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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 functionally complement TarO of S. aureus.(A) S. agalactiae ΔgbcO or S. aureus ΔtarO strains does not take Gram staining. In both species, the Gram staining and morphological phenotypes are restored by introduction of the plasmid pTCVΩgbcO carrying a functional S. agalactiae gbcO gene. (B) PAGE analysis of WTA extracted from S. aureus visualized with the alcyan blue-silver staining protocol. The gel shows the production of WTA in RN4220WT (first lane), the absence of WTA in the S. aureus ΔtarO strain (second lane) and the restoration of the WTA synthesis when the tarO deficiency is complemented in trans with the streptococcal gbcO gene (third lane). The arrowhead indicates the bromophenol blue migration front.
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ppat-1002756-g004: GbcO functionally complement TarO of S. aureus.(A) S. agalactiae ΔgbcO or S. aureus ΔtarO strains does not take Gram staining. In both species, the Gram staining and morphological phenotypes are restored by introduction of the plasmid pTCVΩgbcO carrying a functional S. agalactiae gbcO gene. (B) PAGE analysis of WTA extracted from S. aureus visualized with the alcyan blue-silver staining protocol. The gel shows the production of WTA in RN4220WT (first lane), the absence of WTA in the S. aureus ΔtarO strain (second lane) and the restoration of the WTA synthesis when the tarO deficiency is complemented in trans with the streptococcal gbcO gene (third lane). The arrowhead indicates the bromophenol blue migration front.

Mentions: The sequence homology between the streptococcal GbcO and staphylococcal TarO proteins suggests that they catalyze the same enzymatic reaction (Figure S1). To ascertain this hypothesis, the complementing plasmid pTCVΩgbcO was introduced into the S. aureus RN4220ΔtarO mutant. This complementation experiment revealed that the morphological and Gram staining defects of the S. aureus ΔtarO mutant were corrected by expression of the streptococcal gbcO gene (Figure 4A). To prove that the heterologous complementation of ΔtarO was fully functional, we performed the extraction and analysis of WTA in the three staphylococcal strains following established protocols [25], [26]. The results shown in Figure 4B unambiguously demonstrate WTA production in RN4220 WT and complemented ΔtarOpTCVΩgbcO, but not in RN4220ΔtarO mutant. The fact that GbcO can functionally complement TarO provides further support for the hypothesis that GbcO is an UDP-GlcNAC:lipid phosphate transferase. These results demonstrated that, although the cell wall anionic polymers GBC and polyribitol WTA are structurally and genetically unrelated, the first step of their synthesis involves the same enzymatic reaction.


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 functionally complement TarO of S. aureus.(A) S. agalactiae ΔgbcO or S. aureus ΔtarO strains does not take Gram staining. In both species, the Gram staining and morphological phenotypes are restored by introduction of the plasmid pTCVΩgbcO carrying a functional S. agalactiae gbcO gene. (B) PAGE analysis of WTA extracted from S. aureus visualized with the alcyan blue-silver staining protocol. The gel shows the production of WTA in RN4220WT (first lane), the absence of WTA in the S. aureus ΔtarO strain (second lane) and the restoration of the WTA synthesis when the tarO deficiency is complemented in trans with the streptococcal gbcO gene (third lane). The arrowhead indicates the bromophenol blue migration front.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375309&req=5

ppat-1002756-g004: GbcO functionally complement TarO of S. aureus.(A) S. agalactiae ΔgbcO or S. aureus ΔtarO strains does not take Gram staining. In both species, the Gram staining and morphological phenotypes are restored by introduction of the plasmid pTCVΩgbcO carrying a functional S. agalactiae gbcO gene. (B) PAGE analysis of WTA extracted from S. aureus visualized with the alcyan blue-silver staining protocol. The gel shows the production of WTA in RN4220WT (first lane), the absence of WTA in the S. aureus ΔtarO strain (second lane) and the restoration of the WTA synthesis when the tarO deficiency is complemented in trans with the streptococcal gbcO gene (third lane). The arrowhead indicates the bromophenol blue migration front.
Mentions: The sequence homology between the streptococcal GbcO and staphylococcal TarO proteins suggests that they catalyze the same enzymatic reaction (Figure S1). To ascertain this hypothesis, the complementing plasmid pTCVΩgbcO was introduced into the S. aureus RN4220ΔtarO mutant. This complementation experiment revealed that the morphological and Gram staining defects of the S. aureus ΔtarO mutant were corrected by expression of the streptococcal gbcO gene (Figure 4A). To prove that the heterologous complementation of ΔtarO was fully functional, we performed the extraction and analysis of WTA in the three staphylococcal strains following established protocols [25], [26]. The results shown in Figure 4B unambiguously demonstrate WTA production in RN4220 WT and complemented ΔtarOpTCVΩgbcO, but not in RN4220ΔtarO mutant. The fact that GbcO can functionally complement TarO provides further support for the hypothesis that GbcO is an UDP-GlcNAC:lipid phosphate transferase. These results demonstrated that, although the cell wall anionic polymers GBC and polyribitol WTA are structurally and genetically unrelated, the first step of their synthesis involves the same enzymatic reaction.

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