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Journal of Bacteriology, January 2007, p. 280-283, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01221-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Influence of Wall Teichoic Acid on Lysozyme Resistance in Staphylococcus aureus{triangledown}

Agnieszka Bera,1,{dagger} Raja Biswas,1,{dagger} Silvia Herbert,1 Emir Kulauzovic,2 Christopher Weidenmaier,2 Andreas Peschel,2 and Friedrich Götz1*

Microbial Genetics, University of Tübingen, 72076 Tübingen, Germany,1 Cellular and Molecular Microbiology Division, Medical Microbiology and Hygiene Institute, University of Tübingen, 72076 Tübingen, Germany2

Received 4 August 2006/ Accepted 20 October 2006


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ABSTRACT
 
Staphylococcus aureus peptidoglycan (PG) is completely resistant to the hydrolytic activity of lysozyme. Here we show that modifications in PG by O acetylation, wall teichoic acid, and a high degree of cross-linking contribute to this resistance.


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TEXT
 
The human defense system uses a variety of factors to destroy bacteria that include reactive oxygen substances (7), bacteriolytic enzymes (lysozyme [13] and phospholipase A2 [17]), the complement system, and antimicrobial peptides (9). One important and widespread defense enzyme is lysozyme (5, 10), a component of both phagocytic and secretory granules of neutrophils, monocytes, macrophages, and epithelial cells (9, 13). Pathogenic bacteria, such as Staphylococcus aureus, express a wide variety of virulence factors that enable the organism to cause acute and chronic infections (1, 15). Host-colonizing bacteria must have developed mechanisms to overcome the adaptive and innate immune system. As recently shown, S. aureus is completely resistant to lysozyme, and the primary mechanism for this resistance is the modification of its peptidoglycan (PG) by O acetylation at the C-6 position of the N-acetyl muramic acid (NAM) (3). However, in S. aureus the C-6 position of NAM also carries phosphoester-linked wall teichoic acid (WTA) (19, 21) (Fig. 1), and the question is whether WTA also contributes to lysozyme resistance. Our results indicate that WTA and the degree of PG cross-linking indeed contribute to resistance against the hydrolytic activity of lysozyme.


Figure 1
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  		FIG. 1. Possible modifications at the C-6 position of the NAM in S. aureus PG. The C-6 position of NAM can be unmodified or O acetylated by OatA (every second residue [3]) or carries phosphoester-linked WTA in every ninth residue (14). WTA is composed of three glycerol phosphate and 40 ribitol phosphate units that are modified with D-alanine ester and N-acetylglucosaminyl residues. Lysozyme cleaves the ß-1,4-glycosidic bond between NAM and N-acetylglucosamine (NAG).

Characterization of the double mutant SA{Delta}oatA/tagO. In order to analyze the role of WTA of S. aureus SA113 in lysozyme resistance, we tested a tagO deletion mutant (SA{Delta}tagO::erm) that is completely devoid of WTA (21). Surprisingly, this mutant turned out to be as resistant to lysozyme as the wild type (WT) (Fig. 2). This was astonishing insofar as WTA is a much more bulky residue than the O-acetyl group and should therefore hinder the interaction of lysozyme with PG more efficiently. Therefore, we created a double-deletion mutant (SA{Delta}oatA/tagO) that lacked both WTA and O acetylation. Gene replacement of oatA in the mutant SA{Delta}tagO::erm was performed as described before (6). As a control, the oatA gene in the double mutant SA{Delta}oatA/tagO was complemented with the plasmid pRBoatA (3); the complemented mutant restored the lysozyme-resistant phenotype. In liquid medium (B medium or tryptic soy broth), growth of the wild type and that of the tagO mutant were not inhibited at lysozyme concentrations of >50 mg per ml, growth of the oatA mutant was inhibited by 1 mg per ml, and the double mutant SA{Delta}oatA/tagO showed the highest lysozyme sensitivity (<0.05 mg per ml); the MIC of this mutant was 20-fold lower than that of the mutant SA{Delta}tagO. The increase in the lysozyme inhibition zone of SA{Delta}oatA/tagO over that of SA{Delta}oatA is illustrated in Fig. 2.


Figure 2
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  		FIG. 2. Lysozyme sensitivity by agar diffusion assay. Four milligrams (upper four wells) or only 1 mg (lower row) of lysozyme (L) was applied. Note that the tagO mutant is completely insensitive to lysozyme, whereas the double mutant SA{Delta}oatA/tagO is much more sensitive than the oatA mutant. The presence of sublethal concentrations of penicillin (5 ng P) enhanced lysozyme sensitivity markedly.

