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Role of the Group B antigen of Streptococcus agalactiae: a peptidoglycan-anchored polysaccharide involved in cell wall biogenesis.

Caliot É, Dramsi S, Chapot-Chartier MP, Courtin P, Kulakauskas S, Péchoux C, Trieu-Cuot P, Mistou MY - PLoS Pathog. (2012)

Bottom Line: Furthermore, vancomycin labeling and peptidoglycan structure analysis demonstrated that, in the absence of GBC, cells failed to initiate normal PG synthesis and cannot complete polymerization of the murein sacculus.Collectively, these findings show that GBC is an essential component of the cell wall of S. agalactiae whose function is reminiscent of that of conventional wall teichoic acids found in Staphylococcus aureus or Bacillus subtilis.Furthermore, our findings raise the possibility that GBC-like molecules play a major role in the growth of most if not all beta-hemolytic streptococci.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité des Bactéries Pathogènes à Gram positif, Paris, France.

ABSTRACT
Streptococcus agalactiae (Group B streptococcus, GBS) is a leading cause of infections in neonates and an emerging pathogen in adults. The Lancefield Group B carbohydrate (GBC) is a peptidoglycan-anchored antigen that defines this species as a Group B Streptococcus. Despite earlier immunological and biochemical characterizations, the function of this abundant glycopolymer has never been addressed experimentally. Here, we inactivated the gene gbcO encoding a putative UDP-N-acetylglucosamine-1-phosphate:lipid phosphate transferase thought to catalyze the first step of GBC synthesis. Indeed, the gbcO mutant was unable to synthesize the GBC polymer, and displayed an important growth defect in vitro. Electron microscopy study of the GBC-depleted strain of S. agalactiae revealed a series of growth-related abnormalities: random placement of septa, defective cell division and separation processes, and aberrant cell morphology. Furthermore, vancomycin labeling and peptidoglycan structure analysis demonstrated that, in the absence of GBC, cells failed to initiate normal PG synthesis and cannot complete polymerization of the murein sacculus. Finally, the subcellular localization of the PG hydrolase PcsB, which has a critical role in cell division of streptococci, was altered in the gbcO mutant. Collectively, these findings show that GBC is an essential component of the cell wall of S. agalactiae whose function is reminiscent of that of conventional wall teichoic acids found in Staphylococcus aureus or Bacillus subtilis. Furthermore, our findings raise the possibility that GBC-like molecules play a major role in the growth of most if not all beta-hemolytic streptococci.

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GbcO is required for GBC synthesis.(A) IFM of bacteria harvested in stationary phase and labeled with anti-GBC serum and Wheat Germ Agglutinin lectin to detect GBC (red) or PG (green), respectively. The representative views show the GBC antigen exposed at the surface of NEM316 WT and complemented (ΔgbcOpTCVΩgbcO) strains but not at the surface of the ΔgbcO mutant. (B) Immunoelectron microscopy (IEM) of NEM316 WT, ΔgbcO mutant and complemented (ΔgbcO pTCVΩgbcO) strains. The subcellular localization of GBC was analyzed using IEM on thin sections (<100 nm) of frozen cells; labelled with anti-GBC serum and revealed with colloidal gold particles (black dots). Black dots are clearly visible on the periphery and septa of cells of WT and complemented strains; no labeling can be detected with the ΔgbcO strain. (C) Immunodetection of GBC in mutanolysin cell wall extracts obtained from cultures harvested in stationary phase. Cell wall extracts were treated with pronase, separated on SDS-PAGE, transferred on nitrocellulose and membrane incubated with anti-GBC serum. In this experiment, the GBC-associated signal appeared as a single band that was undetectable in ΔgbcO cell wall extracts. As a loading control, cell wall extracts before pronase treatment were probed with the biotinylated MalII lectin. (D) Analysis of muramic acid, phosphate, and rhamnose content of the cell wall of WT (black bars), ΔgbcO (light gray bars), and complemented (dark gray bars) strains harvested in stationary phase (see Text S1 in supporting information). For each compound the GC-MS analysis result is presented as a percentage of the WT value. Rhamnose, the main GBC sugar, was not detected in the cell wall of the ΔgbcO strain. Error bars represent ± S.E. of two independent experiments.
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ppat-1002756-g002: GbcO is required for GBC synthesis.(A) IFM of bacteria harvested in stationary phase and labeled with anti-GBC serum and Wheat Germ Agglutinin lectin to detect GBC (red) or PG (green), respectively. The representative views show the GBC antigen exposed at the surface of NEM316 WT and complemented (ΔgbcOpTCVΩgbcO) strains but not at the surface of the ΔgbcO mutant. (B) Immunoelectron microscopy (IEM) of NEM316 WT, ΔgbcO mutant and complemented (ΔgbcO pTCVΩgbcO) strains. The subcellular localization of GBC was analyzed using IEM on thin sections (<100 nm) of frozen cells; labelled with anti-GBC serum and revealed with colloidal gold particles (black dots). Black dots are clearly visible on the periphery and septa of cells of WT and complemented strains; no labeling can be detected with the ΔgbcO strain. (C) Immunodetection of GBC in mutanolysin cell wall extracts obtained from cultures harvested in stationary phase. Cell wall extracts were treated with pronase, separated on SDS-PAGE, transferred on nitrocellulose and membrane incubated with anti-GBC serum. In this experiment, the GBC-associated signal appeared as a single band that was undetectable in ΔgbcO cell wall extracts. As a loading control, cell wall extracts before pronase treatment were probed with the biotinylated MalII lectin. (D) Analysis of muramic acid, phosphate, and rhamnose content of the cell wall of WT (black bars), ΔgbcO (light gray bars), and complemented (dark gray bars) strains harvested in stationary phase (see Text S1 in supporting information). For each compound the GC-MS analysis result is presented as a percentage of the WT value. Rhamnose, the main GBC sugar, was not detected in the cell wall of the ΔgbcO strain. Error bars represent ± S.E. of two independent experiments.

