ABSTRACT
We previously reported that Staphylococcus aureus avoids killing within macrophages by exploiting the action of Toll-like receptor 2 (TLR2), which leads to the c-Jun N-terminal kinase (JNK)-mediated inhibition of superoxide production. To search for bacterial components responsible for this event, a series of S. aureus mutants, in which the synthesis of the cell wall was interrupted, were screened for the level of JNK activation in macrophages. In addition to a mutant lacking the lipoproteins that have been suggested to act as a TLR2 ligand, two mutant strains were found to activate the phosphorylation of JNK to a lesser extent than the parental strain, and this defect was recovered by acquisition of the corresponding wild-type genes. Macrophages that had phagocytosed the mutant strains produced more superoxide than those engulfing the parental strain, and the mutant bacteria were more efficiently killed in macrophages than the parent. The genes mutated, dltA and tagO, encoded proteins involved in the synthesis of D-alanylated wall teichoic acid. Unlike a cell wall fraction rich in lipoproteins, D-alanine-bound wall teichoic acid purified from the parent strain by itself did not activate JNK phosphorylation in macrophages. These results suggest that the d-alanylated wall teichoic acid of S. aureus modulates the cell wall milieu for lipoproteins so that they effectively serve as a ligand for TLR2.
Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/metabolism , Macrophages/metabolism , Macrophages/microbiology , Staphylococcal Infections/immunology , Staphylococcus aureus/physiology , Teichoic Acids/immunology , Teichoic Acids/metabolism , Toll-Like Receptor 2/metabolism , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacteriolysis/genetics , Bacteriolysis/immunology , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/immunology , Cell Line , Cell Wall/metabolism , Enzyme Activation/genetics , Genetic Complementation Test , Lipopolysaccharides/chemistry , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Macrophages/immunology , Macrophages/pathology , Mice , Mutagenesis, Site-Directed , Mutation , Phagocytosis/genetics , Phagocytosis/immunology , Staphylococcal Infections/genetics , Staphylococcal Infections/metabolism , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicity , Superoxides/metabolism , Teichoic Acids/chemistry , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/immunologyABSTRACT
Injection of stationary phase culture-supernatants of Staphylococcus aureus and Pseudomonas aeruginosa into the hemolymph of silkworm larvae caused their death, whereas a culture-supernatant of a non-pathogenic strain of Escherichia coli did not. A culture-supernatant of a mutant of agr, a global virulence regulator of S. aureus that is required for exotoxin production, was much less toxic to silkworm larvae. A culture-supernatant of a disruption mutant of the S. aureus beta-toxin gene did not kill larvae, whereas one of a deletion mutant of alpha-toxin, gamma-toxin, or aureolysin killed larvae, indicating that the beta-toxin gene is required for staphylococcal supernatant-mediated killing of silkworm larvae. The 50% lethal doses (LD50) of staphylococcal alpha-toxin and beta-toxin, Pseudomonas exotoxin A and diphtheria toxin were 12 microg/g, 9 microg/g, 0.14 microg/g and 1.1 microg/g, respectively. As the purified toxins killed the larvae, silkworm larvae could be used as a model to study the actions of pathogenic bacterial toxins in animal bodies.
