RNAIII-inhibiting Peptide Research Articles

Preparation, Characterization, and Biological Evaluation of a Hydrophilic Peptide Loaded on PEG-PLGA Nanoparticles.

The encapsulation of peptides and proteins in nanosystems has been extensively investigated for masking unfavorable biopharmaceutical properties, including short half-life and poor permeation through biological membranes. Therefore, the aim of this work was to encapsulate a small antimicrobial hydrophilic peptide (H-Ser-Pro-Trp-Thr-NH2, FS10) in PEG-PLGA (polyethylene glycol-poly lactic acid-co-glycolic acid) nanoparticles (Nps) and thereby overcome the common limitations of hydrophilic drugs, which because they facilitate water absorption suffer from rapid degradation. FS10 is structurally related to the well-known RNAIII inhibiting peptide (RIP) and inhibits S. aureus biofilm formation. Various parameters, including different method (double emulsion and nanoprecipitation), pH of the aqueous phase and polymeric composition, were investigated to load FS10 into PEG-PLGA nanoparticles. The combination of different strategies resulted in an encapsulation efficiency of around 25% for both the double emulsion and the nanoprecipitation method. It was found that the most influential parameters were the pH—which tailors the peptides charge—and the polymeric composition. FS10-PEG-PLGA nanoparticles, obtained under optimized parameters, showed size lower than 180 nm with zeta potential values ranging from −11 to −21 mV. In vitro release studies showed that the Nps had an initial burst release of 48–63%, followed by a continuous drug release up to 21 h, probably caused by the porous character of the Nps. Furthermore, transmission electron microscopy (TEM) analysis revealed particles with a spherical morphology and size of around 100 nm. Antimicrobial assay showed that the minimum inhibitory concentration (MIC) of the FS10-loaded Nps, against S. aureus strains, was lower (>128 µg/mL) than that of the free FS10 (>256 µg/mL). The main goal of this work was to develop polymeric drug delivery systems aiming at protecting the peptide from a fast degradation, thus improving its accumulation in the target site and increasing the drug-bacterial membrane interactions.

Structure-Activity Relationship of New Chimeric Analogs of Mastoparan from the Wasp Venom Paravespula lewisii.

Mastoparan (MP) is an antimicrobial cationic tetradecapeptide with the primary structure INLKALAALAKKIL-NH2. This amphiphilic α-helical peptide was originally isolated from the venom of the wasp Paravespula lewisii. MP shows a variety of biological activities, such as inhibition of the growth of Gram-positive and Gram-negative bacteria, as well as hemolytic activity and activation of mast cell degranulation. Although MP appears to be toxic, studies have shown that its analogs have a potential therapeutic application as antimicrobial, antiviral and antitumor agents. In the present study we have designed and synthesized several new chimeric mastoparan analogs composed of MP and other biologically active peptides such as galanin, RNA III inhibiting peptide (RIP) or carrying benzimidazole derivatives attached to the ε-amino side group of Lys residue. Next, we compared their antimicrobial activity against three reference bacterial strains and conformational changes induced by membrane-mimic environments using circular dichroism (CD) spectroscopy. A comparative analysis of the relationship between the activity of peptides and the structure, as well as the calculated physicochemical parameters was also carried out. As a result of our structure–activity study, we have found two analogs of MP, MP-RIP and RIP-MP, with interesting properties. These two analogs exhibited a relatively high antibacterial activity against S. aureus compared to the other MP analogs, making them a potentially attractive target for further studies. Moreover, a comparative analysis of the relationship between peptide activity and structure, as well as the calculated physicochemical parameters, may provide information that may be useful in the design of new MP analogs.

Role of Daptomycin in Cutaneous Wound Healing: A Narrative Review.

