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.

Fig 1. Tn-seq screening of GBS grown in human amniotic fluid revealed 5 conditionally essential genes.

GBS transposon library in a background of strain A909 was grown in eight human amniotic fluid samples from three individuals (see main text), then subjected to Tn-seq analysis. Each ring of the Circos plot shows a different representation of the A909 circular chromosome. From the center of the plot, rings 1–8 show transposon insertion density detected in the eight amniotic fluid outgrowth samples (lowest detection: red; highest detection: blue). Ring 9 shows essential genes (red), nonessential genes (green), intermediate genes (yellow), and those for which baseline fitness cannot be determined (white; see reference [41]). The outermost ring shows log₂ fold-change values for each gene in amniotic fluid outgrowth as determined by ESSENTIALS. Genes with inward-pointing peaks had fewer than expected transposon insertions. Outward-pointing peaks had more than expected transposon insertions. The peaks are color coded for statistical significance (low adjusted p value: red; high adjusted p value: green). The five labeled peaks are described in the text and are listed in Table 1. Peak 4 is the GntR-class transcription factor, mrvR.

Fig 2. CRISPRi knockdowns of candidate genes and phenotypic characterization of GBS lacking MrvR in amniotic fluid, rich media, and biofilm-promoting growth conditions.

Two CRISPRi targeting protospacers (targeting nucleotides 112 and 213 of cylx, the first gene in the operon) against the cyl operon were developed to knockdown expression of β-hemolysin/cytolysin. The resultant knockdown strains (in a 10/84 background) were compared to wild type in a human erythrocyte hemolysis assay (A). Targeting protospacers against the five candidate conditionally essential genes from amniotic fluid Tn-seq. For each gene, the number indicates the targeted nucleotide; the protospacer sequence is shown in parentheses (B). A qPCR-based multiplex CRISPRi screen using knockdowns of all five candidate genes revealed decreased fitness in amniotic fluid compared to tryptic soy (TS) broth (C); black columns show the competition index of strains bearing individual targeting protospacer plasmids, while red columns show pooled results from both targeting plasmids for each gene. Statistical significance of each knockdown phenotype was determined relative to growth of the knockdowns in TS broth. Growth curves of the ΔmrvR and wild type (WT) 10/84 in broth (D). Colony forming unit enumeration-based competition assays between ΔmrvR and ΔmrvR+pBSU101::mrvR in TS broth (TSB) and amniotic fluid (AF); E). Biofilm assay results comparing WT 10/84, ΔmrvR, and ΔmrvR+pBSU101::mrvR. (*p<0.05, ** p<0.01, *** p<0.005, **** p<0.001; t test, error bars show standard error surrounding mean).

Fig 3. MrvR does not affect establishment or maintenance of vaginal colonization.

Adult nonpregnant C57/BLJ mice were vaginally colonized with wild type (WT) 10/84 or ΔmrvR. Neither the colonization density at the initial swab 48 hours after inoculation (A; t test) nor duration of colonization (B; Mantel-Cox test) showed significant differences (CFU: colony forming units, ns: not significant).

Fig 4. MrvR has a significant effect on the outcome of chorioamnionitis.

Pregnant BALB/c mice were vaginally colonized with wild type (WT) or knockout (KO) 10/84 on pregnancy day 13. Pregnancies were monitored until day 17 or preterm delivery, whichever came first. On day 17, mice that remained pregnant were dissected and evaluated for vaginal colonization and chorioamnionitis. While none of the KO-colonized mice with chorioamnionitis delivered preterm, 8 of 11 WT-colonized mice delivered early. One WT-colonized mouse did not develop chorioamnionitis (A-B; *** p<0.005; Mantel-Cox test). Dissected pregnancy tissue was homogenized and quantified, showing a difference in invasion density in amniotic fluid (AF) but not in fetal or placental tissue (C, ** p<0.01; t test, error bars show interquartile ranges surrounding medians; ns: not significant).

Fig 5. MrvR affects GBS invasiveness and lethality in a sepsis model.

BALB/c mice were infected intraperitoneally with wild type (WT) 10/84 or ΔmrvR, both bearing a plasmid to allow in vivo tracking of a luciferase signal. There was a significant difference in outcome between the two strains (A, **** p<0.001 Mantel-Cox test). Mice were imaged daily (B) until they either died (red boxes) or completely cleared the luciferase signal (green circles). Mice that cleared the luciferase signal were monitored for the following five days and remained well.

Fig 6. Analysis of the MrvR regulon reveals widespread effects with emphasis on genes involved in nucleotide metabolism.

RNA-seq analysis of wild type (WT) and ΔmrvR 10/84 at three growth timepoints showed extensive regulation across the chromosome in all phases of growth (A, expression log₂ fold-change shown relative to WT). A phage island (Φ) with significant differences from WT is noted, as are the udp gene (stars) and mrvR, which was deleted in the knockout strain. Principal component analysis showed isolated clustering of the late log phase replicates of the knockout strain (B). Gene set enrichment analysis showed that, across all three bacterial growth phases tested, the KEGG pathways most influenced by the presence of MrvR were pyrimidine and purine metabolism (C), which is also shown in volcano plots (D-F). Panel G shows specific genes of the de novo and salvage pathways of pyrimidine metabolism in GBS whose expression is significantly affected by the presence of MrvR (KO = ΔmrvR knockout; EL = early log; LL = late log; S = stationary).