In a cell lysis assay, lysozyme (300 µg/ml) was added to exponentially growing cultures. The optical density at 578 nm (OD578) values of SA113 and SA{Delta}tagO were unaffected in the presence of lysozyme, whereas the OD578 of the mutant SA{Delta}oatA continued for 1 h before it declined slowly during the next hours. In contrast to the case with SA{Delta}tagO, the OD578 of the double mutant SA{Delta}oatA/tagO declined rapidly after addition of lysozyme. These results show that growth of SA{Delta}oatA/tagO is much more sensitive to lysozyme than that of the single mutant SA{Delta}oatA (Fig. 3A).


Figure 3
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  		FIG. 3. Lysozyme induced cell lysis during growth in the absence or presence of penicillin. One percent overnight cultures were inoculated into fresh B medium broth. Lysozyme (A, B) was added to the culture during the late exponential phase. Penicillin (B) was added immediately after inoculation. Changes in optical density were measured at hourly intervals. Symbols: SA113 wild type, {square}; SA{Delta}oatA, {blacksquare}; SA{Delta}oatA/tagO, {blacktriangleup}. Like the wild type, the mutant SA{Delta}tagO was completely resistant (data not shown).

Susceptibility of isolated PG to the hydrolytic activity of lysozyme. More than 80% of SA{Delta}oatA/tagO-derived PG was digested rapidly within 1 h, whereas only 6 to 7% of PG from SA{Delta}oatA was degraded within the same time span (Fig. 4). In contrast to the case with these mutants, the PG of SA113 and that of its WTA-less mutant (SA{Delta}tagO) were not solubilized within 3 h; only after 24 h did the turbidity decline a little, probably due to constant release of the rather labile O-acetyl group. Indeed, when we removed the O-acetyl groups by alkaline treatment of the PG of SA113 and SA{Delta}tagO (3 h in 80 mM NaOH at 37°C) (2), both PGs became susceptible to lysozyme. We also removed WTA by hydrofluoric acid treatment in wild-type PG and found that the WTA-less PG was completely resistant to the hydrolytic activity of lysozyme (data not shown). This result indicates that the presence or absence of WTA alone has little effect on lysozyme activity; only in the absence of both WTA and O acetylation does PG become highly susceptible to lysozyme hydrolysis.


Figure 4
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  		FIG. 4. Digestion of the purified peptidoglycan with lysozyme. PG was suspended in 80 mM sodium phosphate-buffered saline (0.5 mg/ml) and digested with 300 µg/ml lysozyme. Aliquots were taken at hourly intervals, and lysis of PG was measured as a decrease in absorbance at 578 nm. Symbols: SA113 wild type, {square}; SA{Delta}tagO, {triangleup}; SA{Delta}oatA, {blacksquare}; SA{Delta}oatA/tagO, {blacktriangleup}.

Comparative analysis of peptidoglycan O acetylation. The lack of effect of lysozyme on the tagO mutant could be due to an increased level of O acetylation owing to a higher abundance of O-acetylation sites in the absence of WTA. Soluble monomeric muropeptides were generated by digestion of isolated PG from all strains with lysostaphin and cellosyl (3) and separated by high-performance liquid chromatography (HPLC). HPLC profiles of SA113 and the mutant SA{Delta}tagO revealed two peaks: P1, comprised of monomeric de-O-acetylated muropeptides, and P2, comprised of monomeric O-acetylated muropeptides (3). The HPLC profile revealed no significant differences between the muropeptides of SA113 and those of the tagO mutant (Fig. 5A and C), and peak P2 is absent in the HPLC profiles of the mutants SA{Delta}oatA and SA{Delta}oatA/tagO, indicating that both mutants possess only de-O-acetylated PG (Fig. 5B and D). These data were further confirmed by determination of acetate (3) that was released by alkaline treatment of PG. This result shows that the insensitivity of SA{Delta}tagO-derived PG cannot be explained by a higher abundance of O acetylation.


Figure 5
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  		FIG. 5. Comparative analysis of O acetylation in monomeric muropeptides isolated from various S. aureus strains. PG was isolated from SA113 (A), SA{Delta}oatA (B), SA{Delta}tagO (C), and SA{Delta}oatA/tagO (D) and digested with lysostaphin and cellosyl. The muropeptides were reduced with NaBH4 (pH 8.0) and analyzed on a C18 column using a linear gradient of 10 mM sodium phosphate buffer to 30% methanol for 190 min. Muropeptides were detected at 210 nm. Peak P1 (retention time, 54 min) represents de-O-acetylated muropeptides and peak P2 (retention time, 67 min) O-acetylated muropeptides. Results illustrate that PG of SA{Delta}oatA and SA{Delta}oatA/tagO contained no O-acetylated muropeptides and that the ratios of de-O-acetylated (P1) to O-acetylated muropeptides (P2) were very similar for SA113 and SA{Delta}tagO.

