The Antimicrobial Activity of Sub3 is Dependent on Membrane Binding and Cell‐Penetrating Ability

Abstract

Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high-throughput screening with bactenecin as the template. Sub3 has potent activity against Gram-negative and Gram-positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ζ potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat.

The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells

“…As each peptide is labeled with Alexa Fluor 488 and its fluorescence emission quantum yield is insensitive to environment (Gadd et al, 2012), the mean fluorescence emission signal is directly proportional to the amount of peptide internalized into cells, whereas the percentage of fluorescent cells represents the fraction of cells that have internalized the peptide. The membrane-impermeable dye trypan blue was added to quench the fluorescence of membrane-bound peptide and/or of cells with compromised membranes (Torcato et al, 2013).…”

“…Single Lys peptides were labeled with Alexa Fluor 488 sulfodichlorophenol ester or pHrodo green sulfotetrafluorophenyl ester (Life Technologies) through amide-bound ligation (Torcato et al, 2013).…”

Identification of survival-promoting OSIP108 peptide variants and their internalization in human cells

“…The gold standard cell-penetrating peptide TAT was included as a positive control, and its derivative TAT-G (in which positively-charged residues are replaced with Gly) was used to evaluate non-specific cellular internalization. Trypan blue (TB), a membrane-impermeable dye, was added to quench the fluorescence of cells with compromised membranes and/or the fraction of membrane-bound peptide (Torcato et al, 2013). Using flow cytometry we found that OSIP108 is internalized into HeLa cells in nonpermeabilising conditions: after one hour incubation all cells (>98%) became fluorescent (Fig.…”

“…OSIP108 and its variants were labeled with Alexa Fluor® 488 sulfodichlorophenol ester (Life technologies) through an amide bond (Torcato et al, 2013). Briefly, peptide …”

Anticancer Peptides: Prospective Innovation in Cancer Therapy

“…HNP-1 (3) interacts with the negatively-charged cell membrane of the tumor cell (4), translocates into the cell-inducing DNA fragmentation (6) and fragilizing the cell's cytoskeleton structure (6) resulting in the collapse of the cell as shown in the 3D projection of the AFM height image (7). For both SVS-1 and HNP-1, cell death precedes membrane neutralization, in contrast to typical AMP action (Alves et al 2010, Torcato et al 2013b. Adapted from references Sinthuvanich et al (2012) and Gaspar et al (2015) folding is the membrane net negative charge but full membrane neutralization is not mandatory for cell death .…”