CRISPR Content Correlates with the Pathogenic Potential of Escherichia coli

Abstract

Guide RNA molecules (crRNA) produced from clustered regularly interspaced short palindromic repeat (CRISPR) arrays, altogether with effector proteins (Cas) encoded by cognate cas (CRISPR associated) genes, mount an interference mechanism (CRISPR-Cas) that limits acquisition of foreign DNA in Bacteria and Archaea. The specificity of this action is provided by the repeat intervening spacer carried in the crRNA, which upon hybridization with complementary sequences enables their degradation by a Cas endonuclease. Moreover, CRISPR arrays are dynamic landscapes that may gain new spacers from infecting elements or lose them for example during genome replication. Thus, the spacer content of a strain determines the diversity of sequences that can be targeted by the corresponding CRISPR-Cas system reflecting its functionality. Most Escherichia coli strains possess either type I-E or I-F CRISPR-Cas systems. To evaluate their impact on the pathogenicity of the species, we inferred the pathotype and pathogenic potential of 126 strains of this and other closely related species and analyzed their repeat content. Our results revealed a negative correlation between the number of I-E CRISPR units in this system and the presence of pathogenicity traits: the median number of repeats was 2.5-fold higher for commensal isolates (with 29.5 units, range 0-53) than for pathogenic ones (12.0, range 0-42). Moreover, the higher the number of virulence factors within a strain, the lower the repeat content. Additionally, pathogenic strains of distinct ecological niches (i.e., intestinal or extraintestinal) differ in repeat counts. Altogether, these findings support an evolutionary connection between CRISPR and pathogenicity in E. coli.

Fig 1. Comparison of CRISPR counts and pathogenic categories.

Median numbers of CRISPR2 units in commensal (CEC), enteric (EnPEC) or extraintestinal (ExPEC) pathogens of the E. coli and related strains analyzed in this study, are indicated by a horizontal line. Light grey boxes represent the interquartile range values for the whole set of 126 strains (with 28, 50 and 43 isolates for each group, respectively). Dark grey boxes comprise the interquartile range values for the reduced subset of 71 strains with intact cas I-E genes (22, 35 and 11 isolates). Vertical lines for each box denote the corresponding CRISPR2 count range. Significant differences of median values (Kruskal-Wallis p-values lower than 0.05) for the comparisons within each of these two sets of strains are indicated by an asterisk (ns, not significant).

Fig 2. Correlation of CRISPR counts and pathogenic categories.

Graphical representation of the number of CRISPR repeats in strains categorized as commensal (CEC) or as pathogens of enteric (EnPEC) or extraintestinal (ExPEC) origins for the whole set of N = 126 strains (A) or the 71 strains with the intact set of cas I-E genes (B). Dotted lines represent the least-square linear regressions, and their corresponding R² values are indicated.

Fig 3. Comparison of the CRISPR counts and the number of UPEC genes.

Median numbers of CRISPR2 units in the strains under study, referred to the number of selected uropathogenicity genes within those strains. For each UPEC number category (x-axis), light grey boxes represent the interquartile range for the median value (horizontal line) of all strains (N = 126, with 63, 23, 22, 7 and 11 isolates for each category, respectively), while dark grey boxes indicate that value for strains with complete cas I-E genes (N = 71 and 49, 12, 9, 1 and 0 isolates, respectively). Vertical lines indicate the CRISPR2 count ranges. Significant differences of median values (Kruskal-Wallis p-values lower than 0.05) for the comparisons within each set of strains are indicated by an asterisk (ns, not significant). The categories compared are indicated in brackets, while categories with an insufficient number of isolates are not considered for comparison (see Materials and Methods).

Fig 4. Correlation of CRISPR counts and the number of UPEC genes.

Graphical representation of the number of CRISPR repeats for strains harboring 0, 1, 2, 3 or 4 UPEC factors for the whole set of N = 126 strains (A) or the 71 strains with the intact set of cas I-E genes (B). Dotted lines represent the least-square linear regressions, and their corresponding R² values are indicated.