Antimicrobial activity of the recombinant designer host defence peptide P-novispirin G10 in infected full-thickness wounds of porcine skin.

OBJECTIVES: The growing number of patients with impaired wound healing and the development of multidrug-resistant bacteria demand the investigation of alternatives in wound care. The antimicrobial activity of naturally occurring host defence peptides and their derivatives could be one alternative to the existing therapy options for topical treatment of wound infection. Therefore, the aim of this study was to investigate the antimicrobial activity of proline-novispirin G10 (P-novispirin G10) in vitro and in the infected porcine titanium wound chamber model. METHODS: The new derived designer host defence peptide P-novispirin G10 was tested in vitro against Gram-positive and Gram-negative bacterial strains. Additionally, cytotoxicity and haemolytic activities of P-novispirin G10 and protegrin-1 were measured. For in vivo studies, six wound chambers were implanted on each flank of Göttinger minipigs (n = 2, female, 6 months old, 15-20 kg). Eleven wound chambers were inoculated 8 days post-operatively with 5 x 10(8) of Staphylococcus aureus; one wound chamber remained uninfected as a system control. After wound infection had been established (4 days after inoculation), each wound chamber was topically treated with P-novispirin G10, protegrin-1 or carrier control. Wound fluid was harvested every hour for a total follow up of 3 h. RESULTS: P-novispirin G10 demonstrated broad-spectrum antimicrobial activity with moderate haemolytic and cytotoxic activities compared with protegrin-1. In the infected wound chamber model P-novispirin G10 demonstrated a 4 log(10) reduction in bacterial counts. CONCLUSIONS: This implicates the potential of P-novispirin G10 as an alternative in future antimicrobial wound care. However, more studies are necessary to further define clinical applications and potential side effects in greater detail.

Improving on nature's defenses: optimization & high throughput screening of antimicrobial peptides.

Antimicrobial peptides (AMPs) are ubiquitous in nature where they play important roles in host defense and microbial control. Despite their natural origin, antimicrobial spectrum and potency, the lead peptide candidates that so far have entered pharmaceutical development have all been further optimized by rational or semi-rational approaches. In recent years, several high throughput screening (HTS) systems have been developed to specifically address optimization of AMPs. These include a range of computational in silico systems and cell-based in vivo systems. The in silico-based screening systems comprise several computational methods such as Quantitative Structure/Activity Relationships (QSAR) as well as simulation methods mimicking peptide/membrane interactions. The in vivo-based systems can be divided in cis-acting and trans-acting screening systems. The cis-acting pre-screens, where the AMP exerts its antimicrobial effect on the producing cell, allow screening of millions or even billions of lead candidates for their basic antimicrobial or membrane-perturbating activity. The trans-acting screens, where the AMP is secreted or actively liberated from the producing cell and interacts with cells different from the producing cell, allow for screening under more complex and application-relevant conditions. This review describes the application of HTS systems employed for AMPs and lists advantages as well as limitations of these systems.

Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards.

A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards for the tested sample by enzymatic hydrolysis of the same sample (with known concentration) in (18)O-water. A mathematical algorithm was developed which uses the isotopic patterns of the substance, the internal standard, and the substance/internal standard mixture for accurate quantitation of the substance. A great advantages of the proposed method is the absence of molecular weight limitation for the protein quantitation and the possibility of quantitation without previous fractionation of proteins and peptides. Using this strategy, the peptide angiotensinogen and two proteins, RNase and its protein inhibitor, were quantified by MALDI-time-of-flight (TOF) mass spectrometry.

Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometry.

We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 microL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB.

Combining MALDI mass spectrometry and biomolecular interaction analysis using a biomolecular interaction analysis instrument.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been combined with biomolecular interaction analysis (BIA) in a Biacore instrument. A method has been developed for the recovery of the affinity-bound molecules from the sensor chip in a few microliters ready for mass spectrometric analysis. The procedure is illustrated with two molecular systems which exemplify antibody-antigen and DNA-protein interactions. In both cases, femtomole quantities of the affinity-bound proteins were eluted and subsequently detected by MALDI-MS. Whereas the Biacore analysis yields the surface concentration of protein bound to the sensor chip, identity of the bound compounds is revealed in the second step by accurate molecular mass determination. Combining the information of the two analyses allows calculation of the total surface molar concentration of affinity-bound molecules.