Metabolic reprogramming is one of the hallmarks of cancer and can be targeted by therapeutic agents. We previously reported that cathelicidin-related or modified antimicrobial peptides, such as FF/CAP18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1, and the colon carcinoma cell line HCT116. Although antimicrobial peptides have potential use in the development of new therapeutic strategies, their effects on the metabolism of cancer cells are poorly understood. Here, we investigated changes in the levels of metabolites in HCT116 cells caused by FF/CAP18, via capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Analysis of the 177 intracellular metabolites and 113 metabolites in conditioned medium that were detected by CE-TOFMS, revealed dramatic changes in the metabolic profile of HCT116 cells after treatment with FF/CAP18. The metabolic profile showed that the levels of most metabolites in the major metabolic pathways supported the rapid proliferation of cancer cells. Purine metabolism, glycolysis, and the TCA cycle, were altered in FF/CAP18-treated cells in a dose-dependent manner. Our present study provides mechanistic insights into the anticancer effects of antimicrobial peptides that show great potential as new therapies for colon cancer.

The placenta is a major barrier that prevents potentially infectious agents from causing fetal diseases or related complications during pregnancy. Therefore, we postulated that the placenta might express a broad repertoire of antimicrobial proteins as well as inflammatory chemokines and cytokines to combat invading microorganisms. Here we demonstrate that placental cells indeed express a wide range of AMPs (antimicrobial peptides and proteins) including bactericidal/permeability-increasing protein (BPI), secretory leukocyte protease inhibitor (SLPI), human β-defensin 2 (hBD2), acyloxyacyl hydrolase (AOAH), and cathelicidin (CAP18). In addition, these cells also secrete pro-inflammatory cytokines and chemokines upon stimulation with bacterial ligands. Notably, we show that BPI expression by placental cells could be completely attributed to granulocytes while highly purified placental trophoblasts expressed only a subset of the AMPs like SLPI. Unexpectedly, trophoblast AMPs did not exhibit inducible secretion in response to various TLR ligands and further investigations showed that the unresponsiveness of trophoblasts to lipopolysaccharide (LPS) was due to a lack of TLR4 expression. In summary, we have shown that the expression of different AMPs can be allocated to various cells in the placenta and the repertoire of the AMPs expressed by placental cells is a result of a cooperation of leukocytes as well as cells from embryonic origin.

The mechanism of interaction of the cationic antimicrobial protein (18 kDa), CAP18, with the outer membrane of Gram-negative bacteria was investigated applying transmission electron microscopy and voltage-clamp techniques on artificial planar bilayer membranes. Electron micrographs of bacterial cells exposed to CAP18 showed damage to the outer membrane of the sensitive Escherichia coli strains F515 and ATCC 11775, whereas the membrane of the resistant Proteus mirabilis strain R45 remained intact. Electrical measurements on various planar asymmetric bilayer membranes, one side consisting of a phospholipid mixture and the other of different phospholipids or of lipopolysaccharide (reconstitution model of the outer membrane), yielded information about the influence of CAP18 on membrane integrity. Addition of CAP18 to the side with the varying lipid composition led to lipid-specific adsorption of CAP18 and subsequent induction of current fluctuations due to the formation of transient membrane lesions at a lipid-specific clamp voltage. We propose that the applied clamp voltage leads to reorientation of CAP18 molecules adsorbed to the bilayer into an active transmembrane configuration, allowing the formation of lesions by multimeric clustering.

BACKGROUND

The antimicrobial peptide PR39 is a porcine cathelicidin with angiogenic and antiapoptotic activities, as it can regulate the expression of vascular endothelial growth factor (VEGF) and inhibitor apoptosis protein-2 (c-IAP-2) in endothelial cells. The human homolog LL-37 has been found to be highly expressed in human keratinocytes from psoriatic patients, but it is not known whether LL-37 can modulate the expression of VEGF and c-IAP-2 in keratinocytes, as both molecules are involved in the overgrowth of psoriatic skin. Therefore, in this work, we studied the possible role of CAP18/LL-37 in the modulation of VEGF and c-IAP-2 expression in human keratinocytes.

