MrvR, a Group BStreptococcusTranscription Factor that Controls Multiple Virulence Traits

Abstract

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted knockout of the mrvR gene, which we found to have a growth defect in amniotic fluid relative to the wild type parent strain. In addition to growing poorly in amniotic fluid, the knockout also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that, while the knockout was able to cause persistent murine vaginal colonization, pregnant mice colonized with the knockout strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. Similarly, in a sepsis model in which 87% of mice infected with wild type GBS died within three days, none of the mice infected with the knockout strain died. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed electrophoresis mobility shift assays with recombinant MrvR and whole-genome transcriptomic analysis on the knockout and wild type strains. We show that MrvR binds to its own promoter region, suggesting likely self-regulation. RNA-seq revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting a linkage between purine or pyrimidine availability and activity of MrvR in multiple GBS virulence traits.

Comparative genomics reveals the diversity of CRISPR-Cas systems among neonatal sepsis causing group BStreptococcus agalactiae

“…Cas9 was found in all 51 genomes. The recent studies indicates that Type II CRISPR-associated protein 9 (cas9) influenced virulence in GBS strains [28,29]. The virulence factors of GBS have been implicated in vaginal colonization and invasive disease through Cas9 based regulators [29].None of the genomes contained any transposon or retrotransposon elements in the CRISPR loci.…”

Experimental and Analytical Approaches for Improving the Resolution of Randomly Barcoded Transposon Insertion Sequencing (RB-TnSeq) Studies

“…T ransposon insertion sequencing (TnSeq) is a widely used systems biology tool that combines the broad genomic modification capability of transposons with the throughput power of next generation sequencing to characterize gene function, 1 as employed for catabolism, 2 virulence, 3,4 stress resistance, 5,6 and environmental adaptation. 7,8 When the transposon insertion density sufficiently saturates the genome, genes contributing to function(s) of interest are identified by enumerating strains before and after a desired growth selection.…”