Figure 2. HvgA is a cell surface protein of GBS ST-17. (a) Structure of the bibA/hvgA locus in GBS strains NEM316 (WT ST-23) and BM110 (WT ST-17). Comparison of the nucleotide sequences of the two loci revealed that only the 5′ and 3′ ends of the two genes were highly conserved, displaying >90% sequence identity, whereas their internal parts displayed low-level (50–60%) or no significant (<20%) sequence identity. The positions of the primers used to carry out in-frame deletion within bibA and hvgA (O1-O2 plus O3-O4), or to clone hvgA (O5-O6), are depicted by small vertical arrows. P, promoter; ter, terminator. The CovR binding site is depicted by a blue box. The pairwise local alignment was performed with LFasta and visualize with LalnView (http://pbil.univ-lyon1.fr/lfasta.php). (b) Western-blot analysis of cell wall–anchored proteins of GBS with anti-HvgA antiserum. Surface proteins extracted by mutanolysin (CW) or hot SDS treatment from GBS BM110 WT ST-17, its ΔhvgA and ΔsrtA mutants, and the complemented ΔhvgA mutant were separated on 10% tris-glycine SDS-PAGE gels and immunoblotted with a specific anti-HvgA antiserum (protein fragment 30–216). HvgA corresponds to 2 ng of purified recombinant protein extracted from E. coli. (c) Cell surface exposure of HvgA in GBS WT ST-17. Immunofluorescence analysis was performed with rabbit polyclonal anti-HvgA antibodies (protein fragment 30–216) revealed with an anti-IgG coupled to Alexa Fluor 488. Bars, 10 µm. (d) Flow cytometry analysis of GBS WT ST-17 and its ΔhvgA mutant, GBS WT ST-23 and its ΔbibA mutant incubated with a polyclonal anti-HvgA, or anti-BibA (protein fragment 34–295) antibodies and stained with a secondary Alexa Fluor 488–conjugated anti–rabbit IgG antibody (solid line) or with secondary only (dotted line). (e) qRT-PCR analysis of hvgA in GBS BM110 in vitro and in vivo. Values are presented as a ratio of expression in blood, brain, and cecum of infected BALB/c mice relative to expression in TH broth medium. Results shown are representative of two independent experiments performed in triplicate. Error bars are the SD of the depicted variable. *, P < 0.05.
Figure 2.

HvgA is a cell surface protein of GBS ST-17. (a) Structure of the bibA/hvgA locus in GBS strains NEM316 (WT ST-23) and BM110 (WT ST-17). Comparison of the nucleotide sequences of the two loci revealed that only the 5′ and 3′ ends of the two genes were highly conserved, displaying >90% sequence identity, whereas their internal parts displayed low-level (50–60%) or no significant (<20%) sequence identity. The positions of the primers used to carry out in-frame deletion within bibA and hvgA (O1-O2 plus O3-O4), or to clone hvgA (O5-O6), are depicted by small vertical arrows. P, promoter; ter, terminator. The CovR binding site is depicted by a blue box. The pairwise local alignment was performed with LFasta and visualize with LalnView (http://pbil.univ-lyon1.fr/lfasta.php). (b) Western-blot analysis of cell wall–anchored proteins of GBS with anti-HvgA antiserum. Surface proteins extracted by mutanolysin (CW) or hot SDS treatment from GBS BM110 WT ST-17, its ΔhvgA and ΔsrtA mutants, and the complemented ΔhvgA mutant were separated on 10% tris-glycine SDS-PAGE gels and immunoblotted with a specific anti-HvgA antiserum (protein fragment 30–216). HvgA corresponds to 2 ng of purified recombinant protein extracted from E. coli. (c) Cell surface exposure of HvgA in GBS WT ST-17. Immunofluorescence analysis was performed with rabbit polyclonal anti-HvgA antibodies (protein fragment 30–216) revealed with an anti-IgG coupled to Alexa Fluor 488. Bars, 10 µm. (d) Flow cytometry analysis of GBS WT ST-17 and its ΔhvgA mutant, GBS WT ST-23 and its ΔbibA mutant incubated with a polyclonal anti-HvgA, or anti-BibA (protein fragment 34–295) antibodies and stained with a secondary Alexa Fluor 488–conjugated anti–rabbit IgG antibody (solid line) or with secondary only (dotted line). (e) qRT-PCR analysis of hvgA in GBS BM110 in vitro and in vivo. Values are presented as a ratio of expression in blood, brain, and cecum of infected BALB/c mice relative to expression in TH broth medium. Results shown are representative of two independent experiments performed in triplicate. Error bars are the SD of the depicted variable. *, P < 0.05.

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