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A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity.

Fedtke I, Mader D, Kohler T, Moll H, Nicholson G, Biswas R, Henseler K, Götz F, Zähringer U, Peschel A - Mol. Microbiol. (2007)

Bottom Line: The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content.However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity.Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Microbiology Division, University of Tübingen, Department of Medical Microbiology and Hygiene, 72076 Tübingen, Germany.

ABSTRACT
Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.

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Related in: MedlinePlus

NMR analyses. A. 1H NMR spectra (600 MHz, D2O, 300 K) of HIC-purified intact LTA. Characteristic resonances for the [Gro-P]n repeating units, fatty acids and Gro-P substituents (d-Ala and α-d-GlcNAc) are indicated. B. 1H NMR spectra (600 MHz, D2O, 300 K) of the putative glycan backbone fragments isolated from HIC-purified LTA after HF treatment, de-O-acylation and GPC. Signals indicated in the glycerol β-gentiobiose pseudotrisaccharide (top) lack in the sample from the S. aureus SA113 ΔypfP::ermB mutant (bottom). Resonances in the region of 3.4–3.9 p.p.m., which appear in both preparations, arise from incompletely degraded LTA repeating units [(Gro-P)n (Gro-P-Ala)n and (Gro-P-GlcNAc)n with n ∼ 1–4] co-eluting in GPC with the glycerol β-gentiobioside.
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fig04: NMR analyses. A. 1H NMR spectra (600 MHz, D2O, 300 K) of HIC-purified intact LTA. Characteristic resonances for the [Gro-P]n repeating units, fatty acids and Gro-P substituents (d-Ala and α-d-GlcNAc) are indicated. B. 1H NMR spectra (600 MHz, D2O, 300 K) of the putative glycan backbone fragments isolated from HIC-purified LTA after HF treatment, de-O-acylation and GPC. Signals indicated in the glycerol β-gentiobiose pseudotrisaccharide (top) lack in the sample from the S. aureus SA113 ΔypfP::ermB mutant (bottom). Resonances in the region of 3.4–3.9 p.p.m., which appear in both preparations, arise from incompletely degraded LTA repeating units [(Gro-P)n (Gro-P-Ala)n and (Gro-P-GlcNAc)n with n ∼ 1–4] co-eluting in GPC with the glycerol β-gentiobioside.

Mentions: 1H nuclear magnetic resonance (NMR) analyses of underivatized and intact LTA preparations from S. aureus SA113 wild-type and ypfP mutant strains were carried out in water (D2O) (Fig. 4A). Diagnostic 1H NMR signals from substituted H-2Gro (5.45 p.p.m.) of the [Gro-P]n repeating unit, as well as resonances assigned to H-1a,bGro, H-3a,bGro (3.7–4.2 p.p.m.), and fatty acids (1.2–0.8 p.p.m.) dominated in the 1H NMR spectrum, which was in very good agreement with data published elsewhere (Morath et al., 2001). In addition to signals from the [Gro-P]n repeating units, resonances assigned to the Gro-P substituents (d-Ala and α-GlcNAc) could also be identified in comparable intensities in both LTA preparations. Our results from the 1H NMR analyses of the intact LTA suggest that both LTA preparations are similar with respect to the structure of the repeating units of the [Gro-P]n as well as to their degree of substitution with d-Ala and α-GlcNAc respectively. Following a previously described 1H NMR approach (Morath et al., 2001), we identified a chain length of n ∼ 18 for the wild-type SA113 and only n ∼ 12 for the ypfP mutant. However, signals of the glycerol β-gentiobiose lipid backbone anchor could not be identified by this method. This is mainly due to limited resolution of the 1H NMR signals of the LTA in water (D2O), in which it is present in micelle-forming aggregates leading to badly resolved proton signals. Moreover, due to the high polymerization degree of the [Gro-P]n repeating units the glyceryl β-gentiobiose moiety represents only ∼3% of the total molecular mass in the intact LTA molecule. This explains why the glycan backbone in the underivatized LTA cannot be analysed by 1H NMR spectroscopy. Therefore, we decided to isolate the target glycan backbone structure of the lipid anchor of LTA of both strains aiming to determine their structure(s) separately and in more detail.


A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity.

