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Microbial Membrane-Permeating Peptides and Their Applications 385
18.3.3 CELL TYPE-SPECIFIC ACTIVITIES
If microbial cell-permeating peptides are to prove useful in therapeutics, their cellpermeating
properties must be largely specific for microbial cell membranes. Cell
type-specificity is an important feature of conventional antibiotics that allows pathogen
killing at concentrations that are not cytotoxic to host cells. 50 The activity of
natural antimicrobial peptides and the safety of foods that contain such peptides
indicates that antimicrobial peptides possess the needed specificity to be useful as
therapeutics. 1 Many antimicrobial peptides and their derivatives display a therapeutic
window in which they can kill microbial cells without damaging host cells.
Bacterial membranes have a more negatively charged outer leaf than mammalian
cells, which provides some basis for selective activities. Tossi et al. studied 150
linear peptides and suggested that both position of the amphipathic structure and
net charge can influence peptide specificity. 35 Also, the differing sterol content
between mammalian and microbial membranes is often regarded as a reason for
selectivity. Cholesterol inhibits antimicrobial peptide activity, possibly by providing
improved membrane stability, but other differences could also affect activity. Furthermore,
many antimicrobial peptides display selective activity against certain
classes of microorganisms, such as Gram-positive bacteria or fungi. 51 Therefore, cell
wall differences between species (Figure 18.1) provide a basis for cell type-specific
killing by membrane active peptides, although the basis for this selectivity remains
unclear.
18.4 METHODS
18.4.1 ANTIMICROBIAL ACTIVITY ASSAYS
Several methods can be used to test antimicrobial activity. The three most common
are the thin agar inhibition zone assay, microdilution assay, and colony forming units
assay. 52,53 These methods are described in detail elsewhere, but a brief description
is appropriate here. 54
The inhibition zone assay is based on bacterial growth on agar plates. Small
holes (usually 3 mm) are punched in the agar and small samples placed within. The
plates are incubated and active peptides will diffuse and prevent microbial growth,
seen as a zone of clearing. The diameter of the zone is proportional (exponential
relationship) to peptide activity and can be given as zone diameter or as activity
units relative to a standard peptide. The lethal concentration (LC) can be determined
as the lowest amount of peptide needed to kill bacteria. The method is simple and
the sensitivity is down to 30 ng for a peptide with an LC-value of approximately
1 µM; however, a disadvantage is that results are influenced by diffusion properties
of the peptide and it is only suitable for rapid bacterial growth.
The microdilution assay involves bacterial growth in broth using microtitre plates
with the addition of a serial dilution of peptides. The plates are incubated overnight
and growth of bacteria is measured by monitoring turbidity. The peptide minimal
inhibitory concentration (MIC) is taken as the lowest peptide concentration that
prevents growth. This gives a value for bacteriostatic activity; for many antimicrobial