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380 Cell-Penetrating Peptides: Processes and Applications
between species. In the case of Gram-negative bacteria, the cell barrier consists of
two phospholipid bilayers, with the inner membrane surrounded by a peptidoglycan
layer and the outer coated on the exterior by a lipopolysaccharide (LPS) layer
(Figure 18.1). 13 The LPS layer provides Gram-negative bacteria with a particularly
stringent sieve against foreign substances.
The cell barrier of eukaryotic cells typically presents less of an obstacle to
permeating peptides because the main structure is the phospholipid bilayer. Most
mammalian cells have little additional coating as protection. In contrast, fungal
plasma membranes are enriched in inositol phosphoceramides and a thick outer
protective cell wall composed of β-1,3 glucan and β-1,6 glucans and chitin. 14 In
some cases the fungal cell wall can amount to as much as 40% of the dry cell mass.
Therefore, fungi are similar to bacteria in having thick and complex barriers against
cell-permeating peptides and other foreign substances.
18.2.2 ORIGINS AND DISCOVERY OF MICROBIAL CELL-PERMEATING PEPTIDES
Antimicrobial peptides were discovered as a group of small, cationic peptides, and
it was soon found that these peptides possessed potent bactericidal and fungicidal
properties. 15-17 Antimicrobial peptides have been isolated from most vertebrate and
invertebrate material studied, such as the blood and epithelia of animals, plants,
hemolymph of insects, and venom of scorpions. The multitude of peptides discovered,
more than 500, are cataloged and updated in a database (www.bbcm.univ.trieste.it/~tossi/antimic.html).
The diverse occurrence and similar activities of antimicrobial
peptides indicate an ancient origin and conserved role in host immunity to
pathogens. 18,19 Indeed, during the last two decades, antimicrobial peptides have
become well recognized as important within the innate and the humoral immune
responses.
18.2.3 COMPOSITION AND STRUCTURE OF MICROBIAL CELL-PERMEATING PEPTIDES
The naturally occurring antimicrobial peptides are normally 12–50 amino acids long
(occasionally up to 80 amino acids) and share two main characteristics. First, they
are polycationic with a positive net charge of more than +2 (due to an excess of
primarily lysine and arginine residues). Second, they have the ability to form amphipathic
structures with both positively charged and hydrophobic regions (see
Figure 18.2b). The hydrophobic content is generally around 50% and this face
promotes interaction with membrane lipids. The positively charged face promotes
interaction with water and negatively charged lipids and cell wall structures. This
organization is ideal for a molecule that needs to be soluble in water and also
lipophylic.
Apart from their positive charge and amphipathic features, antimicrobial peptides
can be categorized into different structural classes (Figure 18.2a), including peptides
composed of α-helices, β-sheet, loops, and extended sequences. The most common
classes are linear peptides that form amphipathic α-helices in contact with membranes
and the cystein-containing peptides, which are usually β-sheet peptides constrained
by two to four disulfide bonds. Less common are linear peptides with a