D-alanylation of teichoic acids promotes group a streptococcus antimicrobial peptide resistance, neutrophil survival, and epithelial cell invasion

Abstract

Group A streptococcus (GAS) is a leading cause of severe, invasive human infections, including necrotizing fasciitis and toxic shock syndrome. An important element of the mammalian innate defense system against invasive bacterial infections such as GAS is the production of antimicrobial peptides (AMPs) such as cathelicidins. In this study, we identify a specific GAS phenotype that confers resistance to host AMPs. Allelic replacement of the dltA gene encoding d-alanine-d-alanyl carrier protein ligase in an invasive serotype M1 GAS isolate led to loss of teichoic acid d-alanylation and an increase in net negative charge on the bacterial surface. Compared to the wild-type (WT) parent strain, the GAS DeltadltA mutant exhibited increased susceptibility to AMP and lysozyme killing and to acidic pH. While phagocytic uptake of WT and DeltadltA mutants by human neutrophils was equivalent, neutrophil-mediated killing of the DeltadltA strain was greatly accelerated. Furthermore, we observed the DeltadltA mutant to be diminished in its ability to adhere to and invade cultured human pharyngeal epithelial cells, a likely proximal step in the pathogenesis of invasive infection. Thus, teichoic acid d-alanylation may contribute in multiple ways to the propensity of invasive GAS to bypass mucosal defenses and produce systemic infection.

FIG. 1.

The dlt operon encodes teichoic acid d-alanylation in GAS and affects bacterial surface charge. (A) Scheme for allelic replacement of the dltA gene in a GAS chromosome. (B) HPLC analysis of the d-alanine content of teichoic acids in WT, ΔdltA, and ΔdltA plus pdltA GAS strains. (C) Binding of the cationic protein cytochrome c to WT and ΔdltA GAS strains compared to analogous mutations in S. aureus strain 113 (24). Data represent the mean values of protein bound by the bacterium ± standard deviations of four independent experiments (**, P < 0.005; ***, P < 0.0005; two-tailed t test).

FIG. 2.

Effect of the ΔdltA mutation on additional GAS phenotypes. (A) β-Hemolytic (streptolysin S) phenotype on sheep's blood agar. (B) M protein and cysteine proteinase SpeB expression by dot blot analysis. (C) Rate of water-induced autolysis activity as determined by decreasing A₆₀₀. (D) Phase-contrast transmission microscopy of WT and ΔdltA mutant GAS in exponential and stationary growth phases. Note the long chains formed by the ΔdltA mutant in stationary phase.

FIG. 3.

GAS ΔdltA mutant exhibits increased pH sensitivity. Exponential phase WT and ΔdltA mutant GAS were incubated up to 4 h in sodium phosphate buffer containing d-arginine buffered to the indicated pH. (***, P < 0.0005; two-tailed paired t test).

FIG. 4.

GAS LTA d-alanylation decreases susceptibility to neutrophil killing. (A) Phagocytosis and killing kinetics of WT and ΔdltA mutant GAS by human neutrophils at a bacteria:cell ratio of 1:10. The values represent the means and standard deviations of three independent experiments (*, P < 0.05, two-tailed t test). (B) Human neutrophil phagocytosis of fluorescence-tagged GAS. The percentage of neutrophils with intracellular bacteria is depicted (200 cells examined per sample); the graph shows mean values ± standard deviations in one representative study of three experiments with similar results.

FIG. 5.

GAS teichoic acid d-alanylation promotes Hep-2 human pharyngeal epithelial cell adherence and invasion. (A) Total cell-associated bacteria after 1 h of incubation. Samples were run in triplicate, and the mean values ± standard deviations of three independent experiments are shown (**, P < 0.005; two-tailed t test). (B) Intracellular bacteria quantified in an antibiotic protection assay after 1 h of incubation followed by 2 h of antibiotic treatment to kill extracellular bacteria. Samples were run in triplicate, and the mean values ± standard deviations of one representative experiment of three performed are shown (***, P < 0.005; two-tailed t test).