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crc press - E-Lib FK UWKS

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Microbial Membrane-Permeating Peptides and Their Applications 383<br />

Although cationic peptide uptake into bacteria is viewed differently by many<br />

researchers, it is accepted that electrostatic attraction is often an important first step<br />

in uptake. Consistent with this, it is generally observed that increased salt concentrations<br />

shield the interaction and reduce peptide potency. Also, resistance can arise<br />

from cell barrier modifications that limit permeation. For example, resistant Salmonella<br />

cells contain modified LPS molecules with reduced membrane negative charge<br />

density. 33 Thus, an initial electrostatic attraction appears to be needed for cellpermeating<br />

peptides to enter cells. However, some variations and exceptions exist<br />

within this rule. For example, certain cationic peptides are more salt tolerant and<br />

some antimicrobial peptides are not cationic. 34<br />

Based on this general view of peptide permeation, numerous synthetic cationic<br />

peptides in the range of 10–20 amino acids have been made with variations in their<br />

amphipathic structure. The peptides have been subjected to a variety of biochemical<br />

analysis with artificial membranes in order to elucidate the mechanism of action, as<br />

reviewed. 28-30,35 However, given uncertainties surrounding the mechanism of action<br />

of cell-permeating peptides and the possibility for several different mechanisms, the<br />

design process remains uncertain.<br />

18.3.1.2 Receptor-Mediated Transport of Peptides and Endocytosis<br />

A variety of peptide sequences are known to enter microbial cells via receptormediated<br />

translocation or permeases; these mechanisms certainly offer opportunities<br />

to deliver foreign substances into microbial cells. Furthermore, receptor-mediated<br />

delivery is a very attractive approach for cellular delivery because of the potential<br />

for cell type specificity. The best known receptor-mediated transport mechanism in<br />

microorganisms involves the oligopeptide permeases, which import di- and trioligopeptides.<br />

There are multiple oligopeptide permeases in microbial cells, and overlapping<br />

substrate specificity between species. For more information on receptormediated<br />

uptake we direct readers to two excellent reviews. 36,37<br />

Endocytosis also offers a range of attractive possibilities for cell delivery; however,<br />

it is very complex and the intracellular fate of internalized vesicles is difficult<br />

to predict. Furthermore, most substances that undergo endocytosis are not released<br />

into the cytoplasm or able to reach the nucleus. Nevertheless, endocytosis is used<br />

by many bacterial toxins to reach the intracellular targets in eukaryotic hosts, so<br />

there are possibilities for practical applications. 38,39 We direct interested readers to<br />

an excellent recent review by Sandvig and van Deurs. 40<br />

18.3.2 CELL-KILLING MECHANISMS<br />

18.3.22.1 Membrane Leakage<br />

Membrane leakage is usually referred to as the main cytotoxic activity of antimicrobial<br />

peptides. However, it is not clear which events actually lead to microbial<br />

death by most antimicrobial peptides. There is no doubt that antimicrobial peptides<br />

interact with membranes, but several different hypotheses explain how this could<br />

lead to cell death. These include physical disturbance of membrane integrity, fatal

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