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. 2024 Jul 3;24(1):240.
doi: 10.1186/s12866-024-03381-7.

CRISPR-Cas3 and type I restriction-modification team up against blaKPC-IncF plasmid transfer in Klebsiella pneumoniae

Yang Yang #  1   2 , Peiyao Zhou #  3 , Dongxing Tian  4 , Weiwen Wang  4 , Ying Zhou  5 , Xiaofei Jiang  6
Affiliations

CRISPR-Cas3 and type I restriction-modification team up against blaKPC-IncF plasmid transfer in Klebsiella pneumoniae

Yang Yang et al. BMC Microbiol. .

Abstract

Objective: We explored whether the Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification (R-M) systems are compatible and act together to resist plasmid attacks.

Methods: 932 global whole-genome sequences from GenBank, and 459 K. pneumoniae isolates from six provinces of China, were collected to investigate the co-distribution of CRISPR-Cas, R-M systems, and blaKPC plasmid. Conjugation and transformation assays were applied to explore the anti-plasmid function of CRISPR and R-M systems.

Results: We found a significant inverse correlation between the presence of CRISPR and R-M systems and blaKPC plasmids in K. pneumoniae, especially when both systems cohabited in one host. The multiple matched recognition sequences of both systems in blaKPC-IncF plasmids (97%) revealed that they were good targets for both systems. Furthermore, the results of conjugation assay demonstrated that CRISPR-Cas and R-M systems in K. pneumoniae could effectively hinder blaKPC plasmid invasion. Notably, CRISPR-Cas and R-M worked together to confer a 4-log reduction in the acquisition of blaKPC plasmid in conjugative events, exhibiting robust synergistic anti-plasmid immunity.

Conclusions: Our results indicate the synergistic role of CRISPR and R-M in regulating horizontal gene transfer in K. pneumoniae and rationalize the development of antimicrobial strategies that capitalize on the immunocompromised status of KPC-KP.

Keywords: Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella Pneumoniae; bla KPC harbouring plasmids; CRISPR-Cas system; Horizontal gene transfer; Type I R-M systems.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Distribution of CRISPR-Cas systems, R-M systems, and blaKPC in 932 completely sequenced global Klebsiella pneumoniae(A) Presence of CRISPR-Cas systems, R-M systems, and blaKPC in 271 blaKPC harbouring isolates in (i), 247 CRISPR-positive isolates, and 360 R-M positive isolates. (B) Distribution of different types of CRISPR-Cas and R-M systems in Klebsiella pneumoniae co-harbouring CRISPR, R-M and blaKPC
Fig. 2
Fig. 2
Characteristics of blaKPC harbouring plasmids extracted from 932 global Klebsiella pneumoniae. (A) The incompatibility type distribution of blaKPC harbouring plasmids. (B) The number of proto-spacers (i) and R-M recognition sites among IncF blaKPC harbouring plasmids (ii)
Fig. 3
Fig. 3
Conjugation frequencies of p187-2 plasmid in strains with or without type I R-M systems. (A) Comparative analysis of IncF blaKPC harbouring plasmids using p187-2 as the reference. The CRISPR and R-M targeted regions are illustrated with red dotted boxes. The detailed information on these plasmids is listed in the dataset. (B) Type I R-M systems influence the conjugation of the p187-2 plasmid. The data represent the mean ± SD for three independent biological replicates. ****p < 0.0001 indicate significant differences between strains and the control group as determined using one-way ANOVA with Dunnett correction
Fig. 4
Fig. 4
Conjugation frequencies of p187-2 in strains with or without CRISPR-Cas or type I R-M systems. The data represent the mean ± SD for three independent biological replicates. ****p < 0.0001 indicates significant differences between strains harbouring CRISPR or R-M systems and the control group as determined using one-way ANOVA with Dunnett correction. “ns” indicates no significant differences between strains harbouring CRISPR and strains harbouring R-M
Fig. 5
Fig. 5
CRISPR-Cas and R-M provide additive defense against pl87-2 plasmid. (A) Schematic representation of donor and recipient strains used to assess the individual and collective contributions of R-M and CRISPR-Cas to genome defense. (B) Conjugation frequencies of p187-2 in different derivatives strains. Results of these experiments show that the combined effects of CRISPR-Cas and R-M outweigh the effect of either system alone. Data represent results of a minimum of three independent conjugations for all experiments shown. The open circle indicates that no valid conjugant was obtained (JS700, CRISPR&ID R-M). ****p < 0.0001 indicate significant differences between strains and corresponding CRISPR & R-M group as determined using one-way ANOVA with Dunnett correction. E.g., the type IA R-M strains were compared with type IA R-M & CRISPR strains; the CRISPR strains were compared with type IA R-M & CRISPR, type IB R-M & CRISPR, and type ID R-M & CRISPR strains
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