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Prophage integration into CRISPR loci enables evasion of antiviral immunity in Streptococcus pyogenes

Nature Microbiology volume 6, pages 1516–1525 (2021)Cite this article

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Abstract

CRISPR loci are composed of short DNA repeats separated by sequences, known as spacers, that match the genomes of invaders such as phages and plasmids. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defence, phages can produce small proteins that inhibit these nucleases, termed anti-CRISPRs (Acrs). Here we demonstrate that the ΦAP1.1 temperate phage utilizes an alternative approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection, this phage expresses a small anti-CRISPR protein, AcrIIA23, that prevents Cas9 function, allowing ΦAP1.1 to integrate into the direct repeats of the CRISPR locus, neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transduction of spacers, enabling a complex modulation of the type II-A CRISPR immune response. A bioinformatic search identified prophages integrated not only in the CRISPR repeats, but also the cas genes, of diverse bacterial species, suggesting that prophage disruption of the CRISPR–cas locus is a recurrent mechanism to counteract immunity.

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Fig. 1: ΦAP1.1 lysogenization into the CRISPR locus.
Fig. 2: Disruption of CRISPR function by ΦAP1.1 lysogenization.
Fig. 3: ΦAP1.1 carries a type II-A CRISPR–Cas inhibitor, AcrIIA23.
Fig. 4: AcrIIA23 is not transcribed from ΦAP1.1 prophages.
Fig. 5: ΦAP1.1 mediates the transduction of CRISPR spacers.
Fig. 6: Prophage integration into other CRISPR–cas loci.

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Data availability

Source data are provided with this paper. The data from this study are available from the corresponding authors upon request. The raw data for the RNA-seq experiments in Figs. 4a,b, 5c,d and the DNA reads of the ΦAP1.1-spec sequence can be found at the Sequence Read Archive (NIH), BioProject accession number PRJNA668016. The sequence of ΦAP1.1-spec is deposited under accession number MW168838. CRISPR–Cas databases were accessed from CRISPRCasFinder and the prophage database was accessed from PHASTER.

Code availability

Custom python scripts are deposited at https://github.com/Marraffini-Lab/Varble_etal_2021.git. Included are scripts that interact with PHASTER to determine CRISPR proximity to prophages (PHASTER_CRISPR.py), search deep sequencing data for spacer sequences in the phage ΦAP1.1 attB site (Phage_attb_II.py) and convert.sam files to.wig files for RNA sequencing analysis (SAM_WIG.py).

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Acknowledgements

We thank A. J. Meeske for bioinformatic assistance with the RNA-seq analysis. Support for this work was provided by the National Institutes of Health Director’s Pioneer Award 1DP1GM128184-01 and the Burroughs Wellcome Fund PATH Award to L.A.M. L.A.M. is an Investigator of the Howard Hughes Medical Institute. A.V. was supported by the Arnold and Mabel Beckman Postdoctoral Fellowship. J.T.R. was supported by the Boehringer Ingelheim Fonds PhD fellowship. Funding for V.A.F. and E.C. was from the Rockefeller University Laboratory Support. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Laboratory of Bacteriology, The Rockefeller University, New York, NY, USA

    Andrew Varble, Pascal Maguin, Albina Kozlova, Jessica Fyodorova, Jakob T. Rostøl & Luciano A. Marraffini

  2. Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, NY, USA

    Edmondo Campisi, Chad W. Euler & Vincent A. Fischetti

  3. Department of Medical Laboratory Sciences, Hunter College, CUNY, New York, NY, USA

    Chad W. Euler

  4. Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA

    Luciano A. Marraffini

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  1. Andrew Varble
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Contributions

A.V. and L.A.M. conceived the study. E.C., C.W.E. and V.A.F. aided with experimental design. A.V. performed all the experiments with the help of J.F. and A.K. E.C. and P.M. assisted with bioinformatic analysis. E.C. and C.W.E. provided assistance, strains and reagents for S. pyogenes experiments. J.T.R. provided reagents. A.V. and L.A.M. wrote the manuscript with the help of the other authors.

