Biology subjects

Chamby, A. B.

Publications and source records attributed to Chamby, A. B..

2 recordsLinked to original sources

bioRxiv2020

MrvR, a group B Streptococcus transcription factor that controls multiple virulence traits

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.

microbiology↗

bioRxiv2018

A counterselectable sucrose sensitivity marker permits efficient and flexible mutagenesis in Streptococcus agalactiae

Streptococcus agalactiae (group B Streptococcus; GBS) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations such as single base pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrate lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations.\n\nImportanceThe ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single base pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.

microbiology↗