Daptomycin is active against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and the on-label indications for its use include complicated skin and skin structure infections (cSSSI). We performed a narrative review of the literature with the aim to evaluate the role of daptomycin in the skin wound healing process, proposing our point of view on the possible association with other molecules that could improve the skin healing process. Daptomycin may improve wound healing in MRSA-infected burns, surgical wounds, and diabetic feet, but further studies in humans with histological examination are needed. In the future, the combination of daptomycin with other molecules with synergistic action, such as vitamin E and derivates, IB-367, RNA III-inhibiting peptide (RIP), and palladium nanoflowers, may help to improve wound healing and overcome forms of antibiotic resistance.

Methicillin-resistant Staphylococcus aureus as a cause of chronic wound infections: Alternative strategies for management.

Biofilm formation at the level of a wound plays an important role in its chronicization. The difficulty of its eradication has driven research toward the discovery and synthesis of new molecules that can act on biofilm to promote wound healing. This narrative review focuses on alternative molecules that can act and promote the eradication of methicillin-resistant Staphylococcus aureus, taking into consideration its antibiotic resistance, virulence, tendency toward the tenacious colonization of wounds by biofilms, and its increased prevalence in both community and hospital settings. A selection of promising studies were reported, analyzing the in vitro and/or in vivo efficacy of bacteriophages, metal nanoparticles, RNAIII inhibiting peptide (RIP), synthetized RIP derivatives, proteinase K and hamamelitannin.

Staphylococcal Biofilm on the Surface of Catheters: Electron Microscopy Evaluation of the Inhibition of Biofilm Growth by RNAIII Inhibiting Peptide.

Staphylococcus aureus and coagulase-negative staphylococci (CoNS) have become the main causative agents of medical device-related infections due to their biofilm-forming capability, which protects them from the host’s immune system and from the action of antimicrobials. This study evaluated the ability of RNA III inhibiting peptide (RIP) to inhibit biofilm formation in 10 strains isolated from clinical materials, including one S. aureus strain, two S. epidermidis, two S. haemolyticus, two S. lugdunensis, and one isolate each of the following species: S. warneri, S. hominis, and S. saprophyticus. The isolates were selected from a total of 200 strains evaluated regarding phenotypic biofilm production and the presence and expression of the ica operon. The isolates were cultured in trypticase soy broth with 2% glucose in 96-well polystyrene plates containing catheter segments in the presence and absence of RIP. The catheter segments were observed by scanning electron microscopy. The results showed inhibition of biofilm formation in the presence of RIP in all CoNS isolates; however, RIP did not interfere with biofilm formation by S. aureus. RIP is a promising tool that might be used in the future for the prevention of biofilm-related infections caused by CoNS.

The use of RNAIII inhibitory peptide in the treatment of elderly patients with staphylococcus aureus infection and construction of fuzzy mathematical model for curative effect evaluation

The study aims to explore application of RNAIII inhibiting peptide (RIP) and image information counting algorithm in the rehabilitation treatment plan of staphylococcus aureus infection in old people’s bone and joint. A fuzzy mathematical evaluation model is established for the clinical efficacy of osteoarthritis infection, which lays a foundation for the establishment of the expert diagnosis and treatment system of the disease efficacy evaluation. The relationship between the ability of Staphylococcus aureus to form membrane and the concentration of RNAIII inhibiting peptide (RIP) was observed. Additionally, the influence of RIP on the adhesion of Staphylococcus aureus was counted and analyzed by image information counting algorithm. Furthermore, the real-time quantitative PCR (RT-q PCR) was used to detect the mRNA levels of five genes related to biofilm formation, including ica A, ica R, fnb A, atl A, and sar A. It was found that the adhesion ability of Staphylococcus aureus decreased under the effect of RIP, and the results showed that the expression level of ica A, ica R, fnb A, and atl A gene in the experimental group was significantly lower than that in the control group, with statistical difference. After culture for 2 h, 8 h, and 24 h, the expression of ica R gene in the experimental group was 1.45, 1.46, and 1.5, respectively, which were significantly higher than those in the control group, showing statistical significance. There was no significant difference between the two groups in gene expression of sar A. Through the image information counting algorithm for Staphylococcus aureus technology, it was found that RIP could inhibit the adhesion of Staphylococcus aureus and the formation of early biofilm, but the specific regulatory mechanism needs further study. To sum up, according to the results of this study, RIP can be applied to old people's bone and joint diseases caused by Staphylococcus aureus infection, such as pyogenic arthritis and osteomyelitis.