Corresponding authors

Correspondence to Andrew Varble or Luciano A. Marraffini.

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Competing interests

L.A.M. and A.V. are co-inventors on a patent application filed by The Rockefeller University relating to work in this study. L.A.M. is a founder and advisor of Intellia Therapeutics, Eligo Biosciences and CRISPR Biotechnologies. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Microbiology thanks Joseph Bondy-Denomy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 ΦAP1.1 integration into S. pyogenes type II-A CRISPR loci.

(a) ΦAP1.1-like phages integrated into the type II-A CRISPR locus of S. pyogenes strains. (b) Schematic of the ΦAP1.1 genome. Targets for the spacers found in different strains are shown on top or bottom depending on the phage DNA strand that is targeted. The acr region and its four ORFs are highlighted in grey. The aadA gene inserted to confer spectinomycin resistance to ΦAP1.1 lysogens is shown in light blue. (c) Spacer duplication events observed in S. pyogenes K56(ΦAP1.1) lysogens. (d) To determine if the tyrosine site-specific recombinase present in the ΦAP1.1 genome is required for its insertion into the CRISPR repeats, we deleted the integrase gene, generating ΦAP1.1Δint. We infected streptococci with both wild-type and int phages and enumerated spectinomycin-resistant colonies. Interestingly, the number of colony-forming units (cfu) obtained was similar for both infections. However, in the absence of the integrase, the colonies lost antibiotic resistance over time, and we were unable to detect PCR products for the attL site or the phage itself, indicating that infection resulted in the transient transfer of antibiotic resistance but failed to produce lysogenic integration into the CRISPR array. Enumeration of spectinomycin-resistant cfu per ml of S. pyogenes CEMΔΦ[Δ2-5] cultures infected with either wild-type ΦAP1.1 or ΦAP1.1Δint. Mean + STD of 3 biological replicates are reported. Interestingly, the number of colony-forming units (cfu) obtained was similar for both infections. However, in the absence of the integrase, the colonies lost antibiotic resistance over time (not shown). (e) Agarose gel electrophoresis of the attL PCR product obtained after the amplification shown in Fig. 1B, using DNA extracted from 24 spectinomycin-resistant colonies that resulted from infection of S. pyogenes CEMΔΦ[Δ2-5] cultures with ΦAP1.1Δint. Collected from three biological replicates. (f) Sequences of the S. pyogenes CEM1ΔΦ spc4 crRNA annealed to its targets in ΦAP1.1 and ΦAP1.1ΔacrIIA23-esc. The PAM nucleotides are boxed. The escape mutation of ΦAP1.1ΔacrIIA23-esc is in red. (g) Base pairs formed between the crRNA and tracrRNA of S. pyogenes (grey dots). Blue sequence, spacer; pink sequence, repeat; black sequence, tracrRNA. Black arrows indicate RNase III cleavage sites required for crRNA processing. (h) Same as (G) but showing the possible base pairs formed by the prophage transcript and the tracrRNA. Lack of annealing with the prophage-derived sequences (orange) prevents RNase III cleavage and thus processing of the upstream crRNA.

Source data

Extended Data Fig. 2 ΦAP1.1 lysogenization location.

We decided to look for integration of ΦAP1.1 in other genomic sites with the 8-bp sequence of the attB present on the CRISPR repeat. Because strain K56 is not sequenced and therefore we could not identify such sequences, we decided to perform experiments with strain SF370, which harbors an additional 27 sites with the attB sequence. To do this, we infected strain CEM1ΔΦ[Δ2-5], which lacks the prophages as well as most spacers of the CRISPR array (in particular spc4, which targets ΦAP1.1) present in SF370. We isolated and analyzed 110 spectinomycin-resistant colonies and looked for the presence of the phage (p) and its integration attL site (i) via PCR. We found that all prophages were inserted into the CRISPR array (in different repeats, generating different sizes for the attL PCR products). This result indicates a strong preference for integration into the repeat sequences and also suggests that regions outside of the 8-bp attB site are important for integration. Agarose gel electrophoresis of the attL (i) or phage (p) PCR products obtained after selection of individual spectinomycin-resistant ΦAP1.1 lysogens. PCRs for individual lysogens are divided by black lines.

