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

Abstract

Pathogenicity islands (PAIs) are mobile integrated genetic elements that contain a diverse range of virulence factors. PAIs integrate into the host chromosome at a tRNA locus that contains their specific bacterial attachment site, attB, via integrase-mediated site-specific recombination generating attL and attR sites. We identified conserved recombination modules (integrases and att sites) previously described in choleragenic Vibrio cholerae PAIs but with novel cargo genes. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins) and a type VI secretion system (T6SS) gene cluster were identified at the Vibrio pathogenicity island 1 (VPI-1) insertion site in 19 V. cholerae strains and contained the same recombination module. Two divergent type I-F CRISPR-Cas systems were identified, which differed in Cas protein homology and content. The CRISPR repeat sequence was identical among all V. cholerae strains, but the CRISPR spacer sequences and the number of spacers varied. In silico analysis suggests that the CRISPR-Cas systems were active against phages and plasmids. A type III secretion system (T3SS) was present in 12 V. cholerae strains on a 68-kb island inserted at the same tRNA-serine insertion site as VPI-2 and contained the same recombination module. Bioinformatics analysis showed that two divergent T3SSs exist among the strains examined. Both the CRISPR and T3SS islands excised site specifically from the bacterial chromosome as complete units, and the cognate integrases were essential for this excision. These data demonstrated that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes, signifying a novel method of acquisition for both CRISPR-Cas systems and T3SSs. IMPORTANCE This work demonstrated the presence of CRISPR-Cas systems and T3SSs on PAIs. Our work showed that similar recombination modules can associate with different cargo genes and catalyze their incorporation into bacterial chromosomes, which could convert a strain into a pathogen with very different disease pathologies. Each island had the ability to excise from the chromosome as distinct, complete units for possible transfer. Evolutionary analysis of these regions indicates that they were acquired by horizontal transfer and that PAIs are the units of transfer. Similar to the case for phage evolution, PAIs have a modular structure where different functional regions are acquired by identical recombination modules.KEYWORDS CRISPR-Cas, type III secretion, Vibrio cholerae, pathogenicity islands V ibrio cholerae is the causative agent of the severe diarrheal disease cholera. The factors necessary for manifestation of this disease were acquired by horizontal gene transfer. The cholera toxin (CT), a potent enterotoxin whose effects are respon-

Analysis of the CRISPR-Cas system in bacteriophages active on epidemic strains of Vibrio cholerae in Bangladesh

“…The CRISPR-Cas region in 7 of 8 CRISPR-Cas positive non-O1 non-O139 strains sequenced in our study were found to be identical with that of strain HC-36A1 described previously 23 . However, comparisons of the entire sequence of the islands in our strains with those of previously described CRISPR-Cas containing genomic islands of strains S12, RC385, RC586 TM 11079-80, and HC-36A1 22 , 23 , showed that the islands found in 3 of our strains were > 95% identical to that found in strain HC-36A1 23 , whereas the islands in 4 of the strains were 70–74% identical to that in strain HC-36A1. The cas genes in one of our strains 173V1015 were found to share no sequence homology with that of the other islands analyzed, although the predicted protein sequence show more than 82% identity with Cas proteins of Salinivibrio costicola (Accession no.…”

“…cholerae non-O1 non-O139 strains were found to have the features of a genomic island (Fig. 6 ) as described previously 22 , 23 .

Figure 6 Schematic diagram showing the organization of the CRISPR-Cas loci carried by different cholera phages and V .

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“…Moreover, the CRISPR-Cas carried by the V . cholerae non-O1 non-O139 strains were found to be located in a chromosomal island which also carry genes for the Type VI secretion system, as described previously 22 , 23 . The marked difference at the nucleotide level and absence of features associated with horizontal transfer, suggest that its highly unlikely that the phage encoded CRISPR-Cas was derived from that found in the V .…”

Genomics of the Argentinian cholera epidemic elucidate the contrasting dynamics of epidemic and endemic Vibrio cholerae

“…These included the T3SS-2α described in V. cholerae AM_19226 (refs. 50 , 51 ), the less-common T3SS-2β system described by Carpenter et al 51 , and a third putative T3SS system which most closely resembles genes present in the genomes of two virulent Chilean Vibrio anguillarum isolates 52 (Supplementary Fig. 10 ).…”

Pathogenicity island excision during an infection by Salmonella enterica serovar Enteritidis is required for crossing the intestinal epithelial barrier in mice to cause systemic infection

“…This is the case of Salmonella sp (ROD21 and SPI-7) [23], Escherichia coli UPEC (PAI I536, PAI II536, and PAI V536) [36], Klebsiella pneumoniae (GIE492) [37], Vibrio cholera (VPI-2 VSP-I and VSP-I) [38], and Pectobacterium atrosepticum (HAI2) [39]. These islands have significant structural conservation, displaying an unusual G + C profile, are inserted downstream a tRNA gene (usually Asp or Ser), have DRS at the 3´end of the PAI and typically encode paralogs of H-NS (hnsB) and/or an H-NS antagonist [26,40]. These data suggest that excisable PAIs are a conserved cluster of genes related to the family Enterobacteriaceae, and that excision might be an important event related with HGT and/or regulation of gene expression [24,35].…”