METHODS

The CAP18/LL-37 gene was cloned into a plasmid that contained green fluorescent protein (GFP). This plasmid was called pGFP-CAP18/LL-37. The expression of LL-37, VEGF, and c-IAP-2 was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting in HaCaT cells transfected with pGFP-CAP18/LL-37. Specific DNAzymes were used to break the CAP18/LL-37 mRNA (DNAz-CAP18/LL-37).

RESULTS

HaCaT cells transfected with pGFP-CAP18/LL-37 showed the upregulation of VEGF and c-IAP-2 mRNAs. Hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA expression did not change during the assays; however, its protein was increased, as well as the VEGF protein. HaCaT cells cotransfected with pGFP-CAP18/LL-37 and DNAz-CAP18/LL-37 showed depleted expression of LL-37, VEGF, and c-IAP-2 mRNAs.

CONCLUSIONS

These results suggest that LL-37 may modulate the expression of VEGF and c-IAP-2 via HIF-1alpha in human keratinocytes.

Screening of a porcine bone marrow cDNA library with a PCR-derived probe from rabbit LPS-binding protein CAP18 led to the discovery of two closely related clones. The longer, full-length cDNA clone encodes a 228 amino acid residue protein similar to the family of antibacterial/LPS-binding cationic peptides. In contrast to other hitherto discovered precursors of Pro/Arg-rich peptides from this family, they have a novel, unique structure of the C-terminal region of 100 amino acid residues with a repeating sequence of ten residues (FPPPNXPGPR, where X = V or F). These precursors could represent a part of the antibacterial peptide repertoire of porcine bone marrow.

Antimicrobial peptides (AMPs) play important roles in the innate immune system of all life forms and recently have been characterized as multifunctional peptides that have a variety of biological roles such as anticancer agents. However, detailed mechanism of antimicrobial peptides on cancer cells is still largely unknown.

miRNA array and real-time qPCR were performed to reveal the behavior of miRNA in colon cancer HCT116 cells during the growth suppression induced by the AMPs. Establishment of miR-663a over-expressing HCT116 cells was carried out for the evaluation of growth both in vitro and in vivo. To identify the molecular mechanisms, we used western blotting analysis.

miR-663a is upregulated by administration of the human cathelicidin AMP, LL-37, and its analogue peptide, FF/CAP18, in the colon cancer cell line HCT116. Over-expression of miR-663a caused anti-proliferative effects both in vitro and in vivo. We also provide evidence supporting the view that these effects are attributed to suppression of the expression of the chemokine receptor CXCR4, resulting in the abrogation of phosphorylation of Akt and cell cycle arrest in G2/M via p21 activation.

This study contributes to the understanding of the AMPs' mediated anti-cancer mechanisms in colon cancer cells and highlights the possibility of using AMPs and miRNAs towards developing future strategies for cancer therapy.

The 18-kDa cationic protein CAP18 is an antimicrobial protein isolated from rabbit granulocytes that binds lipopolysaccharide (LPS) and inhibits many of its biological activities. We covalently coupled a synthetic peptide representing amino acids 106 to 138 of CAP18 to human immunoglobulin G (IgG) by using the heterobifunctional linker N-succinimidyl-3-(2-pyridyidithio)propionate. The ability of CAP18(106-138)-IgG to bind and neutralize LPS in whole blood in the presence and absence of anticoagulants was studied. Both CAP18(106-138) and CAP18(106-138)-IgG significantly suppressed LPS-induced tumor necrosis factor (TNF) production in whole blood in the absence of anticoagulants. EDTA potentiated the ability of CAP18(106-138) and CAP18(106-138)-IgG to decrease LPS-induced TNF production in a dose-dependent manner. In contrast, heparin inhibited the ability of CAP18(106-138) and CAP18(106-138)-IgG to suppress LPS-induced TNF production. EDTA also enhanced LPS capture in a fluid-phase binding assay that utilizes magnetic anti-IgG beads to capture CAP18(106-138)-IgG (and bound [3H]LPS) in whole blood. In contrast, heparin inhibited the binding dose dependently. We conclude that CAP18(106-138)-IgG binds to and neutralizes LPS in whole blood in the absence of anticoagulants. Further studies of its protective efficacy in animal models are warranted. Caution should be used in interpreting assays that measure the binding and neutralization of LPS in whole blood in the presence of calcium-binding anticoagulants or heparin.

