Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription 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 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.

Bacterial and Host Determinants of Group B Streptococcal Vaginal Colonization and Ascending Infection in Pregnancy

“…MrvR; deletion of mrvR reduced GBS survival in amniotic fluid and impaired biofilm formation. Pregnant mice vaginally-infected by MrvRnull GBS exhibited normal vaginal persistence and consistent ascending infection, but these mice did not experience PTB (Dammann et al, 2021), suggesting that MrvR may mainly promote bacterial survival in vivo.…”

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

“…Native CRISPR-Cas systems can be predicted from the sequenced genome or proteome using computer software ( Couvin et al, 2018 ; Chai et al, 2019 ), aiding in CRISPRi/a tool selection for novel strains. Alternatively, the native system can be engineered to create a CRISPRi/a tool via genetic manipulation, such as the deletion of the native cas2/3 or cas3 gene responsible for cleavage in Type I-F systems ( Zheng et al, 2019 ; Qin et al, 2021 ; Xu et al, 2021 ) or mutating the native cas9 sequence for Type II systems ( Shields et al, 2020 ; Dammann et al, 2021 ). Anti-CRISPR proteins, which inhibit CRISPR systems through a variety of mechanisms ( Pawluk et al, 2018 ), may require deletion or disruption before a heterologous CRISPRi/a tool can be expressed ( Xu et al, 2021 ).…”

Functional Genomics Identified Novel Genes Involved in Growth at Low Temperatures in Listeria monocytogenes

“…A previous study identified the nucleotide excision repair gene uvrA as the genetic determinant of desiccation tolerance of the Streptococcus pneumoniae by Tn-seq ( 18 ). Moreover, five novel Streptococcus agalactiae genes that promote survival in the presence of human amniotic fluid were identified by Tn-seq ( 19 ).…”