Mentions: The gbcO gene of the S. agalactiae NEM316 wild-type (WT) strain, which encodes a TagO/TarO ortholog (Figure S1), was inactivated to investigate its role in GBC biosynthesis (Figure 1B). As all attempts to construct an in-frame deletion mutant of gbcO were unsuccessful, we deleted it by allelic replacement with a promoter- and terminator-less kanamycin marker [20], [21]. Thanks to the use of this positive selection system, the strain NEM2772 (ΔgbcO) bearing an inactivated gbcO gene was isolated and a complemented strain (ΔgbcOpTCVΩgbcO) was constructed by re-introducing a functional gbcO gene cloned onto a low-copy-number plasmid. To validate the role of gbcO in the biosynthesis of GBC, S. agalactiae NEM316 WT, the isogenic ΔgbcO mutant, and the complemented strains were probed with a rabbit anti-GBC polyclonal antibody [22]. Immunofluorescence microscopy (IFM) analysis using Wheat Germ Agglutinin to label the whole bacteria and specific GBC antiserum revealed the presence of GBC at the surface of WT strain and its absence in the ΔgbcO mutant (Figure 2A). This defect was complemented in the ΔgbcO mutant transformed with the pTCVΩgbcO plasmid. This result demonstrates that gbcO restores the exposure of GBC at the bacterial surface. Quantification of the immunofluorescence data by flow cytometry using simple immunolabeling with the anti-GBC serum indicated no significant differences between wild-type and complemented strains (data not shown).


Role of the Group B antigen of Streptococcus agalactiae: a peptidoglycan-anchored polysaccharide involved in cell wall biogenesis.

Caliot É, Dramsi S, Chapot-Chartier MP, Courtin P, Kulakauskas S, Péchoux C, Trieu-Cuot P, Mistou MY - PLoS Pathog. (2012)

GbcO is required for GBC synthesis.(A) IFM of bacteria harvested in stationary phase and labeled with anti-GBC serum and Wheat Germ Agglutinin lectin to detect GBC (red) or PG (green), respectively. The representative views show the GBC antigen exposed at the surface of NEM316 WT and complemented (ΔgbcOpTCVΩgbcO) strains but not at the surface of the ΔgbcO mutant. (B) Immunoelectron microscopy (IEM) of NEM316 WT, ΔgbcO mutant and complemented (ΔgbcO pTCVΩgbcO) strains. The subcellular localization of GBC was analyzed using IEM on thin sections (<100 nm) of frozen cells; labelled with anti-GBC serum and revealed with colloidal gold particles (black dots). Black dots are clearly visible on the periphery and septa of cells of WT and complemented strains; no labeling can be detected with the ΔgbcO strain. (C) Immunodetection of GBC in mutanolysin cell wall extracts obtained from cultures harvested in stationary phase. Cell wall extracts were treated with pronase, separated on SDS-PAGE, transferred on nitrocellulose and membrane incubated with anti-GBC serum. In this experiment, the GBC-associated signal appeared as a single band that was undetectable in ΔgbcO cell wall extracts. As a loading control, cell wall extracts before pronase treatment were probed with the biotinylated MalII lectin. (D) Analysis of muramic acid, phosphate, and rhamnose content of the cell wall of WT (black bars), ΔgbcO (light gray bars), and complemented (dark gray bars) strains harvested in stationary phase (see Text S1 in supporting information). For each compound the GC-MS analysis result is presented as a percentage of the WT value. Rhamnose, the main GBC sugar, was not detected in the cell wall of the ΔgbcO strain. Error bars represent ± S.E. of two independent experiments.
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Related In: Results  -  Collection