Electrical Evaluation of Bacterial Virulence Factors Using Nanopores.

In this work, we propose a novel methodology for electrical monitoring using nanoporous alumina membranes of virulence factors secreted by bacterial pathogens. Bacterial hyaluronidase (HYAL), which is produced by a number of invasive Gram-positive bacteria, is selected as a model compound to prove the concept. Our electrochemical setup takes advantage of the flat surface of indium tin oxide/poly(ethylene terephthalate) (ITO/PET) electrodes for their assembly with the nanoporous membrane. The proposed analytical method, based on the electrical monitoring of the steric/electrostatic nanochannels blocked upon formation of an antibody-HYAL immunocomplex, reached detection limits as low as 64 UI/mL (17.3 U/mg) HYAL. The inert surface of the ITO/PET electrodes together with the anti-biofilm properties of the 20 nm pore-sized alumina membranes allows for culturing the bacteria, capturing the secreted enzymes inside the nanochannels, and removing the cells before the electrochemical measurement. Secreted HYAL at levels of 1000 UI/mL (270 U/mg) are estimated in Gram-positive Staphylococcus aureus cultures, whereas low levels are detected for Gram-negative Pseudomonas aeruginosa (used as a negative control). Finally, HYAL secretion inhibition by RNAIII-inhibiting peptide (YSPWTNF-NH2) is also monitored, opening the way for further applications of the developed monitoring system for evaluation of the antivirulence potential of different compounds. This label-free method is rapid and cheap, avoiding the use of the time-consuming sandwich assays. We envisage future applications for monitoring of bacterial virulence/invasion as well as for testing of novel antimicrobial/antivirulence agents.

RNAIII Inhibiting Peptide (RIP) and Derivatives as Potential Tools for the Treatment of S. aureus Biofilm Infections.

S. aureus under the biofilm mode of growth is often related to several nosocomial infections, more frequently associated with indwelling medical devices (catheters, prostheses, portacaths or heart valves). As a biofilm, the biopolymer matrix provides an excellent growth medium, increasing the tolerance to antibiotics and host immune system. To date, the antimicrobial therapy alone is not effective. A novel strategy to prevent biofilm formation is based on the interference with the bacterial cell-cell communication, a process known as quorum sensing (QS) and mediated by the RNA-III-activating peptide (RAP) and its target protein TRAP (Target of RAP). The RNAIII inhibiting peptide (RIP) is able to inhibit S. aureus pathogenesis by disrupting QS mechanism competing with RAP, thus inhibiting the phosphorylation of TRAP. This alteration leads to a reduced adhesion and to the inhibition of RNAIII synthesis, with the subsequent suppression of toxins synthesis. The present paper will provide an overview on the activity and potential applications of RIP as biofilm inhibiting compound, useful in the management of S. aureus biofilm infections. Moreover, medicinal chemistry strategies have been examined to better understand which modifications and/or structure alterations were able to produce new derivatives of this QS inhibitor with an improved antibiofilm activity.

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.

