Antimicrobial peptide PGQ

Antimicrobial peptides are widely distributed in nature and appear to play a role in the host defense of plants and animals. In this study we report the existence of antimicrobial peptides in the stomach of the vertebrate Xenopus laevis, an animal previously shown to store high concentrations of antimicrobial peptides in its skin. Antimicrobial activity was detected in extracts of X. laevis stomach tissue and nine antimicrobial peptides were then purified. A novel 24-amino acid peptide, designated PGQ, was isolated from these extracts, and has the following amino acid sequence: GVLSNVIGYLKKLGTGALNAVLKQ. PGQ is relatively basic and has the potential to form an amphipathic alpha-helix. The other peptides isolated are members of the magainin family of antimicrobial peptides, and include magainins I and II, PGLa, xenopsin precursor fragment, and four caerulein precursor fragments. None of these peptides had been previously identified in tissues other than the skin. The purification of the peptides from stomach extracts and subsequent protein sequence analysis reveals that the peptides have undergone the same processing as their dermal counterparts, and that they are stored in their processed forms. Northern blot analysis indicates that the magainin family of peptides are synthesized in the stomach, and immunohistochemical studies demonstrate that magainin is stored in a novel granular multinucleated cell in the gastric mucosa of Xenopus. This study demonstrates that the magainin family of antimicrobial peptides is found in the gastrointestinal system of X. laevis and offers an opportunity to further define the physiological role of these defense peptides.

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.

Multidrug resistant (MDR) Gram-negative bacteria are an urgent global health threat. We report on the design and evaluation of a xenosiderophore-conjugated cationic random copolymer (pGQ-DG) which exhibits selective antibacterial activity against Pseudomonas aeruginosa (P. aeruginosa) by targeting select outer membrane (OM) receptors for scavenging xenosiderophores such as deferoxamine (DFO), while possessing favorable cytocompatibility and exhibiting low hemolysis, to enhance and safely damage the bacterial OM. pGQ-DG demonstrated synergistic properties in combination with vancomycin (VAN) when evaluated in vitro against P. aeruginosa. In addition, pGQ-DG plus VAN cleared the P. aeruginosa infection and efficiently accelerated healing in a murine wound healing model as effectively as colistin, suggesting that this strategy could serve as an alternative to colistin against MDR bacteria. STATEMENT OF SIGNIFICANCE: P. aeruginosa exhibits intrinsic antibiotic resistance due to limited permeability of its outer membrane (OM). A triple combination antipseudomonal approach was investigated by 1) selectively targeting P. aeruginosa through the complex DFO:gallium, 2) disrupting the OM through a cationic random copolymer, and 3) enhancing bacteria sensitivity to VAN as a result of the OM disruption. Synthesis and characterization of the lead polymer pGQ-DG, mechanism of action, antimicrobial activity, and biocompatibility were investigated in vitro and in vivo. Overall pGQ-DG plus VAN cleared the P. aeruginosa infection and accelerated wound healing in mice as effectively as colistin, suggesting that this strategy could serve as an alternative to colistin against multidrug resistant P. aeruginosa.