojicaFJ (2010) Diversity of CRISPR loci in Escherichia coli. Microbiology 156: 1351–1361.

59. TouchonM, CharpentierS, ClermontO, RochaEP, DenamurE, et al. (2011) CRISPR distribution within the Escherichia coli species is not suggestive of immunity-associated diversifying selection. J Bacteriol 193: 2460–2467.

60. TouchonM, RochaEP (2010) The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella. PLoS ONE 5: e11126 doi:10.1371/journal.pone.0011126.

61. BrodtA, Lurie-WeinbergerMN, GophnaU (2011) CRISPR loci reveal networks of gene exchange in archaea. Biol Direct 6: 65.

62. YosefI, GorenMG, KiroR, EdgarR, QimronU (2011) High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. Proc Natl Acad Sci U S A 108: 20136–20141.

63. Caillet-FauquetP, Maenhaut-MichelG (1988) Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli. Mol Gen Genet 213: 491–498.

64. Roberts JW, Devoret R (1983) Lysogenic induction. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA, editors. Lambda II. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. pp. 123–144.

65. BeloglazovaN, PetitP, FlickR, BrownG, SavchenkoA, et al. (2011) Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference. EMBO J 30: 4616–4627.

66. MulepatiS, BaileyS (2011) Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J Biol Chem 286: 31896–31903.

67. SinkunasT, GasiunasG, FremauxC, BarrangouR, HorvathP, et al. (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 30: 1335–1342.

68. ChayotR, MontagneB, MazelD, RicchettiM (2010) An end-joining repair mechanism in Escherichia coli. Proc Natl Acad Sci U S A 107: 2141–2146.

69. JiangW, BikardD, CoxD, ZhangF, MarraffiniLA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31: 233–239.

70. Seth-SmithHM (2008) SPI-7: Salmonella's Vi-encoding Pathogenicity Island. J Infect Dev Ctries 2: 267–271.

71. OsbornAM, BoltnerD (2002) When phage, plasmids, and transposons collide: genomic islands, and conjugative- and mobilizable-transposons as a mosaic continuum. Plasmid 48: 202–212.

72. Lopez-SanchezMJ, SauvageE, Da CunhaV, ClermontD, Ratsima HariniainaE, et al. (2012) The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol Microbiol 85: 1057–1071.

73. LovellHC, MansfieldJW, GodfreySA, JacksonRW, HancockJT, et al. (2009) Bacterial evolution by genomic island transfer occurs via DNA transformation in planta. Curr Biol 19: 1586–1590.

74. PitmanAR, JacksonRW, MansfieldJW, KaitellV, ThwaitesR, et al. (2005) Exposure to host resistance mechanisms drives evolution of bacterial virulence in plants. Curr Biol 15: 2230–2235.

75. DobrindtU, HochhutB, HentschelU, HackerJ (2004) Genomic islands in pathogenic and environmental microorganisms. Nat Rev Microbiol 2: 414–424.

76. ShenK, SayeedS, AntalisP, GladitzJ, AhmedA, et al. (2006) Extensive genomic plasticity in Pseudomonas aeruginosa revealed by identification and distribution studies of novel genes among clinical isolates. Infect Immun 74: 5272–5283.

77. FineranPC, EversonL, SlaterH, SalmondGP (2005) A GntR family transcriptional regulator (PigT) controls gluconate-mediated repression and defines a new, independent pathway for regulation of the tripyrrole antibiotic, prodigiosin, in Serratia. Microbiology 151: 3833–3845.

78. KanigaK, DelorI, CornelisGR (1991) A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109: 137–141.

79. WilliamsonNR, FineranPC, OgawaW, WoodleyLR, SalmondGP (2008) Integrated regulation involving quorum sensing, a two-component system, a GGDEF/EAL domain protein and a post-transcriptional regulator controls swarming and RhlA-dependent surfactant biosynthesis in Serratia. Environ Microbiol 10: 1202–1217.