Inhibiting PSM\u03b1-induced neutrophil necroptosis protects mice with MRSA pneumonia by blocking the agr system

Given its high resistance, enhanced virulence, and high transmissibility, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pneumonia is highly associated with high morbidity and mortality. Anti-virulence therapy is a promising strategy that bypasses the evolutionary pressure on the bacterium to develop resistance. RNAIII-inhibiting peptide (RIP), as an accessory gene regulator (agr)-specific inhibitor, significantly restricts the virulence of S. aureus and protects infected mice from death by blocking the agr quorum sensing system. The protective effects of RIP on the neutropenic mice completely disappeared in a neutrophil-deleted mouse infection model, but not in the macrophage-deleted mice. This result confirmed that the in vivo antibacterial activity of RIP is highly associated with neutrophil function. Phenol-soluble modulins (PSMs), as major leukocyte lysis toxins of CA-MRSA, are directly regulated by the agr system. In this experiment, PSMα1, 2, and 3 significantly induced neutrophil necroptosis by activating mixed lineage kinase-like protein (MLKL) phosphorylation and increasing lactate dehydrogenase release. The S. aureus supernatants harvested from the agr or psmα mutant strains both decreased the phosphorylation level of MLKL and cell lysis. PSMα1-mediated neutrophil lysis was significantly inhibited by necrosulfonamide, necrostatin-1, TNFα antibody, and WRW4. These results showed PSMα1 induced necroptosis depends on formylpeptide receptor 2 (FPR2)-mediated autocrine TNFα. Moreover, the neutrophil necroptosis induced by S. aureus was significantly suppressed and pneumonia was effectively prevented by the blockage of agrA and psmα expression levels. These findings indicate that PSMα-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest that interfering with the agr quorum sensing signaling pathway is a potential therapeutic strategy.

Efficacy of the Quorum Sensing Inhibitor FS10 Alone and in Combination with Tigecycline in an Animal Model of Staphylococcal Infected Wound.

In staphylococci, quorum sensing regulates both biofilm formation and toxin production, moreover it has been demonstrated to be inhibited by RNAIII inhibiting peptide (RIP). Aim our study was to evaluate the in vitro activity and its in vivo efficacy of the combined administration of FS10, a novel RIP derivative, and tigecycline in an animal model of methicillin-resistant (MR) and methicillin-sensitive (MS) Staphylococcus aureus wound infection. Using a 1.x2 cm template, one full thickness wound was established through the panniculus carnosus on the back subcutaneous tissue of each animal. Infection was determined by inoculation of 5x107 CFU/ml of bacteria, that produced an abscess within 24 h, after this, treatment was initiated. The study included, for each strain, a control group without infection, a control infected group that did not receive any treatment and a control infected group with drug-free foam dressing, and three infected groups treated, respectively, with: FS10-soaked foam dressing (containing 20 μg FS10), daily intraperitoneal tigecycline (7 mg/Kg), FS10-soaked foam dressing (containing 20 μg FS10) and daily intraperitoneal injections of tigecycline (7 mg/Kg). The main outcome measures were quantitative culture and histological examination of tissue repair. The highest inhibition of infection was achieved in the group that received FS10-soaked and parenteral tigecycline reducing the bacterial load from 107 CFU/ml to about 103 CFU/g for MSSA and to about 104 CFU/g for MRSA. The group treated with FS10-soaked foam dressing associated with parenteral tigecycline showed, histologically, better overall healing with epithelialization and collagen scores significantly higher than those of the other groups in both strains. In conclusion, the combined use of topical FS10 with i.p. tigecycline induced positive interaction in vivo, resulting in an enhanced therapeutic benefit versus staphylococcal infections in murine wound models.

Oligomerization of RNAIII-Inhibiting Peptide Inhibits Adherence and Biofilm Formation of Methicillin-Resistant Staphylococcus aureus In Vitro and In Vivo.

Biofilm formation enhances bacterial resistance and complicates treatment. Therefore, an innovative strategy is urgently needed for the treatment of Staphylococcus aureus biofilm infectious diseases. RNAIII-inhibiting peptide (RIP), as a quorum-sensing inhibitor, inhibits S. aureus biofilm formation. However, RIP possesses poor antibiofilm activity when used alone or at a low dose in vivo. The activity and stability of RIP can be enhanced by designing its derivatives through amino acid substitution, terminal modification, or oligomerization. Among the derivatives, 16P-AC significantly decreased the biofilm formation and adherence of methicillin-resistant S. aureus (MRSA) on polystyrene material by inhibiting the expression level of four biofilm formation-related genes in vitro. Moreover, 16P-AC showed excellent protective effects by decreasing the bacterial titers in the urine, kidney, stent, and bladder, as well as by inhibiting intercellular adhesion on the implanted stent, in a rat urinary tract infection model induced by MRSA. This derivative also exhibited a relatively good stability in rat plasma. Therefore, 16P-AC is a potential drug candidate to treat biofilm-associated infections caused by MRSA. The present modification strategy is feasible to improve the metabolic stability and activity of RIP in vivo.

