Table 1.
B. pertussis strains used in this study.
Table 2.
List of forward and reverse primers used in cloning.
Table 3.
List of forward and reverse primers (from 5′ to 3′) used in Real-time PCR analysis.
Figure 1.
Relative transcriptional activity of vrgs and vags in BPSM bacteria recovered from mice lungs versus in vitro BPSM grown in virulent phase.
Mice were infected intranasally with approx. 5×106 CFU of BPSM and the bacteria were recovered from the mice lungs at different time points (3 hours, day 3 and day 7 p.i.). Bacterial RNA extracted and purified from group of 4 mice were pooled and subjected to real-time PCR analysis using primers mapping in the kpsT, vrg6, bvgA, fhaB and ptx genes. RecA gene was used as the endogenous control. Results are expressed as the average RQ ± SD of triplicate versus BPSM inoculum. Dotted line represents RQ equal to 1 relative to BPSM inoculum. Mock-infected mice were used as negative control. The data obtained with two biological replicates are shown. Please refer to S1 Table for Pearson correlation scores between the two independent sets.
Figure 2.
Construction of ΔkpsT, ΔkpsE and ΔvipC B. pertussis mutants.
(A) Schematic organization of B. pertussis capsule operon. The capsule operon of B. pertussis BPSM strain regulated under the capsule promoter is as shown in panel a, black cross represents mutational insertion found in the locus. Black, hashed and open arrows represent genes involved in polysaccharide transport, polysaccharide modification/translocation and polysaccharide biosynthesis respectively. Adapted from GeneDB. Dotted triangle in panel b (ΔkpsT), c (ΔkpsE) and d (ΔvipC) indicate site of deletion that render each mutant non-capsulated. The DIG-labeled probe binding region (black rounded arrow), restriction sites and size of restriction-digested chromosomal DNA for Southern blot analysis are as shown. (B) Southern blot analysis. Restriction-digested chromosomal DNA from BPSM and ΔkpsT, ΔkpsE and ΔvipC were electrophoresed, transferred onto a nitrocellulose membrane and hybridized with the DIG-labeled probe (Fig. 1A for probe binding site). Panel a, EcoRI-restricted BPSM and ΔkpsT DNA yielded 2.7-kb and 3.2-kb respectively. Panel b, HindIII-NcoI restricted BPSM and ΔkpsE DNA yielded 4.8-kb and 3.9-kb respectively. Panel c, HindIII-NcoI restricted BPSM and ΔvipC DNA yielded 4.8-kb and 3.4-kb respectively. (C) Transcription efficacy of downstream gene. Total RNA was extracted from exponential SS liquid cultures of BPSM, KOcaps, ΔkpsT, ΔkpsE and ΔvipC was reverse-transcribed followed by PCR amplification using primers specific to the endogenous control gene recA, and primers mapping to the respective downstream region of the deleted ORFs. The KOcaps strain which was deleted for the entire capsule locus was used as a negative control. (D) Growth kinetic profiles. SS liquid medium was inoculated with BPSM (closed circles), ΔkpsT (open circles), ΔkpsE (closed triangles) and ΔvipC (open triangles) at initial OD600 nm of 0.5 at time-point 0 hour. OD600 nm was monitored for 52 hrs incubation at 37°C. The growth kinetics assay was performed twice independently for each strain and each culture condition. The data shown is representative of two independent experiments.
Figure 3.
Phenotypic characterization of the ΔkpsT, ΔkpsE and ΔvipC mutants.
(A) Detection of the polysaccharide capsule at the bacterial surface. Mouse polyclonal anti-Salmonella typhi Vi antigen immune serum was co-incubated with non-permeabilized BPSM, KOcaps, ΔkpsT, ΔkpsE and ΔvipC bacteria strains as indicated in each flow cytometry plot. All bacteria strains were grown in Bvg- phase. Anti-mouse FITC-conjugated IgG was used as secondary antibody. Isotype-matched controls are incubated with an anti-mouse antibody as shown in black histogram. The fluorescent cells were detected by flow cytometry, with 20,000 events counted for each sample. A representative experiment is shown from three independent experiments, with percentage of fluorescent cells indicated in each panel. (B) Lung colonization profiles. Balb/C mice were infected intranasally with 5×105 CFU of B. pertussis BPSM, KOcaps, ΔkpsT, ΔkpsTcom, ΔkpsE and ΔvipC as indicated at each of graph plot. At the indicated time points, four infected mice per group were euthanized and their lungs were harvested, homogenized and plated on blood agar to determine the total number of CFU per lung. The results are expressed as the mean ± SEM of four mice per group. ** p value<0.01 and * p value<0.05 relative to BPSM. Results are representative of two independent experiments.
Figure 4.
Production of bvg-regulated virulence proteins in ΔkpsT mutant.
BPSM, ΔkpsT and ΔkpsTcom strains were exponentially grown in virulent Bvg+ phase. Western blot analysis was performed on 10x concentrated or non-concentrated culture supernatants, and whole cell extracts using (A) anti-FHA, (B) anti-BrkA or (C) anti-PT primary antibodies. Bars at the bottom of each blot represent protein densitometry quantification expressed as percentage of change relative to parental BPSM and was determined from two independent western blot sets. Molecular weights are indicated on the right side. (D) SDS-PAGE and Coomassie blue staining of BPSM, ΔkpsT and ΔkpsTcom whole cell lysate to estimate equal loading of protein content. Asterisks next to the Coomassie-stained bands indicate for loading control estimate. Bars represent protein densitometry quantification expressed as percentage of change relative to BPSM. Molecular weights are indicated on the left side.
Figure 5.
Trancriptional activity in ΔkpsT mutant.
