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 deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion 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. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which 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 that MrvR may be involved in regulating nucleotide availability.
Highlights
Streptococcus agalactiae is a cause of chorioamnionitis, stillbirth, and neonatal infections including bacteremia, pneumonia, and meningitis [1,2,3,4,5,6,7,8,9,10]
Group B Streptococcus (GBS) can survive in human amniotic fluid, which is low in bacterial nutrients and contains immune molecules that limit microbial persistence, and this ability likely contributes to GBS chorioamnionitis
This study is focused on a single GBS gene that encodes a genetic regulator we called MrvR, which we show is important for GBS resistance to human amniotic fluid
Summary
Streptococcus agalactiae (group B Streptococcus; GBS) is a cause of chorioamnionitis, stillbirth, and neonatal infections including bacteremia, pneumonia, and meningitis [1,2,3,4,5,6,7,8,9,10]. Tn-seq permits genome-wide quantification of bacterial gene fitness through highly parallel sequencing of a saturated transposon library in which transposon insertion within a given gene is expected to result in loss of that gene product [43,44,45,46]. By identifying transposon insertion sites that are relatively absent from the experimental outgrowth condition, it is possible to infer which genes (or potentially intergenic regions) are essential for bacterial survival
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