Discovery of novel RIP derivatives by alanine scanning for the treatment of S. aureus infections

Alanine scanning has been used to define the key residues for the activity of RNA III inhibiting peptide (RIP) against S. aureus. We have found that the threonine residue at position 5 is detrimental to the RIP activity, while the removal of serine (P2) and phenylalanine (P7) afforded the most active RIP derivatives (2 and 7). Moreover, we have identified the small RIP derivative (9), corresponding to the sequence H-Ser-Pro-Trp-Thr-NH2, which displayed the best antistaphylococcal activity.

Molecular Mechanisms of RIP, an Effective Inhibitor of Chronic Infections

Non-healing bacterial infections are often associated with the formation of a biofilm, where bacteria are more resistant to conventional treatment modalities and to host immune responses. We show here that RNAIII inhibiting peptide (RIP), a linear heptapeptide, is very effective in treating severe polymicrobial infections, including drug-resistant staphylococci like MRSA. By functional genomics studies (microarray analysis) on Staphylococcus aureus, we show here that RIP downregulates the expression of genes involved in biofilm formation and toxin production, and upregulates genes involved in stress response. This pattern of gene regulation may explain why RIP has been so effective in treating severe infections and hopefully through the addition of RIP to existing protocols, a new way of tackling chronic persistent infections will be established.

Suppression of Biofilm Related, Device-Associated Infections by Staphylococcal Quorum Sensing Inhibitors

Staphylococcal spp. are notorious for causing biofilm-related device-associated infections, leading to tens of thousands of deaths per year. In this paper, we review quorum sensing inhibitors as potential therapeutics for even the most persistent infections. The animal models reviewed are subcutaneous graft, central venous catheter (CVC), ureteral stent and wound models, and a wound case study. The therapeutic approaches reviewed are the use of RNAIII inhibiting peptide (RIP) and its non-peptide analog. These have been shown to prevent or treat infections caused by any staphylococcal strain tested, including antibiotic-resistant strains like CA-MRSA USA300.

RNAIII-Inhibiting Peptide Enhances Healing of Wounds Infected with Methicillin-Resistant Staphylococcus aureus

Quorum sensing is a mechanism through which a bacterial population receives input from neighboring cells and elicits an appropriate response to enable survival within the host. Inhibiting quorum sensing by RNAIII-inhibiting peptide (RIP) has been demonstrated as a very effective mode of prevention and therapy for device-associated staphylococcal infections and was tested here for healing of wounds that are otherwise resistant to conventional antibiotics. Wounds, established through the panniculus carnosus of BALB/c mice, were inoculated with 5 x 10(7) CFU of methicillin-resistant Staphylococcus aureus. Mice were treated with Allevyn, RIP-soaked Allevyn (containing 20 microg RIP), daily intraperitoneal teicoplanin (7 mg/kg of body weight), Allevyn and teicoplanin, and RIP-soaked Allevyn and daily intraperitoneal teicoplanin. The main outcome measures were quantitative bacterial culture and histological examination with assessment of microvessel density and of vascular endothelial growth factor (VEGF) expression in tissue sections. Treatment with RIP-soaked Allevyn together with teicoplanin injection greatly reduced the bacterial load to 13 CFU/g (control untreated animals had 10(8) CFU/g bacteria). All other treatments were also significantly effective but only reduced the bacterial load to about 10(3) CFU/ml. Histological examination indicated that only treatment with RIP-soaked Allevyn with teicoplanin injection restored epithelial, granulation, and collagen scores, as well as microvessel density and VEGF expression, to the levels found with uninfected mice. In conclusion, we observed that RIP may be useful for the management of infected wounds and that it could represent an exciting and future alternative to the conventional antibiotics, at present considered the gold-standard treatments for methicillin-resistant S. aureus infections.

