The antimicrobial peptide LL-37 is the only known member of the cathelicidin family of peptides expressed in humans. LL-37 is a multifunctional host defense molecule essential for normal immune responses to infection and tissue injury. LL-37 peptide is a potent killer of different microorganisms with the ability to prevent immunostimulatory effects of bacterial wall molecules such as lipopolysaccharide and can therefore protect against lethal endotoxemia. Additional reported activities of LL-37 include chemoattractant function, inhibition of neutrophil apoptosis, and stimulation of angiogenesis, tissue regeneration, and cytokine release (e.g. IL-8). Cellular production of LL-37 is affected by multiple factors, including bacterial products, host cytokines, availability of oxygen, and sun exposure through the activation of CAP-18 gene expression by vitamin D₃. At infection sites, the function of LL-37 can be inhibited by charge-driven interactions with DNA and F-actin released from dead neutrophils and other cells lysed as the result of inflammation. A better understanding of LL-37’s biological properties is necessary for its possible therapeutic application for immunomodulatory purposes as well as in treating bacterial infection.

We aim to develop antibacterial peptide mimics resistant to protease degradation, with broad-spectrum activity at sites of infection. The bactericidal activities of LL-37, ceragenins CSA-13, CSA-90 and CSA-92 and the spermine-conjugated dexamethasone derivative D2S were evaluated using MIC and MBC measurements. Gingival fibroblast counting, interleukin-8 (IL-8) and lactate dehydrogenase (LDH) release from keratinocytes (HaCat) were used to determine effects on cell growth, pro-inflammatory response and toxicity. All tested cationic lipids showed stronger bactericidal activity than LL-37. Incubation of Staphylococcus aureus with half the MIC of LL-37 led to the appearance of bacteria resistant to its bactericidal effects, but identical incubations with CSA-13 or D2S did not produce resistant bacteria. Cathelicidin LL-37 significantly increased the total number of gingival fibroblasts, but ceragenins and D2S did not alter gingival fibroblast growth. Cationic lipids showed no toxicity to HaCat cells at concentrations resulting in bacterial killing. These data suggest that cationic lipids such as ceragenins warrant further testing as potential novel antibacterial agents.

Aims: Ceragenin CSA-13 is a synthetic mimic of cationic antibacterial peptides, with facial amphiphilic morphology reproduced using a cholic acid scaffold. Previous data have shown that this molecule displays broad-spectrum antibacterial activity, which decreases in the presence of blood plasma. However, at higher concentrations, CSA-13 can cause lysis of erythrocytes. This study was designed to assess in vitro antibacterial and haemolytic activity of CSA-13 in the presence of pluronic F-127. Methods and Results: CSA-13 bactericidal activity against clinical strains of bacteria associated with topical infections and in an experimental setting relevant to their pathophysiological environment, such as various epithelial tissue fluids and the airway sputum of patients suffering from cystic fibrosis (CF), was evaluated using minimum inhibitory and minimum bactericidal concentration (MIC/MBC) measurements and bacterial killing assays. We found that in the presence of pluronic F-127, CSA-13 antibacterial activity was only slightly decreased, but CSA-13 haemolytic activity was significantly inhibited. CSA-13 exhibits bacterial killing activity against clinical isolates of Staphylococcus aureus, including methicillin-resistant strains, Pseudomonas aeruginosa present in CF sputa, and biofilms formed by different Gram (+) and Gram (−) bacteria. CSA-13 bactericidal action is partially compromised in the presence of plasma, but is maintained in ascites, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. The synergistic action of CSA-13, determined by the use of a standard checkerboard assay, reveals an increase in CSA-13 antibacterial activity in the presence of host defence molecules such as the cathelicidin LL-37 peptide, lysozyme, lactoferrin and secretory phospholipase A (sPLA). Conclusion: These results suggest that CSA-13 may be useful to prevent and treat topical infection. Significance and Impact of the Study: Combined application of CSA-13 with pluronic F-127 may be beneficial by reducing CSA-13 toxicity.

Type II toxin-antitoxin systems (TAs) are bicistronic operons ubiquitous in prokaryotic genomes, displaying multilevel association with cell physiology. Various possible functions have been assigned to TAs, ranging from beneficial for their hosts, such as a stress response, dormancy and protection against genomic parasites, to detrimental or useless functions, such as selfish alleles. As there is a link between several Escherichia coli features (e.g. virulence, lifestyle) and the phylogeny of this species, we hypothesized a similar association with TAs. Using PCR we studied the distribution of 15 chromosomal and plasmidic type II TA loci in 84 clinical E. coli isolates in relation to their main phylogenetic groups (A, B1, B2 and D). In addition, we performed in silico searching of these TA loci in 60 completely sequenced E. coli genomes deposited in GenBank. The highest number of TA loci per strain was observed in group A (mean 8.2, range 5-12) and the lowest in group B2 (mean 4.2, range 2-8). Moreover, significant differences in the prevalence of nine chromosomal TAs among E. coli phylogroups were noted. In conclusion, the presence of some chromosomal TAs in E. coli is phylogroup-related rather than a universal feature of the species. In addition, their limited collection in group B2 clearly distinguish it from the other E. coli phylogroups.

In addition to its antibacterial activity, the cathelicidin-derived LL-37 peptide induces multiple immunomodulatory effects on host cells. Atomic force microscopy, F-actin staining with phalloidin, passage of FITC-conjugated dextran through a monolayer of lung epithelial cells, and assessment of bacterial outgrowth from cells subjected to Pseudomonas aeruginosa infection were used to determine LL-37’s effect on epithelial cell mechanical properties, permeability, and bacteria uptake. A concentration-dependent increase in stiffness and F-actin content in the cortical region of A549 cells and primary human lung epithelial cells was observed after treatment with LL-37 (0.5–5 μM), sphingosine 1-phosphate (1 μM), or LPS (1 μg/ml) or infection with PAO1 bacteria. Other cationic peptides, such as RK-31, KR-20, or WLBU2, and the antibacterial cationic steroid CSA-13 did not reproduce the effect of LL-37. A549 cell pretreatment with WRW4, an antagonist of the transmembrane formyl peptide receptor-like 1 protein attenuated LL-37’s ability to increase cell stiffness. The LL-37–mediated increase in cell stiffness was accompanied by a decrease in permeability and P. aeruginosa uptake by a confluent monolayer of polarized normal human bronchial epithelial cells. These results suggested that the antibacterial effect of LL-37 involves an LL-37–dependent increase in cell stiffness that prevents epithelial invasion by bacteria.