Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages

Abstract

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I-C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I-C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage.

Keywords: CRISPR-Cas; ST-17; Streptococcus agalactiae; phylogeny; typing.

FIGURE 1

Genetic organization of the CRISPR1-cas locus in Streptococcus agalactiae. cas genes and core genes are shown as black arrows and blue arrows, respectively. The leader sequence is located between the cas gene cluster and the CRISPR array (white box; L) while the trailer sequence is located downstream of the array (white box; T). The direct repeats (DR) are shown as black diamonds and the terminal repeat, which differs from the consensus DR, is shown as a white diamond. Spacers are shown as colored rectangles and unique spacers are represented by unique colors. Below the CRISPR array, the sequence of the first two repeat-spacer units is shown with the DRs in black characters and the spacers in color characters.

FIGURE 2

Graphic representation of CRISPR1 loci for 126 S. agalactiae strains. Internal repeats are not included; only terminal repeats (RT), the leader and trailer end sequences (last six nucleotides and first six nucleotide, respectively) and spacers are represented. Each spacer is represented by a combination of one select character in a particular front color, on a particular background color. The color combination allows unique representation of a particular spacer, whereby squares with similar color schemes (combination of character color and background color) represent identical spacers, whereas different color combinations represent distinguishable spacers. Deleted spacers are represented by crossed squares. Strain names, clonal complexes (CC), sequence type (ST), and capsular serotype are given on the left. NCC indicates strains that do not belong to CC according to MLST. NT indicates strains that are not discriminated on the basis of the variability of capsular polysaccharides. Strains were arranged according to the CRISPR1 content. A double line separates CRISPR1 groups. Broken lines separate distinct subgroups in CRISPR1 groups and a continuous line separates NCC strains in CRISPR1 groups.

FIGURE 3

Number of spacers per locus, represented in box plots, among different CC. The red line is the median. The lower end and the upper end of the rectangle represent the first and third quartile, respectively. The blue and green lines represent maximum and minimum values, respectively. The number of spacers in locus CRISPR1 was significantly lower for strains belonging to CC-17 than for others CC (*p < 0.001).