Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides

Abstract

Staphylococcus aureus cause many diseases by producing toxins, whose synthesis is regulated by quorum-sensing mechanisms. S. aureus secretes a protein termed RNAIII activating protein (RAP) which autoinduces toxin production via the phosphorylation of is target protein TRAP. Mice vaccinated with RAP were protected from S. aureus infection, suggesting that RAP is an useful target for selecting potential therapeutic molecules to inhibit S. aureus pathogenesis. We show here that RAP (native and recombinant) was used to select RAP-binding peptides (RBPs) from a random 12-mer phage-displayed peptide library. Two RBPs were shown to inhibit RNAIII production in vitro (used a marker for pathogenesis). The peptide WPFAHWPWQYPR, which had the strongest inhibitory activity, was chemically synthesized and also expressed in Escherichia coli as a GST-fusion. Both synthetic peptide and GST-fusion peptide decreased RNAIII levels in a dose-dependent manner. The GST-fusion peptide was also shown to protect mice from a S. aureus infection in vivo (tested in a murine cutaneous S. aureus infection model). Our results suggest the potential use of RAP-binding proteins in treating clinical S. aureus infections.

Introduction

Many diseases are caused by the Gram-positive bacteria Staphylococcus aureus, such as pneumonia, endocarditis, meningitis, septicemia, and toxic shock syndrome [11]. Development of resistance to available antibiotics has become a serious problem in treatment of S. aureus infections [12]. There is therefore an imperative need in finding new types of antibacterial agents.

Multiple virulence factors are produced by S. aureus and cause the various disease symptoms [11]. Temporal expression of many of them has been shown to be under the control of the gene locus agr [14]. agr contains two divergent transcription units. The P2 operon consists of four open reading frames, agrACDB, of which AgrA and AgrC encode proteins of a classical two-component signal transduction pathway [14]. The P3 operon transcribes a 512-nucleotide transcript, RNAIII, which is considered an effector molecule of the agr regulation [15]. RNAIII up-regulates the synthesis of exotoxins like hemolysins, enterotoxins, toxic shock syndrome toxin and proteases, while down regulating the expression of cell surface proteins [15]. The synthesis of RNAIII is autoinduced by RAP, which is a 277AA S. aureus protein that is an ortholog of L2 (GenBank accession number AF205220). RAP is continuously secreted by S. aureus and activates the synthesis of RNAIII by inducing the phosphorylation of TRAP (target of RAP). RAP is thus an autoinducer of the quorum-sensing molecule TRAP [2], [3], [4], [9]. Mice vaccinated with native RAP were protected from a S. aureus infection [3]. Similarly, infections induced by a challenge with various strains of S. aureus were prevented by RNAIII inhibiting peptide (RIP), which is a heptapeptide that competes with RAP and inhibits TRAP phosphorylation [1], [2], [3], [7], [8]. Inhibiting quorum-sensing by antibodies or by inhibitory peptides is therefore emerging as a novel approach for therapy alternative to antibiotics. In this paper, we describe the use of a random phage-displayed peptide library to select RAP-binding peptides (RBPs) that can inhibit RAP activity and prevent S. aureus infections.