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Microbial Membrane-Permeating Peptides and Their Applications 387
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GFP peptide-GFP
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FL1-H
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2 103 FIGURE 18.3 (Color Figure 18.3 follows p. 14.) Direct observation of peptide uptake by
using fluorescent probes (a) fluorescence microscopy of a peptide–GFP (green fluorescent
protein) fusion illustrating cellular localization within S. cerevisiae; (b) FACS analysis of a
peptide–GFP fusion showing cellular delivery to a population of S. cerevisiae cells. The right
shifted peak indicates increased GFP signal for the majority of cells.
N-phenol naphalene (NPN) can be used as an indicator of membrane integrity. In
E. coli and other microbes, NPN normally is excluded by the outer membrane LPS
layer, but can enter at points where membrane integrity is compromised. NPN has
low fluorescence quantum yield in aqueous solution, but fluoresces strongly in the
hydrophobic environment of a biological membrane. An alternative is to use propidium
iodide, which fluoresces strongly if able to enter cells and bind nucleic
acids. 55 These probes can be used with most types of microbial cells.
Gram-negative bacteria, which possess two cell membranes, are doubly challenging
when attempting to assess membrane permeabilization. Nevertheless, in cells
that express β-galactosidase and β-lactamase it is possible to monitor permeabilization
of both membranes at the same time. For example, permeabilization of the outer
membrane can be monitored using nitrocefin as a probe. Nitrocefin is normally excluded
by the outer cell membrane but, if able to pass this barrier, can be cleaved by β-lactamase
localized within the periplasmic space. Cleavage results in a color change from yellow
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