RNAIII-activating Protein Research Articles

The novel combinations of CTB, CpG, and aluminum hydroxide significantly enhanced the immunogenicity of clumping factor A 221-550 of Staphylococcus aureus.

CTB: Cholera Toxin B; CpG: Cytosine preceding Guanosine; ODN: Oligodeoxynucleotides; Alum: Aluminum hydroxide; TRAP: Target of RNAIII-activating Protein; TLR9: Toll-like Receptor 9; TMB: 3, 3', 5, 5'-tetramethylbenzidine; mAbs: Monoclonal Antibodies; OD: Optical Densities; S. aureus: Staphylococcus aureus; ClfA: Clumping factor A; FnBPA: Fibronection-binding protein A; IsdB: Iron-regulated surface determinant B; SasA: Staphylococcus aureus Surface Protein A; GapC: Glycer-aldehyde-3-phosphate dehydrogenase-C.

Comparative homology model building and docking evaluation for RNA III inhibiting peptide of Multi drug resistant Staphylococcus aureus strain MRSA252

Since last several years, infection caused by Staphylococcus aureus is challenging to cure using conventional antibiotics. The organism is a Gram-positive bacterial pathogen that can cause serious diseases not only in humans but also in animals, such as various skin infections, pneumonia, endocarditis and toxin shock syndrome. This bacterium causes such diseases by producing macromolecules such as hemolysins, enterotoxins, proteases and toxic shock syndrome toxin (TSST-1). This organism had developed the multidrug resistance by acquiring MEC-A gene. This account for made organism to come into the category of Superbug. Several studies showed that, the toxin production is induced by AIP and RAP via the phosphorylation of TRAP. TRAP is a 21 kDa protein and was believed to be associated with the membrane via SvrA Phosphoamino acid analysis revealed that TRAP is histidine phosphorylated in a signal transduction pathway that is activated by RAP. The inhibition of TRAP could be done by RIP (RNAIII-inhibiting peptide). The structure for RIP is still undiscovered to be used as inhibitor.Present work has been carried out to get the structural insight with various online and offline homology modeling techniques such as SWISS-MODEL, MODBASE, GENO3D, CPHmodels and I-TASSER for getting unknown structural information target of RNAIII-activating protein from Staphylococcus aureus strain MRSA252 origin for their future exploration as a target in drug discovery process against MRSA.

Structure of the signal transduction protein TRAP (target of RNAIII-activating protein).

The crystal structure of the signal transduction protein TRAP is reported at 1.85 Å resolution. The structure of TRAP consists of a central eight-stranded β-barrel flanked asymmetrically by helices and is monomeric both in solution and in the crystal structure. A formate ion was found bound to TRAP identically in all four molecules in the asymmetric unit.

OpuC – an ABC Transporter that is Associated with Staphylococcus Aureus Pathogenesis

RIP is a novel antibiotic against staphylococci. It acts at least in part by competing with RNAIII activating protein (RAP) by downregulating TRAP histidine phosphorylation, and by downregulating the expression of the acessory gene regulator (agr). While much is known about the function of the agr as a quorum sensing system that regulates virulence, not much is known about TRAP. TRAP is a 167-kDa protein that is highly conserved among staphylococci and is involved in DNA protection from stress. TRAP is membrane-associated but does not have a transmembrane domain, and thus it may be bound to the membrane through other proteins. To search for these proteins, protein-protein interaction studies were carried out using a bacterial two-hybrid system, and OpuCA was discovered as a TRAP-binding protein. OpuCA is an ATP binding-cytoplasmic (ABC) domain of an OpuC ABC transporter. S. aureus OpuC- mutant strain was constructed and shown to be less tolerant to salt stress, and was defective in choline uptake. OpuC- cells were less pathogenic and showed reduced TRAP phosphorylation and agr activity, did not respond to RAP, and were defective in biofilm formation in vitro and in vivo. These results suggest that OpuC acts as a transporter and also plays a role in S. aureus pathogenesis.