Fedtke I, Mader D, Kohler T, Moll H, Nicholson G, Biswas R, Henseler K, Götz F, Zähringer U, Peschel A - Mol. Microbiol. (2007)

NMR analyses. A. 1H NMR spectra (600 MHz, D2O, 300 K) of HIC-purified intact LTA. Characteristic resonances for the [Gro-P]n repeating units, fatty acids and Gro-P substituents (d-Ala and α-d-GlcNAc) are indicated. B. 1H NMR spectra (600 MHz, D2O, 300 K) of the putative glycan backbone fragments isolated from HIC-purified LTA after HF treatment, de-O-acylation and GPC. Signals indicated in the glycerol β-gentiobiose pseudotrisaccharide (top) lack in the sample from the S. aureus SA113 ΔypfP::ermB mutant (bottom). Resonances in the region of 3.4–3.9 p.p.m., which appear in both preparations, arise from incompletely degraded LTA repeating units [(Gro-P)n (Gro-P-Ala)n and (Gro-P-GlcNAc)n with n ∼ 1–4] co-eluting in GPC with the glycerol β-gentiobioside.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169524&req=5

fig04: NMR analyses. A. 1H NMR spectra (600 MHz, D2O, 300 K) of HIC-purified intact LTA. Characteristic resonances for the [Gro-P]n repeating units, fatty acids and Gro-P substituents (d-Ala and α-d-GlcNAc) are indicated. B. 1H NMR spectra (600 MHz, D2O, 300 K) of the putative glycan backbone fragments isolated from HIC-purified LTA after HF treatment, de-O-acylation and GPC. Signals indicated in the glycerol β-gentiobiose pseudotrisaccharide (top) lack in the sample from the S. aureus SA113 ΔypfP::ermB mutant (bottom). Resonances in the region of 3.4–3.9 p.p.m., which appear in both preparations, arise from incompletely degraded LTA repeating units [(Gro-P)n (Gro-P-Ala)n and (Gro-P-GlcNAc)n with n ∼ 1–4] co-eluting in GPC with the glycerol β-gentiobioside.
Mentions: 1H nuclear magnetic resonance (NMR) analyses of underivatized and intact LTA preparations from S. aureus SA113 wild-type and ypfP mutant strains were carried out in water (D2O) (Fig. 4A). Diagnostic 1H NMR signals from substituted H-2Gro (5.45 p.p.m.) of the [Gro-P]n repeating unit, as well as resonances assigned to H-1a,bGro, H-3a,bGro (3.7–4.2 p.p.m.), and fatty acids (1.2–0.8 p.p.m.) dominated in the 1H NMR spectrum, which was in very good agreement with data published elsewhere (Morath et al., 2001). In addition to signals from the [Gro-P]n repeating units, resonances assigned to the Gro-P substituents (d-Ala and α-GlcNAc) could also be identified in comparable intensities in both LTA preparations. Our results from the 1H NMR analyses of the intact LTA suggest that both LTA preparations are similar with respect to the structure of the repeating units of the [Gro-P]n as well as to their degree of substitution with d-Ala and α-GlcNAc respectively. Following a previously described 1H NMR approach (Morath et al., 2001), we identified a chain length of n ∼ 18 for the wild-type SA113 and only n ∼ 12 for the ypfP mutant. However, signals of the glycerol β-gentiobiose lipid backbone anchor could not be identified by this method. This is mainly due to limited resolution of the 1H NMR signals of the LTA in water (D2O), in which it is present in micelle-forming aggregates leading to badly resolved proton signals. Moreover, due to the high polymerization degree of the [Gro-P]n repeating units the glyceryl β-gentiobiose moiety represents only ∼3% of the total molecular mass in the intact LTA molecule. This explains why the glycan backbone in the underivatized LTA cannot be analysed by 1H NMR spectroscopy. Therefore, we decided to isolate the target glycan backbone structure of the lipid anchor of LTA of both strains aiming to determine their structure(s) separately and in more detail.

Bottom Line: The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content.However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity.Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Microbiology Division, University of Tübingen, Department of Medical Microbiology and Hygiene, 72076 Tübingen, Germany.

ABSTRACT
Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.

Show MeSH
Related in: MedlinePlus