CAP18 (cationic antimicrobial protein, 18kDa) is a 142 amino acid protein originally isolated from rabbit granulocytes using agglutination of LPS-coated erythrocytes as an assay. CAP-18 is composed of an N-terminal domain of unknown function (CAP18CAP18CAP18CAP18CAP18CAP18CAP18CAP18CAP18CAP18CAP18CAP18 family protein from human granulocytes. The cloned cDNA encoded 140 amino acid residues. Human CAP18 (CAP18CAP18CAP18 and the derived peptides bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation. In this regard, CAP may act as host defense protein against infectious diseases, and have therapeutic potential for sepsis and endotoxin shock.

The saccharide constituents of lipopolysaccharides (LPS) of Proteus spp. vary with the strain and contain unique components about which little is known. The biological activities of LPS and lipid A from S- and R-forms of 10 Proteus strains were examined. LPS from all S-form Proteus strains was lethal to D-(+)-galactosamine (GalN)-loaded, LPS-responsive, C3H/HeN mice, but not to LPS-hypo-responsive C3H/HeJ mice. P. vulgaris 025 LPS evoked strong anaphylactoid reactions in N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)-primed C3H/HeJ mice. LPS from S- and R-form Proteus strains induced production of nitric oxide (NO) and tumour necrosis factor (TNF) by macrophages isolated from C3H/HeN but not C3H/HeJ mice. Lipid A from Proteus strains also induced NO and TNF production, although lipid A was less potent than LPS. The effects of LPS were mainly dependent on CD14; LPS-induced NO and TNF production in CD14+ J774.1 cells was significantly greater than in CD14-J7.DEF.3 cells. All LPS from Proteus strains, and especially from P. vulgaris 025, exhibited higher anti-complementary activity than LPS from Escherichia coli or Pseudomonas aeruginosa. Polymyxin B inactivated proteus LPS in a dose-dependent manner, but these LPS preparations were more resistant to polymyxin B than E. coli LPS. CAP18(109-135), a granulocyte-derived peptide, inhibited proteus LPS endotoxicity only when the LPS:CAP18(109-135) ratio was appropriate, which suggests that CAP18(109-135) acts through a different mechanism than polymyxin B. The results indicate that LPS from Proteus spp. are potently endotoxic, but that the toxicity is different from that of LPS from E. coli or Salmonella spp. and even varies among different Proteus strains. The variation in biological activities among proteus LPS may be due to unique components within the respective LPS.

We have investigated the mechanism of action of the cationic antimicrobial protein (18 kDa) CAP18 on liposomes and monolayers made from phospholipids and enterobacterial lipopolysaccharides (LPS). CAP18 intercalates into lipid matrices composed of LPS from sensitive strains, weaker into those made of LPS from a resistant strain (Proteus mirabilis strain R45) or negatively charged phospholipids, but not into those composed of neutral phosphatidylcholine. From the combination of data obtained with fluorescence resonance energy transfer and Fourier-transform infrared spectroscopy and film balance measurements, it can be concluded that structural differences in the LPS determine the depth of intercalation of CAP18 into the respective lipid matrices. Thus, we identified the L-Arap4N linked to the first Kdo of the LPS of P. mirabilis strain R45 to be responsible for the CAP18 resistance of this strain. These data provide insight into CAP18-mediated effects on the integrity of the outer membrane of Gram-negative bacteria and led to an improved model for rabbit CAP18 membrane interaction.

