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386 Cell-Penetrating Peptides: Processes and Applications
peptides this value is close to the LC values obtained. The minimal bactericidal
concentration (MBC) is calculated by determining the number of colony-forming
units during treatment. An aliquot from the microdilution assay is dispersed on agar
plates and incubated. The MBC is usually defined as the lowest peptide concentration
that prevents colony formation.
18.4.2 CELL UPTAKE ASSAYS
Peptide uptake into cells can be assayed by direct and indirect methods. Early studies
on microorganisms showed that the growth response of amino acid auxotrophic mutants
to peptides can be used to indicate peptide entry; however, it is difficult to obtain
quantitative data from the growth response. A more direct approach is to use radioactively
labeled peptides and cell fractionation to assess localization and uptake kinetics.
However, cell fractionation can be difficult and uncertain, especially with small microbial
cells. Many researchers now prefer uptake assays based on colorimetric or fluorescent
probes, which are easier to handle and better suited to measurements of uptake
kinetics and cell localization.
18.4.2.1 Fluorescence Microscopy and FACS Analysis
There is a range of flexible fluorescent probes that can be attached covalently to
peptides enables direct observation of peptide entry by using fluorescence microscopy
and fluorescence-activated cell sorting (FACS). In broad terms, fluorescence
microscopy can be used to indicate localization within a relatively small number of
cells and FACS analysis can be used to profile uptake into a large number of cells.
In our efforts to discover peptides that permeate into microbial cells, we have
attached peptides to fluorescent probes and studied treated cells using fluorescence
microscopy and FACS analysis. Figure 18.3 shows the microscopy image of GFP
following peptide-mediated delivery into Saccharomyces cerevisisae and FACS analysis
of the same cell population. In this example, the addition of the carrier peptide
clearly improved cellular delivery.
18.4.2.2 Cell Permeabilization Assays
As mentioned earlier in the chapter, most antimicrobial peptides have potent cell wall
activity, which often appears to be the major mechanism involved in cell killing. In
other cases, killing also appears to involve binding to intracellular targets, but such
peptides first must permeate the outer barriers to reach their internal targets (see
Table 18.1). Therefore, whether cell wall activity involves permeabilization to cause
cell leakage or permeation to gain access to the cell interior, some degree of membrane
disturbance is involved. The most straightforward method to assess this disturbance is
to use small fluorescent molecules or chromogenic probes that normally are excluded
by cells but can gain access at points where the membrane is disturbed. By monitoring
the cell entry and chemical conversion of such small molecule probes it is possible to
gain insight into permeabilization kinetics and conditions that affect activity.
Several different fluorescent probes can be used to probe microbial membrane
disturbance or permeabilization. For example, the hydrophobic fluorescent probe