Functional Analysis of Bacteriophage Immunity through a Type I-E CRISPR-Cas System in Vibrio cholerae and Its Application in Bacteriophage Genome Engineering

Abstract

The classical and El Tor biotypes of Vibrio cholerae serogroup O1, the etiological agent of cholera, are responsible for the sixth and seventh (current) pandemics, respectively. A genomic island (GI), GI-24, previously identified in a classical biotype strain of V. cholerae, is predicted to encode clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins); however, experimental evidence in support of CRISPR activity in V. cholerae has not been documented. Here, we show that CRISPR-Cas is ubiquitous in strains of the classical biotype but excluded from strains of the El Tor biotype. We also provide in silico evidence to suggest that CRISPR-Cas actively contributes to phage resistance in classical strains. We demonstrate that transfer of GI-24 to V. cholerae El Tor via natural transformation enables CRISPR-Cas-mediated resistance to bacteriophage CP-T1 under laboratory conditions. To elucidate the sequence requirements of this type I-E CRISPR-Cas system, we engineered a plasmid-based system allowing the directed targeting of a region of interest. Through screening for phage mutants that escape CRISPR-Cas-mediated resistance, we show that CRISPR targets must be accompanied by a 3= TT protospacer-adjacent motif (PAM) for efficient interference. Finally, we demonstrate that efficient editing of V. cholerae lytic phage genomes can be performed by simultaneously introducing an editing template that allows homologous recombination and escape from CRISPR-Cas targeting. IMPORTANCECholera, caused by the facultative pathogen Vibrio cholerae, remains a serious public health threat. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) provide prokaryotes with sequence-specific protection from invading nucleic acids, including bacteriophages. In this work, we show that one genomic feature differentiating sixth pandemic (classical biotype) strains from seventh pandemic (El Tor biotype) strains is the presence of a CRISPR-Cas system in the classical biotype. We demonstrate that the CRISPR-Cas system from a classical biotype strain can be transferred to a V. cholerae El Tor biotype strain and that it is functional in providing resistance to phage infection. Finally, we show that this CRISPR-Cas system can be used as an efficient tool for the editing of V. cholerae lytic phage genomes. Vibrio cholerae is a Gram-negative facultative pathogen that causes the acute diarrheal disease cholera. The current cholera pandemic, the seventh in recorded history, began in 1961 and is caused by V. cholerae O1 of the El Tor biotype (1). This pandemic has affected much of the developing world, including many countries in Asia and Africa and most recently in the Caribbean (2). The sixth cholera pandemic, however, was caused by V. cholerae O1 of the classical biotype. Classical biotype strains declined following the emergence of El Tor strains and are now believed to be extinct, after last being seen in 1990 in Bangladesh (3). The mechanisms underpinn...

CRISPR-Cas and Contact-Dependent Secretion Systems Present on Excisable Pathogenicity Islands with Conserved Recombination Modules

“…The CRISPR-Cas system in strain O395 consisted of eight Cas proteins, a 2,407-bp CRISPR sequence comprised of a 28-bp repeat (GTCTTCCCCACGCAGGTGG GGGTGTTTC), and 39 unique spacer sequences (Table 1). This CRISPR-Cas system was shown to be a functional type I-E system that was confined to classical strains of V. cholerae (31). This system showed no homology to the CRISPR-Cas system identified here in RC385, and the integrase shared Ͻ27% (E value, 2eϪ32) amino acid identity with IntV1 from strain N16961, indicating they are unrelated.…”

“…In evolutionary terms, these data demonstrate that there is ongoing recombination and acquisition of CRISPR-Cas systems among closely related isolates and horizontal transfer among distantly related species. In support of this, a recent study showed that a type I-E CRISPR-Cas system could be transferred by natural transformation between V. cholerae isolates (31). A dynamic system would be expected in a region that is required for adaptive immunity to protect against constant attack from phages.…”

“…All CRISPR-Cas systems identified to date contain Cas1 and Cas2 proteins (28). Type I CRISPR-Cas systems, specifically type I-E, have been described in V. cholerae strains, and type I-F has been described in bacteriophages that infect V. cholerae isolates (29)(30)(31). A signature of type I CRISPR-Cas systems is the presence of a Cas3 protein, which has a helicase domain and, in many Cas3 proteins, an HD family endonuclease domain.…”

DominantVibrio choleraephage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite

“…To test if LidI PLE 1 -mediated accelerated lysis is enough to impact diversity through the acquisition of random mutations in the resulting progeny phage population, we exposed PLE (-) V. cholerae with and without lidI PLE 1 to ICP1, collected the population of progeny phage, and looked for plaque formation on PLE (-) V. cholerae encoding a Type I-E CRISPR-Cas system endogenous to V. cholerae classical biotype strains engineered to harbor various anti-ICP1 spacers (Fig. 6C) (27). We see that for each spacer, fewer phages are able to overcome targeting in the phage progeny from lidI PLE 1 V. cholerae infections than the infections of strains without lidI PLE 1 (Fig.…”

“…As no infectious ICP1 are produced, PLE activity ultimately defends populations of V. cholerae from ICP1 attack, functionally acting as an abortive infection system. In the face of this assault, ICP1 has acquired a functional Type I-F CRISPR-Cas adaptive immune system to target PLE in a sequence-specific manner, restoring ICP1 progeny phage production and overcoming PLE (24,27). While the full extent of ICP1, PLE, and V. cholerae interactions are unknown and continuing to evolve, efforts to understand how the PLE completely restricts ICP1 production have yet to identify any single PLE-encoded gene product that is necessary for inhibition, suggesting that multiple inhibitory strategies act synergistically to fully eliminate phage production (22,23,26).…”