Anne-Claire Coûté-Monvoisin

Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.

CRISPR (clustered regularly interspaced short palindromic repeats) together with CAS (RISPR-associated) genes form the CRISPR-Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel 'spacers' into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR-Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.

OBJECTIVES: Vasoactive intestinal peptide (VIP) is an immunomodulatory neuropeptide with therapeutic properties in multiple murine models of inflammatory disease including the trinitrobenzene-sulfonic acid (TNBS)-colitis model of Crohn's disease. Understanding the spectrum of biological actions of endogenously produced VIP may help us dissect the complex and multifactorial pathogenesis of such inflammatory diseases. Our goal was to determine the contribution of endogenously produced VIP to TNBS-colitis by using VIP knockout (KO) mice. METHODS: TNBS was intracolonically administered to wild-type (WT) and VIP KO mice, and weight loss and colitis were assessed over time. Colon histopathological changes and myeloperoxidase activities were analyzed and the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in colon and serum quantified. The proliferative response in vitro of splenocytes from TNBS WT and VIP KO administered mice to anti-CD3 and anti-CD28 was determined. RESULTS: VIP KO mice did not exhibit the predicted exacerbated response to TNBS. Instead, they developed a milder clinical profile than WT mice, with lower TNF-α and IL-6 levels. Such potential defects seem selective, because other parameters such as the histopathological scores and the cytokine levels in the colon did not differ between the two strains of mice. Moreover, splenocytes from TNBS-treated VIP KO mice exhibited an enhanced proliferative response to anti-CD3/CD28 stimulation in vitro. CONCLUSION: Chronic loss of VIP in mice leads to a disruption of certain but not all immunological compartments, corroborating recent findings that VIP KO mice exhibit reduced mortality in the lipopolysaccharide-induced endotoxemia model and attenuated clinical development of experimental autoimmune encephalomyelitis while developing robust T-cell responses.