Cell penetrating peptides and cationic antibacterial peptides: two sides of the same coin

Abstract

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.

Keywords: Antimicrobial Peptides; Cell-penetrating Peptides; Computational Biology; Membrane Biophysics; Receptor Endocytosis.

FIGURE 1.

Iztli peptide 1. Embedding of the α-pheromone sequence into a cationic antibacterial peptide sequence is shown. Note that the residues added to the α-pheromone (bold letters) do not present any antibacterial or antifungal activity and were added to the pheromone to match the physicochemical properties of cationic antibacterial peptides (11).

FIGURE 2.

Cell death effect of six null mutants preventing internalization of Ste2p. Six strains carrying gene null mutants (YKL002W (www.ncbi.nlm.nih.gov), YML001W, YCR009C, YMR231W, YHR135C, and YNL154C) reported to reduce the internalization of Ste2p were compared in their sensitivity to be killed by IP-1 with the wild-type strain (BY4741). To estimate the number of cells remaining in the presence of IP-1, the figure presents the area under the growth curves (summation of A₆₀₀ values recorded every hour is presented as the percentage) of these six mutants plus the wild-type strain in the presence (ORF name+IP-1; gray bars) and in the absence (ORF name; black bars) of IP-1. The figures present the average data obtained from four independent experiments; the lines on top of each bar represent the S.D. calculated for each data set. The optical density was followed in the Synergy MX plate-reader (BioTek) incubated at 30 °C.

FIGURE 3.

Models for Iztli peptides internalization on yeast cells. A, the model assuming an endocytic import of Iztli peptide via Ste2p (× denotes degradation). B, the model assuming permeation of Iztli peptide where the peptide may cross the membrane independent of Ste2p-mediated endocytosis. C, comparison of the two models fitted to the measured colony-forming unit time course of dead cells (n = 8, bars denote S.D.). The model assuming direct import generally performed better, noticeable for early time points. Mio, millions of cells.

FIGURE 4.

Analysis of two fluorogenic derivates of Iztli peptide 1. The antifungal activity (A) and the ability to get internalized by S. cerevisiae MATa cells (B) are shown for the TAMRA-IP-1 (left side, red) and Hilytefluor 488-IP-1 (right side, green). The change in cell density over time is presented in the presence (black symbols) or in the absence of these peptides (white symbols): squares, TAMRA-IP-1; triangles, Hilytefluor 488-IP-1. Scale bars in B are 5 μm.

FIGURE 5.

Time-dependent internalization of Iztli peptide 1 in yeast cells. TAMRA-IP1 was used to follow the internalization of the peptide in MATa cells. The sites where the peptide is found near or within cells appear as red dots. Scale bars are 5 μm. DIC, differential interference contrast.

FIGURE 6.

Iztli peptide 1 gets internalized by different cell types. S. cerevisiae MATα cells (A), C. glabrata (left in panel B), A. nidulans (right in panel B), HEK239 (left in panel C), and NL20 (right in panel C) human cells were exposed to TAMRA-IP-1 (red) to test for its internalization.

FIGURE 7.

Iztli peptide 1 makes pores. A, example of a 5-min record of electrical activity for IP-1 (10 μm) in POPC membrane with 30% mol cholesterol in a transmembrane electric potential of 200 mV. The inset corresponds to a 10-s section showing an example of the variability of the opening times of the pores formed. B, average all point histograms for five experiments at the concentrations of 5 μm (black), 10 μm (red), and 15 μm (blue) on the same bilayer and at 200 mV of applied potential; the duration of each experiment was 5 min. The all points histogram is constructed by filling the histogram buckets with the number of points, i.e. the number of times that a particular current was registered during the experiment with a 100-μs sampling. This histogram is normalized by the time duration of the experiment, and therefore, the ρ value corresponds to the number of counts in each bucket divided by the total number of counts. The records were base-line-corrected and filtered for peaks with duration of <30 μs (to suppress electric noise of the bath) with a homemade suite of programs. The inset corresponds to large conductance channels that present small occurrence, which depends on peptide concentration.