Source data

Extended Data Fig. 3 ΦAP1.1 lysogenization into the S. pyogenes NCTC13743 type II-A CRISPR locus.

(a) S. pyogenes NCTC13743 type II-A CRISPR-cas locus, wild-type and the mutant lacking spacer-repeat units 1 through 10, [Δ1-10]. The ‘t’ indicates three degenerate repeats that contain mutations from the repeat consensus sequence. (b) Sequences of the S. pyogenes NCTC13743 spc2, spc6 and spc9 crRNAs annealed to their targets in ΦAP1.1. The PAM nucleotides are boxed. (c) We verified that the type II-A CRISPR system present in NCTC13743 is capable of restricting a plasmid containing the ΦAP1.1 target sties. Transformation efficiency of the pC194 plasmid or a modified version harboring the three targets for spc2, spc6 and spc9, pTgt2-6-9 from ΦAP1.1, after electroporation of wild-type or [Δ1-10] S. pyogenes NCTC13743 competent cells. Mean + STD of 3 biological replicates are reported. These results demonstrate that the type II-A CRISPR system present in this strain is capable of restricting a plasmid containing the three target sequences present in ΦAP1.1 (d) Diagrams of the type II-A locus of 25 ΦAP1.1 lysogens in S. pyogenes NCTC13743, reconstructed after sequencing of their attL and attR sites.

Source data

Extended Data Fig. 4 AcrIIA23 inhibits the type II-A CRISPR-Cas response in S. pyogenes NCTC13743 and S. aureus RN4220.

(a) Lysogenization rates of ΦAP1.1 or ΦAP1.1ΔacrIIA23 after infection of wild-type or [Δ1-10] S. pyogenes NCTC13743 cells. Mean + STD of 3 biological replicates are reported. (b) Integration of the S. pyogenes CEM1ΔΦ type II-A system into the lipase gene of S. aureus RNA4220 using the integrative vector pCL55, generating strain JAV17. A CRISPR locus with a spacer matching the gp68 gene from the virulent staphylococcal phage ΦNM4γ4 was also integrated, generating JAV16. (c) JAV17 and JAV16 strains were transformed with each of the individual ΦAP1.1 ORFs under the control of an anhydrotetracycline (aTc)-inducible promoter, on the pJTR162 staphylococcal plasmid backbone, with the exception of ORF3, for which we did not obtain transformants. Strains harboring the pJTR-ORF1, pJTR-ORF2 and pJTR-ORF4 constructs were then infected with ΦNM4γ4 in the presence or absence of aTc, and pfus were counted. We found that none of the ORFs affected the immunity of the JAV16 strain in the absence of the inducer, and ΦNM4γ4 propagation was limited. In the presence of aTc, however, ORF2 expression enabled high levels of viral replication, similar to those observed in the absence of type II-A CRISPR-Cas immunity after infection of the JAV17 strain. Efficiency of plaquing of phage ΦNM4γ4 on soft agar lawns of S. aureus JAV17 or JAV16, in the presence or absence of aTc, carrying the pJTR162 control plasmid or versions that express the different ORFs of the ΦAP1.1 anti-CRISPR locus. Mean + STD of 3 biological replicates are reported. Two-tailed unpaired t-test was used to calculate P value.

Source data

Extended Data Fig. 5 ΦAP1.1ΔacrIIA23-esc integration pattern.

