Abstract
Staphylococcus aureus cause infections by producing toxins, a process regulated by cell-cell communication (quorum sensing) through the histidine-phosphorylation of the target of RNAIII-activating protein (TRAP). We show here that TRAP is highly conserved in staphylococci and contains three completely conserved histidine residues (His-66, His-79, His-154) that are phosphorylated and essential for its activity. This was tested by constructing a TRAP(-) strain with each of the conserved histidine residues changed to alanine by site-directed mutagenesis. All mutants were tested for pathogenesis in vitro (expression of RNAIII and hemolytic activity) and in vivo (murine cellulitis model). Results show that RNAIII is not expressed in the TRAP(-) strain, that it is non hemolytic, and that it does not cause disease in vivo. These pathogenic phenotypes could be rescued in the strain containing the recovered traP, confirming the importance of TRAP in S. aureus pathogenesis. The phosphorylation of TRAP mutated in any of the conserved histidine residues was significantly reduced, and mutants defective in any one of these residues were non-pathogenic in vitro or in vivo, whereas those mutated in a non-conserved histidine residue (His-124) were as pathogenic as the wild type. These results confirm the importance of the three conserved histidine residues in TRAP activity. The phosphorylation pattern, structure, and gene organization of TRAP deviates from signaling molecules known to date, suggesting that TRAP belongs to a novel class of signal transducers.
Highlights
Staphylococcus aureus are Gram-positive bacteria that are part of the normal flora but can become pathogenic and cause disease in humans and animals once they produce toxic exomolecules [1]
Strains and Species—The traP gene in various clinical isolates of S. aureus and S. epidermidis was amplified by PCR, and its sequence was determined
Translation of all known traP nucleotide sequences of S. aureus and S. epidermidis indicates that the encoded protein contains three conserved histidine residues (His-66, His-79, His-154), which are indicated in bold (Fig. 1A)
Summary
Staphylococcus aureus are Gram-positive bacteria that are part of the normal flora but can become pathogenic and cause disease in humans and animals once they produce toxic exomolecules [1]. S. aureus pathogenesis is regulated by quorum sensing mechanisms [2,3,4], where molecules (autoinducers) produced and secreted by the bacteria accumulate as a function of cell density. SQS 1 consists of the autoinducer RNAIII-activating protein (RAP) and its target molecule TRAP [5, 6]. RAP is a 277-amino acid protein that is orthologous to the ribosomal protein L2 and is secreted by S. aureus [7]. TRAP is a 167-amino acid protein that is constitutively expressed but is histidine-phosphorylated in a RAP-dependent manner [5] and is, suggested to act as the sensor of RAP. RNAIII acts as a regulatory RNA molecule to activate various toxin genes and encodes for ␦-hemolysin [15,16,17]
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