From liposomes to cells: Filling the gap between physicochemical and microbiological studies of the activity and selectivity of host‐defense peptides

Abstract

Host‐defense peptides (HPDs) are bactericidal and immunomodulatory molecules, part of the innate immune system of many organisms, including man. They kill bacteria mostly by perturbing their membranes, and for this reason they are a promising class of molecules to fight drug‐resistant microbes. However, their success towards clinical application is still limited, partly due to many unanswered questions on their activity and function. Our current understanding of HDPs has been reached by two parallel, but largely independent, approaches: microbiological studies on HDP effects on cells, and physicochemical investigations on model membranes. All current models for the mechanisms of HDP membrane perturbation and cell selectivity were derived from the latter kind of studies, but their relevance for real cells still had to be demonstrated. In the last few years, several studies led to quantitative insights into HDP behavior directly in cells: membrane‐binding and peptide‐induced pores in bacteria and liposomes were compared; the number of cell‐bound peptide molecules needed to kill a bacterium was determined; the variation of peptide activity and toxicity with the density of cells was characterized; selectivity was examined in a mixture of target and host cells; the sequence of events leading to bacterial death was observed in real time by microscopy on single cells. Overall, these approaches led to a new understanding of HDPs that will be helpful for their development into effective antibiotic drugs.

Heterogeneous Absorption of Antimicrobial Peptide LL37 inEscherichia coliCells Enhances Population Survivability

“…Our quantification of peptide retention (~3.8 × 10 7 peptides/cell) is within the range reported using various methods Steiner et al (1988); Savini et al (2018); Starr et al (2016); Tran et al (2002); Melo et al (2011); Roversi et al (2014). Yet, this large number raises the question of which molecules inside of the cells are interacting with LL37.…”

“…At the cellular and molecular scales, a distinct feature of the AMP’s mechanism of action is its collective nature, where a large number of AMPs are required to first bind to and then disrupt the cell membranes to kill the cell. AMP’s absorption has been discussed and quantified previously for different microorganisms and peptides using various techniques Steiner et al (1988); Savini et al (2018); Starr et al (2016); Tran et al (2002); Melo et al (2011); Roversi et al (2014). Utilizing live, single-cell microscopy, we observed the temporal and cell-to-cell heterogeneity in the peptide absorption into E. coli cells, which goes beyond the membrane binding.…”

“…Early exponential cultures contain ~10 6 cells/ml, which amounts to the ratio of ~10 8 AMPs/cell. At such a high ratio, only binding or degradation of AMPs of the same order of magnitude per individual cells can lead to the inoculum effect Starr et al (2016); Savini et al (2018).…”

“…AMP absorption into bacteria has been previously discussed and quantified using various techniques. Different prokaryotes were reported to absorb 1–20 × 10 7 AMPs/cell Steiner et al (1988); Savini et al (2018); Starr et al (2016); Tran et al (2002); Melo et al (2011); Roversi et al (2014), which is high enough to initiate the inoculum effect. Here we also quanti1ed the absorption of a dye-tagged analogue of LL37 (5-FAM-LC-LL37, AnaSpec) by colorimetric measurement of the 14 μM of peptide concentration before and after incubation with E. coli and separation by centrifugation (see Fig.…”

Aggregation determines the selectivity of membrane-active anticancer and antimicrobial peptides: The case of killerFLIP

“…In addition, cancer cells have a higher content of other anionic moieties on their surface, such as sialic acid and heparan sulphate [3,[18][19][20]. A net negative charge is a property shared by bacterial membranes, which contain significant fractions of the anionic lipids phosphatidylglycerol (PG) and cardiolipin (CL); additional negative moieties are present in other components of the cell envelope (teichoic and teichuronic acids and lipopolysaccharides) [8,21]. The cationic charge of HDPs is an obvious determinant of their selectivity.…”