(A) Relative transcriptional activity of vags. Total RNA was extracted from BPSM (solid bars), ΔkpsT (dotted bars) and ΔkpsTcom (stripped bars) strains grown in virulent Bvg+ phase. Real-time PCR analysis was performed using primers mapping in the brkA, ptx, sphB1, fhaB, bvgA and bvgR genes. recA gene was used as the endogenous control. Results are expressed as average relative quantification (RQ) vs wild type BPSM (RQ = 1). Results are representative of 3 independent experiments. (B) Microarray analysis. Total RNA was extracted from BPSM and ΔkpsT strains grown in virulent (Bvg+) phase. Microarray gene expression values were selected based on log2 fold change <-0.8, with adjusted p value<0.01. Results are expressed as average fold change ΔkpsT compared to BPSM, negative value indicates gene repression. Solid bars represent standard deviation of 2 independent experiments.
Figure 6.
Characterization of the BvgS-VFT2-ΔkpsT mutant. (A) Production of bvg-regulated virulence proteins.
BPSM, BvgS-VFT2, ΔkpsT and BvgS-VFT2-ΔkpsT strains were exponentially grown in virulent (Bvg+) and avirulent (Bvg-) phase. Western blot analysis was performed on whole cell extract (panel a) and 10x concentrated (panel b) or non-concentrated (panel c) culture supernatants using anti-BrkA (a), anti-PT (b) or anti-FHA (c) primary antibodies. Bars at the right of the blots represent protein densitometry quantification expressed as percentage of change relative to parental BPSM in Bvg+ phase. Results are representative of three independent experiments. Molecular weights are indicated on the right side. (B) Relative transcriptional activity of vags. Total RNA was extracted from BPSM, BvgS-VFT2, ΔkpsT and BvgS-VFT2-ΔkpsT strains grown in virulent Bvg+ phase. Real-time PCR analysis was performed using primers mapping in the brkA, ptx, and sphB1 genes. recA gene was used as the endogenous control. Results are expressed for each target gene as average fold change ± SD of triplicate Ct values obtained with BvgS-VFT2, ΔkpsT and BvgS-VFT2-ΔkpsT versus the Ct value obtained with BPSM strain. The results are representative of two independent experiments. (C) Lung colonization profile. Balb/C mice were infected intranasally with 5×105 CFU of B. pertussis BvgS-VFT2, ΔkpsT and BvgS-VFT2-ΔkpsT. At the indicated time points, four infected mice per group were euthanized and their lungs were harvested, homogenized and plated on blood agar to determine the total number of CFU per lung. The results are expressed as the mean ± SEM of four mice per group. ** p value<0.01 relative to BPSM. Results are representative of two independent experiments.
Figure 7.
Expression and purification of His-BvgS from B. pertussis strains.
(A) Coomassie blue analysis. 5 mg of solubilize cell lysate harvested from BPSM (untagged control) and BPSH was mixed with Ni-NTA agarose beads prior to loading onto a chromatography column. Lysate input, flow-through and batch eluted fractions were heated to 95°C for 15 min and analyzed under reducing 10% SDS-PAGE and stained with Coomassie blue. Lane IP; Input, FT; Flow through, E1; Eluted fraction 1, E2; Eluted fraction 2, E3; Eluted fraction 3, E4; Eluted fraction 4. Molecular weights are indicated on the right side. (B) Western blot analysis of purified fractions with anti-His and anti-BvgS antibodies. Lane IP; Input, FT; Flow through, E2; Eluted fraction 2, E3; Eluted fraction 3. Molecular weights are indicated on the right side. (C) Detection of BvgS associated oligomers and BvgS monomer. Purified His-BvgS from BPSH cells were mixed with equal volume of Laemmli SDS-PAGE buffer containing either no reducing agent (0% β-mercaptoethanol) or increasing concentrations (2.5% and 5% β-mercaptoethanol). The proteins samples were then subjected to either no heat or heat denaturation at 95°C for 15 min prior to SDS-PAGE analysis and Western blotted with anti-His or anti-BvgS antibody. Molecular weights are indicated on the right side. (D) Detection of BvgS associated oligomers and BvgS monomer in BPSH and BPSH-KOcaps. Equal amount of purified His-BvgS from BPSH and BPSH-KOcaps cells were mixed with Laemmli SDS-PAGE buffer containing either no reducing agent or with 5% β-mercaptoethanol. The proteins samples were then subjected to either no heat or heat denaturation at 95°C for 15 min. Equal amount of protein were loaded for each well for SDS-PAGE analysis and Western blotted with anti-BvgS antibody. Molecular weights are indicated on the right side. (E) Detection of BvgS associated oligomers and BvgS monomer in BPSH, BPSH-ΔkpsT and BPSH-ΔkpsTcom. Equal amount of purified His-BvgS from BPSH, BPSH-ΔkpsT and BPSH-ΔkpsTcom cells were mixed with Laemmli SDS-PAGE buffer containing either no reducing agent or with 5% β-mercaptoethanol. The proteins samples were then subjected to either no heat or heat denaturation at 95°C for 15 min. Equal amounts of protein were loaded for each well for SDS-PAGE analysis and Western blotted with anti-BvgS antibody. Molecular weights are indicated on the right side.
Figure 8.
Lung colonization profile of B. pertussis KOcaps:kpsT and KOcaps:kpsMT strains.
Balb/C mice were infected intranasally with 5×105 CFU of B. pertussis BPSM (solid bars), KOcaps (striped bars), KOcaps:kpsT (dotted bars) and KOcaps:kpsMT (open bars). At the indicated time points, four infected mice per group were euthanized and their lungs were harvested, homogenized and plated on blood agar to determine the total number of CFU per lung. The results are expressed as the mean ± SEM of four mice per group. ** p value<0.01 relative to KOcaps. Results are representative of two independent experiments.