Wall teichoic acid deficiency in Staphylococcus aureus confers selective resistance to mammalian group IIA phospholipase A(2) and human beta-defensin 3

Abstract

Wall teichoic acids (WTAs) and membrane lipoteichoic acids (LTAs) are the major polyanionic polymers in the envelope of Staphylococcus aureus. WTAs in S. aureus play an important role in bacteriophage attachment and bacterial adherence to certain host cells, suggesting that WTAs are exposed on the cell surface and could also provide necessary binding sites for cationic antimicrobial peptides and proteins (CAMPs). Highly cationic mammalian group IIA phospholipase A(2) (gIIA PLA(2)) kills S. aureus at nanomolar concentrations by an action(s) that depends on initial electrostatic interactions, cell wall penetration, membrane phospholipid (PL) degradation, and activation of autolysins. A tagO mutant of S. aureus that lacks WTA is up to 100-fold more resistant to PL degradation and killing by gIIA PLA(2) and CAMP human beta-defensin 3 (HBD-3) but has the sensitivity of the wild type (wt) to other CAMPs, such as Magainin II amide, hNP1-3, LL-37, and lactoferrin. In contrast, there is little or no difference in either gIIA PLA(2) activity toward cell wall-depleted protoplasts of the wt and tagO strains of S. aureus or in binding of gIIA PLA(2) to wt and tagO strains. Scanning and transmission electron microscopy reveal increased surface protrusions in the S. aureus tagO mutant that might account for reduced activity of bound gIIA PLA(2) and HBD-3 toward the tagO mutant. In summary, the absence of WTA in S. aureus causes a selective increase in bacterial resistance to gIIA PLA(2) and HBD-3, the former apparently by reducing access and/or activity of bound antibacterial enzyme to the bacterial membrane.

FIG. 1.

Sensitivities of wt, tagO, and tagO complemented strains to gIIA PLA₂, HBD-3, and Magainin II amide. S. aureus (wt, tagO, and complemented strains) at 1 × 10⁶/ml were incubated for 2 h as described in Materials and Methods in the presence of increasing concentrations of gIIA PLA₂ (A), HBD-3 (B), and Magainin II amide (C), as indicated. Bacterial viability was measured as CFU in TSA and is expressed as a percentage of the initial inoculum. The lowest doses tested of gIIA PLA₂, HBD-3, and Magainin II amide were 10 ng/ml, 1.25 μg/ml, and 0.4 μg/ml, respectively. Growth of all three bacterial strains during 2 h of incubation without gIIA PLA₂, HBD-3, and Magainin II amide was similar, resulting in >100% CFU. The results shown represent the means of three experiments ± standard errors of the means (error bars). Where indicated, the asterisks denote statistically significant (P < 0.05) greater resistance of the S. aureus tagO mutant to killing by gIIA PLA₂ and by HBD-3 versus that of the wt and S. aureus tagO complemented strains.

FIG. 2.

Effects of gIIA PLA₂ on lipid release and killing of wt and tagO strains of S. aureus. S. aureus wt strain (A and D), S. aureus tagO strain (B and E), and S. aureus tagO complemented strain (C and F) lipids were prelabeled with [1-¹⁴C]oleic acid as described in Materials and Methods before incubation with increasing concentrations of gIIA PLA₂. Samples were taken at 1 and 2 h to measure accumulation of 1-¹⁴C-labeled lipid breakdown products in the extracellular medium and CFU in TSA. Increasing size of symbols corresponds to increasing gIIA PLA₂ concentrations (0, 10, 100, and 1,000 ng/ml). The results shown represent the means of three experiments ± standard errors of the means (error bars). Where indicated, the asterisks denote statistically significant (P < 0.05) greater resistance of the S. aureus tagO mutant to the phospholipolytic and bactericidal activities of gIIA PLA₂ versus those of the wt and S. aureus tagO complemented strains.

FIG. 3.

Sensitivity of protoplasts from wt and tagO S. aureus strains to gIIA PLA₂. Membrane protoplasts derived from 10⁷ bacteria prelabeled with [1-¹⁴C]oleic acid were incubated with increasing concentrations of gIIA PLA₂ (10 and 100 ng/ml) in RPMI supplemented with 10 mM HEPES, 1 mM CaCl₂, and 1% BSA and osmotically stabilized with 30% raffinose. After 60 min of incubation at 37°C in the presence or absence of gIIA PLA₂, lipids were extracted from the protoplasts and separated by TLC as described in Materials and Methods. (A) Loss of [1-¹⁴C]PL; (B) accumulation of gIIA PLA₂-mediated PL degradation products ([1-¹⁴C]lysophospholipid [lyso-PL] and 1-¹⁴C-labeled free fatty acid [FFA]), as quantified by densitometric analysis. The results shown are from one experiment, representative of two similar experiments.

FIG. 4.

Initial binding of gIIA PLA₂ to S. aureus (wt and tagO strains). S. aureus (1 × 10⁸ bacteria/ml) was incubated with catalytically inactive D49S gIIA PLA₂ (1 μg/ml) for 15 min at 37°C as described in Materials and Methods. Unbound gIIA PLA₂ was separated from bacterium-associated PLA₂ by sedimentation of bacteria. Bound gIIA PLA₂ was analyzed by SDS-PAGE and immunoblotting before and after treatment with 1 M NaCl as described in Materials and Methods. (A) Immunoblot of samples containing 2 × 10⁷, 1 × 10⁷, and 5 × 10⁶ (from left to right) bacterial equivalents of wt and tagO strains of S. aureus. (B) Immunoblot of samples containing 2 × 10⁷ bacterial equivalents before and after treatment with 1 M NaCl. gIIA PLA₂ recovered in the 1 M NaCl eluate and, for comparison, purified gIIA PLA₂ standard (20 ng) are shown in the three rightmost lanes. The results shown are representative of three similar experiments.

FIG. 5.

Sensitivity of wt and tagO S. aureus strains to Triton X-100. Bacteria (wt and tagO strains) were grown until the mid-logarithmic growth phase and then exposed to increasing concentrations of Triton X-100 (twofold dilutions ranging from 1% to 0.001% [vol/vol]). Bacterial absorbance (OD₅₅₀) was measured after 2 h of incubation at 37°C. The values represent the percentages of OD₅₅₀ of initial inoculum after 2 h of incubation of bacteria with or without Triton X-100. The results shown are the means of two independent experiments ± standard errors of the means (error bars), each done in duplicate samples. Where indicated, the asterisks denote statistically significant (P < 0.05) differences in sensitivity of wt and tagO S. aureus strains to Triton X-100.

FIG. 6.

Morphology of wt and tagO strains of S. aureus as examined by electron microscopy. (Top) TEM; (bottom) SEM. Note that wt cocci are round and have a smooth surface, whereas S. aureus tagO cocci have a rough surface with many surface protrusions seen in all cocci.