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382 Cell-Penetrating Peptides: Processes and Applications
example, the recently discovered theta-defensin from Rhesus macaque is a macrocyclic
peptide with three disulfide bonds and a structure similar to protegrin from
pig. 21 The same motif is also found in macrocyclic peptides from plants, although
the cross-linking arrangement of disulfide bonds differs. 22 Interestingly, another
macrocylic peptide AS-48, a plasmid-encoded peptide expressed by Enterococcus
faecalis, shows remarkable structural similarities with porcine NK-lysin in terms of
size and α-helical fold. 23 Therefore, among natural antimicrobial peptides, a range
of structural arrangements can provide stable and active peptides.
When designing antimicrobial peptides, another strategy is to incorporate nonnatural
amino acids. For example, peptides containing D-amino acids can display
improved antimicrobial activities because they retain their activity but are more
resistant to proteases. 24-26 In our studies (Andersson et al., unpublished), we have
noticed that Mycobacterium smegmatis is insensitive to certain α-helical peptides,
while the corresponding D-forms have higher activities. Furthermore, it is possible
to synthesize short cyclic amphiphilic peptides with both L- and D-amino acids, and
these modified circular peptides display good protease resistance. 27
18.3 MECHANISMS OF ACTION
18.3.1 CELL UPTAKE
18.3.1.1 Cell Permeation by Cationic Peptides
In broad terms, microbial cell permeation by peptides can be seen as a three-step
process. The first event is an electrostatic attraction of the peptide to the membrane
surface (Figure 18.2b). The second involves membrane disruption and penetration
and, possibly, some reorganization of the peptide structure. As peptides accumulate
at the surface and start to displace lipids, this would eventually lead to local disruptions
of the membrane (“carpet model”) or perhaps formation of transient peptidelipid
“wormhole” pores. 28-30 Such disorganization or pore formation could allow
cellular components to leak out and weaken the bacterial wall, creating opportunities
for peptides to translocate into the inner membrane and subsequently into the
cytoplasm. It also seems possible for peptides to translocate without causing significant
membrane disruption. 31,32 Finally, in the third step the peptide passes the
membrane and gains access to intracellular processes.
In a more specific example, cationic peptide uptake into Gram-negative bacteria
can be seen as a “self promoted” process, reflecting the fact that uptake is receptor
independent. 5 According to this view, cationic peptides would contact the outer
membrane through electrostatic attraction between positively charged antimicrobial
peptides and negatively charged membrane lipids and the LPS layer. At this point
peptides could compete for divalent cation binding sites within the outer cell membrane.
In Escherichia coli, such competition would disrupt stabilizing cross-linking
between LPS molecules and therefore reduce outer membrane integrity, which would
provide opportunities for peptides to enter the membrane. The membrane charge
gradient (inside negative) would then force cationic peptides into cells.