(a) Frequency of ΦAP1.1ΔacrIIA23-esc lysogens (n = 50) carrying attL or attR sites in each of the DRs of the CEM1ΔΦ CRISPR locus. (b) Same as (A) but showing the combined distribution of attL or attR sites. (c) Northern blot analysis of spc4 crRNAs, as well as 5 S rRNA, produced by wild-type S. pyogenes CEM1ΔΦ or its different ΦAP1.1 (DR1-5, 1-t) or ΦAP1.1ΔacrIIA23-esc (DR6, DRt) lysogens. (d) Transformation efficiency of the pC194 plasmid or a modified version harboring a target for spc4 from ΦAP1.1, pTgt4, after electroporation of the strains used in (C). Mean + STD of 3 biological replicates are reported.

Source data

Extended Data Fig. 6 ΦAP1.1-mediated transduction of spacers from the CEM1ΔΦ to the K56 CRISPR locus.

(a) Agarose gel electrophoresis of the attL or attR PCR products obtained after treatment of S. pyogenes CEM1ΔΦ(ΦAP1.1::DR5) lysogens with mitomycin C (MMC). Primers used and the expected products are shown in the diagrams to the right. Black arrowheads indicate PCR products that lack a number of the expected spacer-repeat units. Representative of three independent replicates. (b) Example of aberrant ΦAP1.1 excision in S. pyogenes CEM1ΔΦ(ΦAP1.1::DR3) lysogens, where DR1 or DR2 are used instead of attL for recombination with attR. ΦAP1.1-, spc1- and spc2-specific primers used to detect attP sites containing either of these spacers are shown as orange, red and yellow arrows, respectively. (c) Same as (B) but for a ΦAP1.1::DR5 lysogen. (d) Agarose gel electrophoresis of PCR products obtained after the amplification of the attP site of ΦAP1.1 phages induced with MMC from ΦAP1.1::DR3, ΦAP1.1::DR5 and ΦAP1.1::DR1-t lysogens, using spc1-specific primers. (e) Same as (D) but using spc2-specific primers. In both cases we found PCR products that indicated the presence of spacer-repeat units in the DNA of the viral particles, with the intensity of the amplicons indicating more spc1- than spc2-containing viral particles. These results suggest a possible preference for certain excision products. (f) Example of possible recombination of the 5’ end (top) or 3’ end (bottom) attP site of the ΦAP1.1spc1-2 phage with the three attB sites present in the DR1, DR2 and DRt of S. pyogenes K56.

Source data

Extended Data Fig. 7 Spacers targeting prophages integrated into other CRISPR-cas loci.

Alignment of spacers described in Fig. 6 with their phage target sites; in the 5’ to 3’ orientation.

Supplementary information

Supplementary Information

Supplementary Tables 1–4 and synthetic DNA.

Reporting Summary.

Source data

Source Data Fig. 1

Original unprocessed DNA gel images.

Source Data Fig. 1

Raw numerical data.

Source Data Fig. 2

Original unprocessed northern blot images.

Source Data Fig. 2

Raw numerical data.

Source Data Fig. 3

Raw numerical data.

Source Data Fig. 5

Original unprocessed DNA gel images.

Source Data Fig. 5

Raw numerical data.

Source Data Extended Data Fig. 1

Original unprocessed DNA gel images.

Source Data Extended Data Fig. 1

Raw numerical data.

Source Data Extended Data Fig. 2

Original unprocessed DNA gel images.

Source Data Extended Data Fig. 3

Raw numerical data.

Source Data Extended Data Fig. 4

Raw numerical data.

Source Data Extended Data Fig. 5

Original unprocessed northern blot images.

Source Data Extended Data Fig. 5

Raw numerical data.

Source Data Extended Data Fig. 6

Original unprocessed DNA gel images.

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Varble, A., Campisi, E., Euler, C.W. et al. Prophage integration into CRISPR loci enables evasion of antiviral immunity in Streptococcus pyogenes. Nat Microbiol 6, 1516–1525 (2021). https://doi.org/10.1038/s41564-021-00996-8

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