Article Figures & Data
Figures
- FIG 1
(A) Schematic diagram of LytTR-containing two-component system transcriptional regulator proteins. Numbers above the diagram correspond to the amino acid positions. The percent identity of the primary amino acid sequences of the Rr2 and RgfA REC and LytTR domains to LtdR is indicated. (B) Schematic of the ltdR-, Rr2-, and rgfA-containing gene loci. Gene locus tags published in the NCBI reference sequence with GenBank accession number NZ_HG939456.1 are indicated above the gene annotations.
- FIG 2
(A and B) Growth curves for WT GBS and the ΔltdR mutant in THB (A) and CDM (B) at 37°C. (C to E) Flow cytometry using serial dilutions of a monoclonal antibody (MAb) to the serotype III capsule to determine the presence of capsule in WT GBS (C) and the ΔltdR mutant (D). A monoclonal antibody to the serotype Ia capsule was used as the isotype control. (F) Hemolysis assay comparing the hemolysis of sheep blood cells by WT GBS and the ΔltdR mutant. Representative data from 1 of at least 2 independent experiments are shown. (G and H) Scanning electron microscopy images of WT GBS (G) and ΔltdR mutant (H) strains. (I) Aggregation assay comparing aggregation of WT GBS, the ΔltdR mutant, and the complemented strain in THB. (J) Clumping assay comparing clumping of the WT GBS, the ΔltdR mutant, and the complemented strain in THB containing 0.1% fibrinogen. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
- FIG 3
Mouse model of GBS meningitis. (A to C) At 72 h after infection, mice were euthanized and bacterial loads in the brain (A), blood (B), and lung (C) were assessed. (D to F) Representative images of hematoxylin-eosin-stained brain sections from mice inoculated with the WT (D) or the ΔltdR mutant (E and F) GBS strain. Arrows, areas of neutrophil infiltration and meningeal thickening. Representative data from 1 of 3 independent experiments are shown. *, P < 0.05.
- FIG 4
LtdR regulation influences GBS invasion into the brain endothelium. (A and B) Transmission electron micrographs of hBMEC infected with WT (A) or ΔltdR mutant (B) GBS. (C) Invasion of WT GBS, the ΔltdR mutant, and the complemented strain into hCMEC was quantified after a 2-h infection. (D) Adherence of WT GBS and the ΔltdR mutant strain to hCMEC was assessed after a 30-min incubation. (E) The intracellular survival of WT GBS and the ΔltdR mutant strain relative to that of the WT and the ΔltdR mutant at 2 h postinfection was determined up to 8 h postinfection. Experiments were performed at least 3 times in triplicate, and error bars represent SDs; the results of a representative experiment are shown. *, P < 0.05; **, P < 0.005; n.s., not significant.
- FIG 5
LtdR impacts cytokine expression by infected hCMEC. (A to C) hCMEC were infected with GBS for 5 h, and then the cells were collected and the transcript levels of IL-8 (A), CXCL-1 (B), and IL-6 (C) were quantified by RT-qPCR. (D to F) The hCMEC supernatant was collected for detection of IL-8 (D), CXCL-1 (E), and IL-6 (F) protein secretion during GBS infection. Experiments were performed at least 3 times in triplicate, and error bars represent SDs; the results of a representative experiment are shown. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005.
- FIG 6
LtdR plays a role in GBS persistence and inflammation in the vaginal tract. (A) Murine vaginal colonization model. Mice were inoculated with either the WT or the ΔltdR mutant GBS strain, and the bacterial load was monitored daily. (B) Invasion of WT GBS, the ΔltdR mutant, and the complemented strain into hVEC was quantified after a 2-h infection. (C) The adherence of WT GBS and the ΔltdR mutant was assessed after a 30-min incubation. (D to F) hVEC were infected with GBS for 5 h, and then the transcript levels of IL-8 (D), CXCL-1 (E), and IL-6 (F) were assessed by RT-qPCR. (G to I) An ELISA to quantify the IL-8 (G), CXCL-1 (H), and IL-6 (I) secreted by hVEC was performed following a 5-h infection with GBS strains. Experiments were performed at least 3 times in triplicate, and error bars represent SDs; the results of a representative experiment are shown. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005; n.s., not significant.
- FIG 7
RNA sequencing to identify LtdR-regulated processes. (A to C) MA plots highlighting genes differentially expressed between WT GBS and the ΔltdR mutant at different growth phases. Significantly differentially expressed genes (adjusted P < 0.1) are indicated in red. An OD600 of 0.2 (A), an OD600 of 0.5 (B), and an OD600 of 1.0 (C) correspond to late lag phase, exponential phase, and early stationary phase, respectively. (D and E) Venn diagrams of genes expressed at significantly (adjusted P < 0.05) higher levels in WT GBS than in the ΔltdR mutant (D) and transcripts that were present at significantly larger amounts in the ΔltdR mutant strain than in the WT (E). (F) Significantly differentially expressed genes (adjusted P < 0.05) were classified according to the cluster of orthologous groups (COG) of genes.
Additional Files
Supplemental material
- Supplemental file 1 -
Table S1. Summary of significantly differentially expressed genes.
XLSX, 54K
- Supplemental file 2 -
Table S2. Results of RT-qPCR to confirm select RNA sequencing hits.
XLSX, 56K
- Supplemental file 3 -
Fig. S1. RT-qPCR to compare gene expression between WT, the ΔltdR mutant, and the complemented strains.
PDF, 407K
- Supplemental file 4 -
Legend for Fig. S1. Captions for Tables S1 to S3.
PDF, 51K
- Supplemental file 5 -
Table S3. Primer sequences used for RT-qPCR to validate GBS RNA sequencing hits.
XLSX, 26K
- Supplemental file 1 -