A novel peptide isolated from phage library to substitute a complex system for a vaccine against staphylococci infection

Staphylococcus aureus is a major human pathogen and many of the strains are resistant to conventional antibacterial treatment. The bacteria cause disease largely due to the production of multiple toxins, whose synthesis is controlled by an RNA molecule termed RNAIII. The production of RNAIII is induced by quorum sensing systems, one of them containing the protein RNAIII activating protein (RAP). Here we show that we partially purified supernatant of S. aureus, used this fraction to vaccinate mice, and selected antibody-binding peptides by phage display. Mice were vaccinated with the various peptides and challenged with S. aureus. One of the binding peptide termed R13 induced a protective immune response. Western blot analysis showed that the anti-R13 antibodies specifically bind to native or recombinant RAP. Mice vaccinated with R13 were protected and protection was sustained for the duration of the 6-month study period. Our results show that R13 could be used as a long-term effective protective-peptide-vaccine to prevent S. aureus infections and once again show that targeting the RAP quorum sensing system is an effective approach to preventing staphylococcal infections. In addition our studies show that selection of specific protective peptides by phage display using sera induced by complex antigens is a rapid and effective way to identify the protective antigen and select for a peptide vaccine.

Transcriptional Profiling of Target of RNAIII-Activating Protein, a Master Regulator of Staphylococcal Virulence

Staphylococcus aureus is a gram-positive bacterium that is part of the normal healthy flora but that can become virulent and cause infections by producing biofilms and toxins. The production of virulence factors is regulated by cell-cell communication (quorum sensing) through the histidine phosphorylation of target of RNAIII-activating protein (TRAP), which is a 21-kDa protein that is highly conserved among staphylococci. Using microarray analysis, we show here that the expression and phosphorylation of TRAP upregulate the expression of most, if not all, toxins known to date, as well as their global regulator agr. In addition, we show here that the expression and phosphorylation of TRAP are also necessary for the expression of genes known to be necessary for the survival of the bacteria in a biofilm, like arc, pyr, and ure. TRAP is thus demonstrated to be a master regulator of staphylococcal pathogenesis.

A Novel Peptide Screened by Phage Display Can Mimic TRAP Antigen Epitope against Staphylococcus aureus Infections

Staphylococcus aureus is a major human pathogen. Pathogenic effects are largely due to production of bacterial toxins, whose synthesis is controlled by an mRNA molecule termed RNAIII. The S. aureus protein called RAP (RNAIII-activating protein) is secreted and activates RNAIII production by inducing the phosphorylation of its target protein TRAP (target of RAP). Antibodies to TRAP have been shown to suppress exotoxin production by S. aureus in vitro, suggesting that TRAP may be a useful vaccine target site. Here we showed that a peptide TA21 was identified by screening a phage display library using anti-TRAP antibodies. Mice vaccinated with Escherichia coli engineered to express TA21 on their surface (FTA21) were protected from S. aureus infections, using sepsis and cellulitis mice models. By sequence analysis, it was found that the TA21 is highly homologous to the C-terminal sequence of TRAP which is conserved among S. aureus and Staphylococcus epidermidis, suggesting that peptide TA21 may be a useful broad vaccine to protect from infection caused by various staphylococcal strains.

The target of RNAIII-activating protein (TRAP) from Staphylococcus aureus: purification, crystallization and preliminary X-ray analysis

The target of the RNAIII-activating protein (TRAP) is a 21 kDa protein in which phosphorylation is activated by the RNAIII-activating protein (RAP), which causes an increase in RNAII and RNAIII synthesis and the production of the virulence factors. In an attempt to examine the structural role of TRAP in the signal transduction pathway, TRAP from Staphylococcus aureus was overexpressed, purified and crystallized using PEG 8000 and 5% Jeffamine M600 (pH 7.0), as precipitants by hanging-drop vapour diffusion methods at 287 K. The crystals belong to the orthorhombic space group, P2 12 12 1, with unit cell parameters of a=39.68, b=50.41, c=85.45 Å. There is one monomer of TRAP per crystallographic asymmetric unit with a crystal volume per protein mass ( V M) of 2.06 Å 3 Da −1 and a solvent content of 40.3%. A complete data set diffracting to 1.9 Å resolution was collected from a single crystal at 100 K using a synchrotron-radiation source.

Quorum Sensing in Staphylococci Is Regulated via Phosphorylation of Three Conserved Histidine Residues

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.

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

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.

Characterization of RAP, a quorum sensing activator of Staphylococcus aureus

Staphylococcus aureus are Gram-positive bacteria and cause diverse serious diseases in humans and animals through the production of toxins. The production of toxins is regulated by quorum sensing mechanisms, where proteins such as RNAIII activating protein (RAP) are secreted by the bacteria and induce virulence. Antibodies to RAP have been shown to protect mice from infection, but the molecular structure of RAP was not known and hindered vaccine development. To characterize RAP, recombinant protein was made and tested for its ability to induce genes important for pathogenesis ( agr). In addition, monoclonal antibodies were produced to identify its cellular localization. Results shown here indicate that RAP is a 277-aa protein that is an ortholog of the ribosomal protein L2. Like the native molecule, recombinant RAP activates the production of RNAIII (encoded by agr). Using RAP specific monoclonal antibodies we demonstrate that RAP is continuously secreted and while RAP is expressed also in other bacteria (like Staphylococcus epidermidis, Staphylococcus xylosus and Escherichia coli), it is secreted to the culture medium only by S. aureus. Our results show that the ribosomal protein L2 has an extraribosomal function and that when secreted RAP acts as an autoinducer of virulence to regulate S. aureus pathogenesis.