Discovery of a Quorum-Sensing Inhibitor of Drug-Resistant Staphylococcal Infections by Structure-Based Virtual Screening

Staphylococci are a major health threat because of increasing resistance to antibiotics. An alternative to antibiotic treatment is preventing virulence by inhibition of bacterial cell-to-cell communication using the quorum-sensing inhibitor RNAIII-inhibiting peptide (RIP). In this work, we identified 2',5-di-O-galloyl-d-hamamelose (hamamelitannin) as a nonpeptide analog of RIP by virtual screening of a RIP-based pharmacophore against a database of commercially available small-molecule compounds. Hamamelitannin is a natural product found in the bark of Hamamelis virginiana (witch hazel), and it has no effect on staphylococcal growth in vitro; but like RIP, it does inhibit the quorum-sensing regulator RNAIII. In a rat graft model, hamamelitannin prevented device-associated infections in vivo, including infections caused by methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains. These findings suggest that hamamelitannin may be used as a suppressor to staphylococcal infections.

RNAIII-Inhibiting Peptide Affects Biofilm Formation in a Rat Model of Staphylococcal Ureteral Stent Infection

Ureteral stents coated with the quorum-sensing inhibitor RNAIII-inhibiting peptide (RIP) were implanted in rat bladders and shown to suppress Staphylococcus aureus formation on the stent and in urine and was especially effective when combined with teicoplanin. Coating ureteral stents with RIP thus increases the efficacy of teicoplanin in preventing ureteral stent-associated staphylococcal infections.

Bacterial Communications in Implant Infections: A Target for an Intelligence War

The status of population density is communicated among bacteria by specific secreted molecules, called pheromones or autoinducers, and the control mechanism is called ""quorum-sensing"". Quorum-sensing systems regulate the expression of a panel of genes, allowing bacteria to adapt to modified environmental conditions at a high density of population. The two known different quorum systems are described as the LuxR-LuxI system in gram-negative bacteria, which uses an N-acyl-homoserine lactone (AHL) as signal, and the agr system in gram-positive bacteria, which uses a peptide-tiolactone as signal and the RNAIII as effector molecules. Both in gram-negative and in gram-positive bacteria, quorum-sensing systems regulate the expression of adhesion mechanisms (biofilm and adhesins) and virulence factors (toxins and exoenzymes) depending on population cell density. In gram-negative Pseudomonas aeruginosa, analogs of signaling molecules such as furanone analogs, are effective in attenuating bacterial virulence and controlling bacterial infections. In grampositive Staphylococcus aureus, the quorum-sensing RNAIII-inhibiting peptide (RIP), tested in vitro and in animal infection models, has been proved to inhibit virulence and prevent infections. Attenuation of bacterial virulence by quorum-sensing inhibitors, rather than by bactericidal or bacteriostatic drugs, is a highly attractive concept because these antibacterial agents are less likely to induce the development of bacterial resistance.

RNAIII-Inhibiting-Peptide-Loaded Polymethylmethacrylate Prevents In Vivo Staphylococcus aureus Biofilm Formation

Staphylococci, common orthopedic pathogens, form antibiotic-resistant biofilms. Polymethylmethacrylate (PMMA) beads loaded with the quorum-sensing inhibitor RNAIII-inhibiting peptide (RIP) were implanted in rats and shown to prevent methicillin-resistant Staphylococcus aureus infection. RIP release was bimodal, typical of previously-tested antibiotics. These results suggest that RIP-PMMA warrants further evaluation for management of orthopedic infections caused by staphylococci.