Content of WTA in the oatA mutant. The PG of the oatA mutant showed a much lower susceptibility to the hydrolytic activity of lysozyme than that of the mutant SA{Delta}oatA/tagO (Fig. 4). One possible reason could be that the content of WTA is increased in the PG of SA{Delta}oatA. As shown in Fig. 1, both WTA and O-acetyl modification occur at the C6Formula OH positions of NAM. It might therefore be possible that in SA{Delta}oatA-derived PG, some of the free C6Formula OH positions of NAM are occupied by WTA. However, we found no significant difference in the content of phosphate (354.4 nmol/mg cell wall for the wild type and 335.8 nmol/mg cell wall for SA{Delta}oatA) or N-acetylglucosamine (394.4 nmol/mg cell wall for the wild type and 306.9 nmol/mg cell wall for SA{Delta}oatA) in PG or WTA preparations (8, 16) of the wild type and the SA{Delta}oatA mutant. Apparently the WTA biosynthesis machinery does not use the free C6Formula OH positions of NAM in SA{Delta}oatA PG.

Activity of endogenous autolysins in presence of lysozyme. To rule out the possibility that the increased sensitivity to lysozyme of SA{Delta}oatA/tagO is a consequence of increased endogenous autolysis activity, we compared the autolysin patterns of the wild type and the mutants in a zymogram gel (4, 11); however, there was no difference. Increased autolysin activity very likely does not account for the high lysozyme susceptibility of SA{Delta}oatA/tagO.

Effect of penicillin on lysozyme resistance. The number of ß-1,4-glycosidic linkages in PG depends on the degree of cross-linking of muropeptides (18). It is well known that penicillin decreases cross-linkages by inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis (20). We incubated SA113 and mutants with lysozyme and a sublethal concentration of penicillin (5 ng/ml) (Fig. 3B). Indeed, in the presence of penicillin the sensitivity against lysozyme was 10-fold higher with SA{Delta}oatA/tagO (MIC, 12 µg/ml) than with SA{Delta}oatA (MIC, 100 µg/ml), while the wild type and the SA{Delta}tagO mutant stayed completely lysozyme resistant. Lysozyme has two modes of action: hydrolysis of PG and antimicrobial peptide activity (12). We questioned to which activity the penicillin effect was due. To answer this question, penicillin was added to a freshly inoculated growth medium, and after 5 h of cultivation, lysozyme was added. While the growth of the wild type and that of SA{Delta}tagO were unaffected, the OD578 values of the SA{Delta}oatA mutant decreased gradually and that of SA{Delta}oatA/tagO rapidly after addition of lysozyme (Fig. 3B). In the absence of lysozyme, no cell lysis was observed with any of the strains (data not shown).

The contribution of WTA in lysozyme resistance is not easy to understand. We see no difference in lysozyme susceptibility whether WTA is present (wild type) or absent ({Delta}tagO mutant); neither growth nor hydrolysis of isolated PG is affected by lysozyme. The contribution of WTA in lysozyme resistance became visible only for the SA{Delta}oatA/tagO double mutant, which is 10-fold more susceptible than SA{Delta}oatA. We have ruled out that the relative insensitivity of SA{Delta}oatA is due to an increased integration of WTA in PG. Apparently, the WTA biosynthesis machinery does not use the excess of free C6Formula OH positions in SA{Delta}oatA PG to increase the content of WTA; conversely, the OatA enzyme does not incorporate more O-acetyl groups in SA{Delta}tagO than in the wild type. We are still left with the question of why WTA shows its effect in lysozyme resistance only in the double mutant SA{Delta}oatA/tagO and not in the WTA-less mutant. One explanation could be that in S. aureus only every ninth NAM residue contains phosphoester-linked WTA (14) while approximately every second is O acetylated (3). Even if WTA is missing, binding of lysozyme, which recognizes a hexameric glycan strand, is still blocked by the O-acetyl groups. If O-acetyl groups are missing, lysozyme still may bind to the glycan strand between the WTA residues, albeit interaction might still be hampered by the WTA residues. Optimal binding and highest hydrolytic activity can be displayed by lysozyme only if both modifications are missing. This might explain why the double mutant SA{Delta}oatA/tagO and its isolated PG are more susceptible to lysozyme than SA{Delta}oatA alone.


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ACKNOWLEDGMENTS
 
We thank Mulugeta Nega for help in HPLC analysis and Christiane Zell for technical support.

This work was supported by the DFG: Graduate College "Infection biology" (GKI 685) and Forschergruppe (FOR 449/1).


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FOOTNOTES
 
* Corresponding author. Mailing address: Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Phone: 0049 7071 2974636. Fax: 0049 7071 295039. E-mail: friedrich.goetz{at}uni-tuebingen.de. Back

{triangledown} Published ahead of print on 3 November 2006. Back

{dagger} Both authors contributed equally to this work. Back


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Journal of Bacteriology, January 2007, p. 280-283, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01221-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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