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388 Cell-Penetrating Peptides: Processes and Applications
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FIGURE 18.4 Indirect monitoring of peptide uptake into microorganisms. Concurrent measurements
of E. coli outer and inner cell membrane permeabilization by polymyxin B. The
symbols represent trials in the presence (�) and absence (�) of polymyxin B. For the left
panel (a), increased absorbance indicates nitrocefin cleavage and permeabilization of the outer
cell membrane. For the right panel (b), increased absorbance indicates ONPG cleavage and
permeabilization of the inner cell membrane.
to red, which can be used to monitor outer membrane permeabilization. In a similar
way, permeabilization of the inner membrane can be monitored by using the βgalactosidase
substrate o-nitrophenyl-β-galactoside (ONPG) as a probe. In E. coli
strain ML35p, which lacks lac permease, ONPG is blocked from cell entry by the
inner membrane, but if able to pass this barrier, ONPG can be cleaved by βgalactosidase
localized within the cytoplasm; this results in a color change from
clear to yellow. 56 Figure 18.4 illustrates examples of E. coli cell permeabilization
by the well known outer cell-permeabilizing peptide polymyxin B.
Permeabilization assays based on small molecule probes are relatively simple and
provide useful information about peptide activities and permeabilization kinetics in a
variety of environments. However, membrane disturbance is indicated only indirectly.
Also, it is not possible to distinguish between permeabilization and permeation by
peptides. This can be a disadvantage because certain peptides appear to cause very little
membrane disturbance during membrane transport. Clearly there are shortcomings with
the available direct and indirect methods to assess peptide permeation into microbial
cells; however, in most studies the main objective is to compare a range of peptides in
a variety of microorganisms.
18.5 APPLICATIONS
18.5.1 ANTIMICROBIAL PEPTIDES AS A NEW SOURCE OF ANTI-INFECTIVE
AGENTS FOR MEDICINE
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There is an increasing need for new antimicrobial agents to combat resistant strains
in the clinic, and antimicrobial peptides appear to offer attractive alternatives. Most
importantly, antimicrobial peptides are active against strains that show antibiotic
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