Regulation of Staphylococcus aureus Pathogenesis via Target of RNAIII-activating Protein (TRAP)

Staphylococcus aureus can cause disease through the production of toxins. Toxin production is autoinduced by the protein RNAIII-activating protein (RAP) and by the autoinducing peptide (AIP), and is inhibited by RNAIII-inhibiting peptide (RIP) and by inhibitory AIPs. RAP has been shown to be a useful vaccine target site, and RIP and inhibitory AIPs as therapeutic molecules to prevent and suppress S. aureus infections. Development of therapeutic strategies based on these molecules has been hindered by a lack of knowledge of the molecular mechanisms by which they activate or inhibit virulence. Here, we show that RAP specifically induces the phosphorylation of a novel 21-kDa protein, whereas RIP inhibits its phosphorylation. This protein was termed target of RAP (TRAP). The synthesis of the virulence regulatory molecule, RNAIII, is not activated by RAP in the trap mutant strain, suggesting that RAP activates RNAIII synthesis via TRAP. Phosphoamino acid analysis shows that TRAP is histidine-phosphorylated, suggesting that TRAP may be a sensor of RAP. AIPs up-regulate the synthesis of RNAIII also in trap mutant strains, suggesting that TRAP and AIPs activate RNAIII synthesis via distinct signal transduction pathways. Furthermore, TRAP phosphorylation is down-regulated in the presence of AIP, suggesting that a network of signal transduction pathways regulate S. aureus pathogenesis.

Regulation of Staphylococcus aureuspathogenesis via target of RNAIII-activating protein (TRAP).

Regulation of Staphylococcus aureuspathogenesis via target of RNAIII-activating protein (TRAP).

Response from Balaban

I would like to expand on one point raised by Jean Lee: how inhibiting the autoinducer of virulence, RNAIII-activating protein (RAP), can act to suppress staphylococcal infections. The expression of many virulence factors by Staphylococcus aureus is controlled by RNAIII, a regulatory RNA molecule that is encoded by the global regulatory locus agr. RNAIII can be activated by two different autoinducers: by RAP, which is agr independent[1, 2], and by an octapeptide, which is encoded by the agr locus itself[3]. Because agr is active only from the mid-exponential phase of growth, whereas RAP is continuously produced and secreted, we hypothesize that there may be a two-step rnaiii activation process (Fig. 1). During cell growth, the concentration of RAP (signal A) builds to a threshold level[1]. It then binds to its receptor, causing a cascade of events that leads to the phosphorylation of a protein called `target of RAP' (TRAP) (N. Balaban et al., unpublished). These events lead to upregulation of sarA, which then upregulates agr (Ref. [4]). Once agr is activated, agrD is expressed, and an octapeptide encoded by this gene (signal B) is produced and secreted[3]. This peptide then binds to its receptor, AgrC (Ref. [5]), resulting in its phosphorylation, in downregulation of TRAP phosphorylation (N. Balaban et al., unpublished) and in upregulation of P3 to transcribe RNAIII. RNAIII induces the genes that encode various virulence factors, leading to production of toxins. RNA-inhibiting protein (RIP) has been proposed to act as an antagonist[1], blocking RAP from binding to its receptor. Therefore, in the presence of RIP or anti-RAP antibodies, TRAP is not phosphorylated (N. Balaban et al., unpublished), the signal transduction of virulence is not activated and toxins regulated by RNAIII are not produced.

Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus.

Staphylococcus aureus causes pathologies ranging from minor skin infections to life-threatening diseases. Pathogenic effects are largely due to production of bacterial toxin, which is regulated by an RNA molecule, RNAIII. The S. aureus protein called RAP (RNAIII activating protein) activates RNAIII, and a peptide called RIP (RNAIII inhibiting peptide), produced by a nonpathogenic bacteria, inhibits RNAIII. Mice vaccinated with RAP or treated with purified or synthetic RIP were protected from S. aureus pathology. Thus, these two molecules may provide useful approaches for the prevention and treatment of diseases caused by S. aureus.