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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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Electron microscopy imaging of NEM316 WT, ΔgbcO mutant, and complemented strains.Bacteria were harvested in mid-log phase (OD600 nm = 0.5), fixed, and prepared as described in Supporting Materials and Methods (see Text S1) (A) Representative views of scanning electron microcopy analysis illustrating the morphological alterations (size, form, and cell division abnormalities) due to gbcO inactivation. (B, C) Transmission electron microscopy views of uranyl acetate stained thin cryosections at two magnifications (see scale bars). The presence of the pellicle (electron dense outer layer) at the surface of WT and complemented strains observed at the higher magnification is highlighted with black arrows. An open triangle depicts the equatorial ring (EqR), a zone of active peptidoglycan synthesis seen in almost all WT and complemented cells but absent in the ΔgbcO mutant cells.
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ppat-1002756-g006: Electron microscopy imaging of NEM316 WT, ΔgbcO mutant, and complemented strains.Bacteria were harvested in mid-log phase (OD600 nm = 0.5), fixed, and prepared as described in Supporting Materials and Methods (see Text S1) (A) Representative views of scanning electron microcopy analysis illustrating the morphological alterations (size, form, and cell division abnormalities) due to gbcO inactivation. (B, C) Transmission electron microscopy views of uranyl acetate stained thin cryosections at two magnifications (see scale bars). The presence of the pellicle (electron dense outer layer) at the surface of WT and complemented strains observed at the higher magnification is highlighted with black arrows. An open triangle depicts the equatorial ring (EqR), a zone of active peptidoglycan synthesis seen in almost all WT and complemented cells but absent in the ΔgbcO mutant cells.

Mentions: In standing cultures, the ΔgbcO mutant strain tends to flocculate rapidly (Figure 5A) and phase contrast microscopy observations revealed the presence of large cellular aggregates instead of small typical chains of ovococci (Figure 5B). A careful examination of ΔgbcO cell clusters (Figure 5B) suggested that they each originated from the folding of a unique chain. To confirm this observation, we followed the growth of GBS ΔgbcO mutant cells in time-lapse experiments under light microscopy. This experiment revealed that clusters of ΔgbcO mutant cells arose from the growth of a single chain that does not break whereas the WT strain forms short individual chains (see Video S1 for NEM316 WT and Video S2 for ΔgbcO in supporting information). This observation indicated that the cell separation process of S. agalactiae was strongly altered in the absence of GBC. The cell and chain morphology of WT, mutant, and complemented strains were then examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As expected, NEM316 WT and complemented strains cells displayed regular size and were assembled in typical ovococci chains, with septa formed in successive parallel planes perpendicular to the chain axis (see Figure 6A, 6B and Figure S3) [27], [28]. By contrast, no regular pattern of division can be observed for the ΔgbcO mutant: cells were heterogeneous both in size and form and the septa localization seemed to occur randomly (Figure 6A 6B; Figure S3). Furthermore, in the mutant strain, the septation process was incomplete and cells were poorly individualized explaining the abnormal growth mode observed in time-lapse experiments.


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)

Electron microscopy imaging of NEM316 WT, ΔgbcO mutant, and complemented strains.Bacteria were harvested in mid-log phase (OD600 nm = 0.5), fixed, and prepared as described in Supporting Materials and Methods (see Text S1) (A) Representative views of scanning electron microcopy analysis illustrating the morphological alterations (size, form, and cell division abnormalities) due to gbcO inactivation. (B, C) Transmission electron microscopy views of uranyl acetate stained thin cryosections at two magnifications (see scale bars). The presence of the pellicle (electron dense outer layer) at the surface of WT and complemented strains observed at the higher magnification is highlighted with black arrows. An open triangle depicts the equatorial ring (EqR), a zone of active peptidoglycan synthesis seen in almost all WT and complemented cells but absent in the ΔgbcO mutant cells.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002756-g006: Electron microscopy imaging of NEM316 WT, ΔgbcO mutant, and complemented strains.Bacteria were harvested in mid-log phase (OD600 nm = 0.5), fixed, and prepared as described in Supporting Materials and Methods (see Text S1) (A) Representative views of scanning electron microcopy analysis illustrating the morphological alterations (size, form, and cell division abnormalities) due to gbcO inactivation. (B, C) Transmission electron microscopy views of uranyl acetate stained thin cryosections at two magnifications (see scale bars). The presence of the pellicle (electron dense outer layer) at the surface of WT and complemented strains observed at the higher magnification is highlighted with black arrows. An open triangle depicts the equatorial ring (EqR), a zone of active peptidoglycan synthesis seen in almost all WT and complemented cells but absent in the ΔgbcO mutant cells.
Mentions: In standing cultures, the ΔgbcO mutant strain tends to flocculate rapidly (Figure 5A) and phase contrast microscopy observations revealed the presence of large cellular aggregates instead of small typical chains of ovococci (Figure 5B). A careful examination of ΔgbcO cell clusters (Figure 5B) suggested that they each originated from the folding of a unique chain. To confirm this observation, we followed the growth of GBS ΔgbcO mutant cells in time-lapse experiments under light microscopy. This experiment revealed that clusters of ΔgbcO mutant cells arose from the growth of a single chain that does not break whereas the WT strain forms short individual chains (see Video S1 for NEM316 WT and Video S2 for ΔgbcO in supporting information). This observation indicated that the cell separation process of S. agalactiae was strongly altered in the absence of GBC. The cell and chain morphology of WT, mutant, and complemented strains were then examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As expected, NEM316 WT and complemented strains cells displayed regular size and were assembled in typical ovococci chains, with septa formed in successive parallel planes perpendicular to the chain axis (see Figure 6A, 6B and Figure S3) [27], [28]. By contrast, no regular pattern of division can be observed for the ΔgbcO mutant: cells were heterogeneous both in size and form and the septa localization seemed to occur randomly (Figure 6A 6B; Figure S3). Furthermore, in the mutant strain, the septation process was incomplete and cells were poorly individualized explaining the abnormal growth mode observed in time-lapse experiments.

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