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ppat-1002756-g002: GbcO is required for GBC synthesis.(A) IFM of bacteria harvested in stationary phase and labeled with anti-GBC serum and Wheat Germ Agglutinin lectin to detect GBC (red) or PG (green), respectively. The representative views show the GBC antigen exposed at the surface of NEM316 WT and complemented (ΔgbcOpTCVΩgbcO) strains but not at the surface of the ΔgbcO mutant. (B) Immunoelectron microscopy (IEM) of NEM316 WT, ΔgbcO mutant and complemented (ΔgbcO pTCVΩgbcO) strains. The subcellular localization of GBC was analyzed using IEM on thin sections (<100 nm) of frozen cells; labelled with anti-GBC serum and revealed with colloidal gold particles (black dots). Black dots are clearly visible on the periphery and septa of cells of WT and complemented strains; no labeling can be detected with the ΔgbcO strain. (C) Immunodetection of GBC in mutanolysin cell wall extracts obtained from cultures harvested in stationary phase. Cell wall extracts were treated with pronase, separated on SDS-PAGE, transferred on nitrocellulose and membrane incubated with anti-GBC serum. In this experiment, the GBC-associated signal appeared as a single band that was undetectable in ΔgbcO cell wall extracts. As a loading control, cell wall extracts before pronase treatment were probed with the biotinylated MalII lectin. (D) Analysis of muramic acid, phosphate, and rhamnose content of the cell wall of WT (black bars), ΔgbcO (light gray bars), and complemented (dark gray bars) strains harvested in stationary phase (see Text S1 in supporting information). For each compound the GC-MS analysis result is presented as a percentage of the WT value. Rhamnose, the main GBC sugar, was not detected in the cell wall of the ΔgbcO strain. Error bars represent ± S.E. of two independent experiments.
Mentions: The gbcO gene of the S. agalactiae NEM316 wild-type (WT) strain, which encodes a TagO/TarO ortholog (Figure S1), was inactivated to investigate its role in GBC biosynthesis (Figure 1B). As all attempts to construct an in-frame deletion mutant of gbcO were unsuccessful, we deleted it by allelic replacement with a promoter- and terminator-less kanamycin marker [20], [21]. Thanks to the use of this positive selection system, the strain NEM2772 (ΔgbcO) bearing an inactivated gbcO gene was isolated and a complemented strain (ΔgbcOpTCVΩgbcO) was constructed by re-introducing a functional gbcO gene cloned onto a low-copy-number plasmid. To validate the role of gbcO in the biosynthesis of GBC, S. agalactiae NEM316 WT, the isogenic ΔgbcO mutant, and the complemented strains were probed with a rabbit anti-GBC polyclonal antibody [22]. Immunofluorescence microscopy (IFM) analysis using Wheat Germ Agglutinin to label the whole bacteria and specific GBC antiserum revealed the presence of GBC at the surface of WT strain and its absence in the ΔgbcO mutant (Figure 2A). This defect was complemented in the ΔgbcO mutant transformed with the pTCVΩgbcO plasmid. This result demonstrates that gbcO restores the exposure of GBC at the bacterial surface. Quantification of the immunofluorescence data by flow cytometry using simple immunolabeling with the anti-GBC serum indicated no significant differences between wild-type and complemented strains (data not shown).

Bottom Line: Furthermore, vancomycin labeling and peptidoglycan structure analysis demonstrated that, in the absence of GBC, cells failed to initiate normal PG synthesis and cannot complete polymerization of the murein sacculus.Collectively, these findings show that GBC is an essential component of the cell wall of S. agalactiae whose function is reminiscent of that of conventional wall teichoic acids found in Staphylococcus aureus or Bacillus subtilis.Furthermore, our findings raise the possibility that GBC-like molecules play a major role in the growth of most if not all beta-hemolytic streptococci.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité des Bactéries Pathogènes à Gram positif, Paris, France.

ABSTRACT
Streptococcus agalactiae (Group B streptococcus, GBS) is a leading cause of infections in neonates and an emerging pathogen in adults. The Lancefield Group B carbohydrate (GBC) is a peptidoglycan-anchored antigen that defines this species as a Group B Streptococcus. Despite earlier immunological and biochemical characterizations, the function of this abundant glycopolymer has never been addressed experimentally. Here, we inactivated the gene gbcO encoding a putative UDP-N-acetylglucosamine-1-phosphate:lipid phosphate transferase thought to catalyze the first step of GBC synthesis. Indeed, the gbcO mutant was unable to synthesize the GBC polymer, and displayed an important growth defect in vitro. Electron microscopy study of the GBC-depleted strain of S. agalactiae revealed a series of growth-related abnormalities: random placement of septa, defective cell division and separation processes, and aberrant cell morphology. Furthermore, vancomycin labeling and peptidoglycan structure analysis demonstrated that, in the absence of GBC, cells failed to initiate normal PG synthesis and cannot complete polymerization of the murein sacculus. Finally, the subcellular localization of the PG hydrolase PcsB, which has a critical role in cell division of streptococci, was altered in the gbcO mutant. Collectively, these findings show that GBC is an essential component of the cell wall of S. agalactiae whose function is reminiscent of that of conventional wall teichoic acids found in Staphylococcus aureus or Bacillus subtilis. Furthermore, our findings raise the possibility that GBC-like molecules play a major role in the growth of most if not all beta-hemolytic streptococci.

Show MeSH
Related in: MedlinePlus