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Evidence for a role of the polysaccharide capsule transport proteins in pertussis pathogenesis.

Hoo R, Lam JH, Huot L, Pant A, Li R, Hot D, Alonso S - PLoS ONE (2014)

Bottom Line: Using a BvgS phase-locked mutant, we demonstrated a functional link between KpsT and BvgA/S-mediated signal transduction.Whereas pull-down assays do not support physical interaction between BvgS sensor and any of the capsule locus encoded proteins, absence of KpsT impaired BvgS oligomerization, necessary for BvgS function.Furthermore, complementation studies indicated that instead of KpsT alone, the entire PS capsule transport machinery spanning the cell envelope likely plays a role in BvgS-mediated signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology Programme, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Life Science #03-05, 28 Medical Drive, Singapore 117597, Singapore.

ABSTRACT
Polysaccharide (PS) capsules are important virulence determinants for many bacterial pathogens. Bordetella pertussis, the agent of whooping cough, produces a surface associated microcapsule but its role in pertussis pathogenesis remained unknown. Here we showed that the B. pertussis capsule locus is expressed in vivo in murine lungs and that absence of the membrane-associated protein KpsT, involved in the transport of the PS polymers across the envelope, but not the surface-exposed PS capsule itself, affects drastically B. pertussis colonization efficacy in mice. Microarray analysis revealed that absence of KpsT in B. pertussis resulted in global down-regulation of gene expression including key virulence genes regulated by BvgA/S, the master two-component system. Using a BvgS phase-locked mutant, we demonstrated a functional link between KpsT and BvgA/S-mediated signal transduction. Whereas pull-down assays do not support physical interaction between BvgS sensor and any of the capsule locus encoded proteins, absence of KpsT impaired BvgS oligomerization, necessary for BvgS function. Furthermore, complementation studies indicated that instead of KpsT alone, the entire PS capsule transport machinery spanning the cell envelope likely plays a role in BvgS-mediated signal transduction. Our work thus provides the first experimental evidence of a role for a virulence-repressed gene in pertussis pathogenesis.

No MeSH data available.


Related in: MedlinePlus

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.
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pone-0115243-g002: 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.

Mentions: To characterize the role of the capsule locus during pertussis infection, non-polar unmarked single gene deletion mutants were constructed by targeting the capsule transport-export and biosynthesis open reading frames (ORFs) in the capsule operon, namely kpsT; which is predicted to encode the putative polysialic acid transport ATP binding protein, kpsE; the putative capsular polysaccharide export inner membrane protein and vipC; the capsular polysaccharide biosynthesis protein as assessed from GeneDB database. The resulting BPSM-derivative knockout mutant strains, designated as ΔkpsT, ΔkpsE and ΔvipC respectively, were screened by PCR and verified by Southern blot analysis (Fig. 2, A and B). Reverse transcription (RT)-PCR performed on purified total RNA from ΔkpsT, ΔkpsE and ΔvipC strains using primers mapping in downstream ORFs of the respective deleted regions confirmed that deletion of these individual ORFs did not lead to polar effects on the transcription of downstream genes within the capsule operon (Fig. 2 C). The KOcaps mutant strain for which the entire capsule operon has been deleted [17] was used as negative control. Furthermore, the ΔkpsT, ΔkpsE and ΔvipC mutant strains displayed hemolytic and domed colony morphology on blood agar plates (data not shown) and in vitro growth kinetics comparable to the parental BPSM strain (Fig. 2 D), implying that these single gene deletions within the capsule locus did not affect the in vitro fitness of the bacteria.


Evidence for a role of the polysaccharide capsule transport proteins in pertussis pathogenesis.

Hoo R, Lam JH, Huot L, Pant A, Li R, Hot D, Alonso S - PLoS ONE (2014)

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.
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pone-0115243-g002: 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.
Mentions: To characterize the role of the capsule locus during pertussis infection, non-polar unmarked single gene deletion mutants were constructed by targeting the capsule transport-export and biosynthesis open reading frames (ORFs) in the capsule operon, namely kpsT; which is predicted to encode the putative polysialic acid transport ATP binding protein, kpsE; the putative capsular polysaccharide export inner membrane protein and vipC; the capsular polysaccharide biosynthesis protein as assessed from GeneDB database. The resulting BPSM-derivative knockout mutant strains, designated as ΔkpsT, ΔkpsE and ΔvipC respectively, were screened by PCR and verified by Southern blot analysis (Fig. 2, A and B). Reverse transcription (RT)-PCR performed on purified total RNA from ΔkpsT, ΔkpsE and ΔvipC strains using primers mapping in downstream ORFs of the respective deleted regions confirmed that deletion of these individual ORFs did not lead to polar effects on the transcription of downstream genes within the capsule operon (Fig. 2 C). The KOcaps mutant strain for which the entire capsule operon has been deleted [17] was used as negative control. Furthermore, the ΔkpsT, ΔkpsE and ΔvipC mutant strains displayed hemolytic and domed colony morphology on blood agar plates (data not shown) and in vitro growth kinetics comparable to the parental BPSM strain (Fig. 2 D), implying that these single gene deletions within the capsule locus did not affect the in vitro fitness of the bacteria.

Bottom Line: Using a BvgS phase-locked mutant, we demonstrated a functional link between KpsT and BvgA/S-mediated signal transduction.Whereas pull-down assays do not support physical interaction between BvgS sensor and any of the capsule locus encoded proteins, absence of KpsT impaired BvgS oligomerization, necessary for BvgS function.Furthermore, complementation studies indicated that instead of KpsT alone, the entire PS capsule transport machinery spanning the cell envelope likely plays a role in BvgS-mediated signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology Programme, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Life Science #03-05, 28 Medical Drive, Singapore 117597, Singapore.

ABSTRACT
Polysaccharide (PS) capsules are important virulence determinants for many bacterial pathogens. Bordetella pertussis, the agent of whooping cough, produces a surface associated microcapsule but its role in pertussis pathogenesis remained unknown. Here we showed that the B. pertussis capsule locus is expressed in vivo in murine lungs and that absence of the membrane-associated protein KpsT, involved in the transport of the PS polymers across the envelope, but not the surface-exposed PS capsule itself, affects drastically B. pertussis colonization efficacy in mice. Microarray analysis revealed that absence of KpsT in B. pertussis resulted in global down-regulation of gene expression including key virulence genes regulated by BvgA/S, the master two-component system. Using a BvgS phase-locked mutant, we demonstrated a functional link between KpsT and BvgA/S-mediated signal transduction. Whereas pull-down assays do not support physical interaction between BvgS sensor and any of the capsule locus encoded proteins, absence of KpsT impaired BvgS oligomerization, necessary for BvgS function. Furthermore, complementation studies indicated that instead of KpsT alone, the entire PS capsule transport machinery spanning the cell envelope likely plays a role in BvgS-mediated signal transduction. Our work thus provides the first experimental evidence of a role for a virulence-repressed gene in pertussis pathogenesis.

No MeSH data available.


Related in: MedlinePlus