Identification and evaluation of a novel peptide binding to the cell surface of Staphylococcus aureus.

Identification of short peptides that serve as specific ligands to biological materials such as microbial cell surfaces has major implications in better understanding the molecular recognition of cell surfaces. In this study we screened a commercially available random phage-display library against Staphylococcus aureus cells and identified peptides specifically binding to the bacteria. A synthetic peptide (SA5-1) representing the consensus sequence (VPHNPGLISLQG) of the bacteria-binding peptide was evaluated for its binding potential against S. aureus. Dot-blot, immunoblot assay and ELISA results revealed the SA5-1 peptide to be highly specific to S. aureus. The SA5-1 peptide binding was optimal between pH 6.0 and 8.0. Nanogold Transmission Electron Microscopy demonstrated that the SA5-1 binds to the outer membrane surface of S. aureus. Diagnostic potential of the SA5-1 peptide was evaluated in human platelet samples spiked with S. aureus and specific detection of the bacteria by biotinylated-SA5-1 and streptavidin-conjugated fluorescent quantum dots. Fluorometry results indicated that the peptide was able to detect ∼100 organisms per ml in a spiked biological sample providing a proof-of-concept towards potential of this peptide as a S. aureus diagnostic tool that can be of use in different detection platforms.

Detection technologies for Bacillus anthracis: prospects and challenges.

Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological agent of acute infectious disease anthrax, a lethal but rare disease of animals and humans in nature. With recent use of anthrax as a bioweapon, a number of techniques have been recently developed and evaluated to facilitate its rapid detection of B. anthracis in the environment as well as in point-of-care settings for humans suspected of exposure to the pathogen. Complex laboratory methods for B. anthracis identification are required since B. anthracis has similarities with other Bacillus species and its existence in both spore and vegetative forms. This review discusses current challenges and various improvements associated with anthrax agent detection.

Antiviral activity of selected antimicrobial peptides against vaccinia virus.

Antimicrobial peptides (AMPs) are gaining importance as effective therapeutic alternatives to conventional antibiotics. Recently we have shown that a set of nine synthetic antimicrobial peptides, four originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1-PD4 and five synthetic repeats of arginine-tryptophan (RW) repeats (RW1-5) demonstrate antibacterial activity in plasma and platelets. Using WR strain of vaccinia virus (VV) as a model virus for enveloped virus in the present study, we tested the same nine synthetic peptides for their antiviral activity. A cell culture-based standard plaque reduction assay was utilized to estimate antiviral effectiveness of the peptides. Our analysis revealed that peptides PD3, PD4, and RW3 were virucidal against VV with PD3 demonstrating the highest antiviral activity of 100-fold reduction in viral titers, whereas, PD4 and RW3 peptide treatments resulted in 10-30-fold reduction. The EC(50) values of PD3, PD4 and RW3 were found to be 40 microg/ml, 50 microg/ml and 6.5 microM, respectively. In VV-spiked plasma samples, the virucidal activity of PD3, PD4 and RW3 was close to 100% (90-100-fold reduction). Overall, the present study constitutes a new proof-of-concept in developing peptide therapeutics for vaccinia virus infections in biothreat scenarios and as in vitro viral reduction agents.

Evaluation of antimicrobial peptides as novel bactericidal agents for room temperature-stored platelets.

BACKGROUND: A single cost-effective pathogen inactivation approach would help to improve the safety of our nation's blood supply. Several methods and technologies are currently being studied to help reduce bacterial contamination of blood components. There is clearly need for simple and easy-to-use pathogen inactivation techniques specific to plasma, platelets (PLTs), and red blood cells. STUDY DESIGN AND METHODS: In this report, we introduce a novel proof of concept: using known therapeutic antimicrobial peptides (AMPs) as bactericidal agents for room temperature-stored PLT concentrates (PCs). Nine synthetic AMPs, four from PLT microbicidal protein-derived peptides (PD1-4) and five Arg-Trp (RW) repeat peptides containing one to five repeats, were tested for bactericidal activity in plasma and PC samples spiked with Staphylococcus aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus cereus. A 3-log reduction of viable bacteria was considered as the bactericidal activity of a given peptide. RESULTS: In both plasma alone and PCs, RW3 peptide demonstrated bactericidal activity against S. aureus, S. epidermidis, E. coli, P. aeruginosa, and K. pneumoniae; PD4 and RW2 against P. aeruginosa; and RW4 against K. pneumoniae. The activity of each of these four peptides against the remaining bacterial species in the test panel resulted in less than a 3-log reduction in the number of viable bacteria and hence considered ineffective. CONCLUSIONS: These findings suggest a new approach to improving the safety of blood components, demonstrating the potential usefulness of screening therapeutic AMPs against selected bacteria to identify suitable bactericidal agents for stored plasma, PCs, and other blood products.

In vitro activities of the newly synthesised ER-2 against clinical isolates of Mycobacterium tuberculosis susceptible or resistant to antituberculosis drugs.

Two hundred isolates of Mycobacterium tuberculosis were evaluated for their susceptibility to a newly synthesised quinolone derivative, ER-2, compared with ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin. ER-2 and moxifloxacin showed the greatest activity [MIC for 90% of strains tested (MIC(90))=0.5 microg/mL], although levofloxacin and ciprofloxacin showed good activity with an MIC(90) of 1 microg/mL. More importantly, ER-2 showed excellent activity against M. tuberculosis H37Rv both in the lungs and spleen of mice, indicating the potential therapeutic value of ER-2 against M. tuberculosis.

Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR.

Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of gene expression of E. coli gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of E. coli, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. RT-PCR experiment was done to investigate the down regulation of gyrase A gene. RT-PCR results demonstrated a significant decrease of gyrase A gene after 18 h of induction of the transformants. These experiments showed that the down regulation of the gene was seen after 18 h of induction than earlier hours of induction with IPTG suggesting inhibition of gyrase A gene with profound effect on cell viability. These results demonstrate the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins.