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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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Alterations of the murein sacculus properties and of the PG synthesis in GBC-depleted cells.(A,B) Cell lysis assays: exponentially growing cells were harvested and resuspended in PBS buffer (OD600 nm = 1) containing (A) 1 mg/ml lysozyme or (B) 20 units/ml mutanolysin. Lysis of NEM316 WT (black circles), ΔgbcO mutant (white squares), and ΔgbcOpTCVΩgbcO complemented (black diamonds) strains was recorded spectrophotometrically at 600 nm. Error bars represent ± S.E. of three independent experiments (C) Comparative analysis of the muropeptides resulting from mutanolysin-digested peptidoglycan (see Text S1) of NEM316 WT (upper panel), ΔgbcO (median panel), and complemented (bottom panel) strains. Muropeptides were separated by RP-HPLC and the peaks were collected and analyzed by MALDI-TOF. (D) Fluorescent vancomycin staining of exponentially growing NEM316 WT, ΔgbcO mutant and complemented strains. Fluorescent vancomycin (2 mg/ml) was added to exponential-phase cultures for 10 min at 37°C. Cells were harvested, transferred to glass slides, fixed, and observed by fluorescent microscopy as described in Supporting Materials and Methods (see Text S1).
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ppat-1002756-g007: Alterations of the murein sacculus properties and of the PG synthesis in GBC-depleted cells.(A,B) Cell lysis assays: exponentially growing cells were harvested and resuspended in PBS buffer (OD600 nm = 1) containing (A) 1 mg/ml lysozyme or (B) 20 units/ml mutanolysin. Lysis of NEM316 WT (black circles), ΔgbcO mutant (white squares), and ΔgbcOpTCVΩgbcO complemented (black diamonds) strains was recorded spectrophotometrically at 600 nm. Error bars represent ± S.E. of three independent experiments (C) Comparative analysis of the muropeptides resulting from mutanolysin-digested peptidoglycan (see Text S1) of NEM316 WT (upper panel), ΔgbcO (median panel), and complemented (bottom panel) strains. Muropeptides were separated by RP-HPLC and the peaks were collected and analyzed by MALDI-TOF. (D) Fluorescent vancomycin staining of exponentially growing NEM316 WT, ΔgbcO mutant and complemented strains. Fluorescent vancomycin (2 mg/ml) was added to exponential-phase cultures for 10 min at 37°C. Cells were harvested, transferred to glass slides, fixed, and observed by fluorescent microscopy as described in Supporting Materials and Methods (see Text S1).

Mentions: In line with this last hypothesis, we consistently observed that the GBS ΔgbcO mutant was more susceptible to mutanolysin-induced lysis than WT cells whereas its sensitivity to lysozyme was not affected (Figure 7A and 7B). To test further this hypothesis, RP-HPLC separation of the PG-derived muropeptides from WT, ΔgbcO mutant, and complemented strains was carried out. More than 50 peaks were analyzed by MALDI-TOF mass spectrometry to deduce the structure of the separated muropeptides (Figure 7C). The chromatograms revealed that while the monomeric forms of PG were more abundant in extracts from the mutant strain as compared to the WT (Figure 7Cmiddle row), the amount of the remaining categories (dimers, trimers, and unresolved high MW oligomers) was lower (Figure 7Cupper row). Quantitative analysis of the chromatograms confirmed this observation and revealed that the cross-linking index plummeted from 34% (WT and complemented strains) to 24% (ΔgbcO mutant) (see Table S3 in supporting information file Text S1). The highly cross-linked PG components accumulated in an unresolved peak eluting between 180–220 min. As the area of the peak was strongly reduced in the ΔgbcO mutant, the decrease of the PG cross-linking was likely underestimated.


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)

Alterations of the murein sacculus properties and of the PG synthesis in GBC-depleted cells.(A,B) Cell lysis assays: exponentially growing cells were harvested and resuspended in PBS buffer (OD600 nm = 1) containing (A) 1 mg/ml lysozyme or (B) 20 units/ml mutanolysin. Lysis of NEM316 WT (black circles), ΔgbcO mutant (white squares), and ΔgbcOpTCVΩgbcO complemented (black diamonds) strains was recorded spectrophotometrically at 600 nm. Error bars represent ± S.E. of three independent experiments (C) Comparative analysis of the muropeptides resulting from mutanolysin-digested peptidoglycan (see Text S1) of NEM316 WT (upper panel), ΔgbcO (median panel), and complemented (bottom panel) strains. Muropeptides were separated by RP-HPLC and the peaks were collected and analyzed by MALDI-TOF. (D) Fluorescent vancomycin staining of exponentially growing NEM316 WT, ΔgbcO mutant and complemented strains. Fluorescent vancomycin (2 mg/ml) was added to exponential-phase cultures for 10 min at 37°C. Cells were harvested, transferred to glass slides, fixed, and observed by fluorescent microscopy as described in Supporting Materials and Methods (see Text S1).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375309&req=5

ppat-1002756-g007: Alterations of the murein sacculus properties and of the PG synthesis in GBC-depleted cells.(A,B) Cell lysis assays: exponentially growing cells were harvested and resuspended in PBS buffer (OD600 nm = 1) containing (A) 1 mg/ml lysozyme or (B) 20 units/ml mutanolysin. Lysis of NEM316 WT (black circles), ΔgbcO mutant (white squares), and ΔgbcOpTCVΩgbcO complemented (black diamonds) strains was recorded spectrophotometrically at 600 nm. Error bars represent ± S.E. of three independent experiments (C) Comparative analysis of the muropeptides resulting from mutanolysin-digested peptidoglycan (see Text S1) of NEM316 WT (upper panel), ΔgbcO (median panel), and complemented (bottom panel) strains. Muropeptides were separated by RP-HPLC and the peaks were collected and analyzed by MALDI-TOF. (D) Fluorescent vancomycin staining of exponentially growing NEM316 WT, ΔgbcO mutant and complemented strains. Fluorescent vancomycin (2 mg/ml) was added to exponential-phase cultures for 10 min at 37°C. Cells were harvested, transferred to glass slides, fixed, and observed by fluorescent microscopy as described in Supporting Materials and Methods (see Text S1).
Mentions: In line with this last hypothesis, we consistently observed that the GBS ΔgbcO mutant was more susceptible to mutanolysin-induced lysis than WT cells whereas its sensitivity to lysozyme was not affected (Figure 7A and 7B). To test further this hypothesis, RP-HPLC separation of the PG-derived muropeptides from WT, ΔgbcO mutant, and complemented strains was carried out. More than 50 peaks were analyzed by MALDI-TOF mass spectrometry to deduce the structure of the separated muropeptides (Figure 7C). The chromatograms revealed that while the monomeric forms of PG were more abundant in extracts from the mutant strain as compared to the WT (Figure 7Cmiddle row), the amount of the remaining categories (dimers, trimers, and unresolved high MW oligomers) was lower (Figure 7Cupper row). Quantitative analysis of the chromatograms confirmed this observation and revealed that the cross-linking index plummeted from 34% (WT and complemented strains) to 24% (ΔgbcO mutant) (see Table S3 in supporting information file Text S1). The highly cross-linked PG components accumulated in an unresolved peak eluting between 180–220 min. As the area of the peak was strongly reduced in the ΔgbcO mutant, the decrease of the PG cross-linking was likely underestimated.

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