We have previously characterized an 18-19 kDa cationic protein, SOB3, that was detected in the epididymis and localized within the acrosome and on the neck region of human spermatozoa. We suggested that it is involved in secondary sperm binding to the zona pellucida. The present study describes its purification to homogeneity by preparative electrophoresis and non-equilibrium pH gradient electrophoresis. Degenerate primers deduced from microsequencing were used to amplify a specific fragment from human epididymal RNA by reverse transcription-polymerase chain reaction (RT-PCR). This 164 bp fragment was extended by 5' and 3'-RACE to obtain the 548 bp full length cDNA. The open reading frame encodes a 170 amino acid protein. SOB3 is a single copy gene. It is 98% identical to prepro-FALL39 and 100% identical to CAP18, two human genes which were initially identified by screening a human bone marrow (lambda)gt11 library, and which encode an antimicrobial protein. Northern blots of human tissues revealed a 1 kb transcript in corpus and cauda epididymis only, while RT-PCR showed presence of the mRNA in the three epididymal regions and also in round spermatids. The above results suggest that SOB3 has two roles in sperm protection and fertilization, depending on its dual origin and final sperm localization.

Cationic antimicrobial protein of 18 kD (CAP18) was identified and purified from rabbit granulocytes and shown to inhibit various activities of lipopolysaccharide (LPS). We investigated the effect of a 32-amino-acid C-terminal fragment of CAP18 (CAP18-derived peptide, CDP) on the pathogenesis of acute lung injury caused by intravenous endotoxin. Guinea pigs were divided into six groups: (I) saline control (n = 8), (2) CDP-alone (n = 8), (3) LPS-alone (n = 8), (4) LPS+CDP0m (n = 8), (5) LPS+CDP10m (n = 8), and (6) LPS+CDP60m (n = 8). A CDP dose of 0.2 mg/kg was injected at various time points after LPS injection. Lung wet-to-dry weight ratio, [125I]albumin leakage in lung tissue and bronchoalveolar lavage (BAL) fluid, differential cell count in BAL fluid, and histopathologic features were examined 4 h after intravenous administration of 0.02 mg/kg of LPS. The LPS+CDP0m and the LPS+CDP10m groups showed significantly attenuated lung injury compared to that seen in the LPS-alone group, however the LPS+CDP60m group revealed no attenuation of lung injury. The accumulation of peripheral white blood cells into pulmonary vasculature was attenuated only in the LPS+CDP0m but not in the LPS+CDP10m groups. We examined the effect of CDP on the expression of adhesion molecules using human umbilical vein endothelial cells, the result of which showed that CDP suppressed the LPS-induced expression of adhesion molecules in a dose-dependent manner. We conclude that CDP attenuates inflammatory cell migration into alveoli resulting in the attenuation of lung injury.

CAP18 is a cationic antimicrobial protein originally isolated from rabbit neutrophils, of which a 32-mer sequence from its C-terminal and (CAP18(106-137)) has been found to be the most active. The bactericidal action of this peptide has been characterized by conventional culture techniques and flow cytometry. Cultures of Escherichia coli NCTC10418 were exposed to the MBC (12 microM) of the peptide for up to 60 min and stained with a fluorochrome sensitive to changes in either membrane potential (bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)), or membrane integrity (propidium iodide [PI]) before flow cytometric analysis. Addition of CAP18(106-137) to E. coli in broth culture resulted in immediate collapse of membrane potential [as determined by uptake of DiBAC4(3)] and loss of membrane integrity (as indicated by uptake of PI), with a corresponding 6- to 8-log decrease in viable counts as determined by colony formation on solid media. In identical experiments, the presence of Mg2+ (1 to 10 mM), K+ (50 to 250 mM), or EDTA (5 mM) or incubation in nutrient-free buffer or at 4 degrees C had no effect on peptide-induced dye uptake. In contrast, addition of Ca2+ (1 to 10 mM) or the respiratory chain poison carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 microM) inhibited the uptake of both dyes. These findings, however, did not relate to bacterial recovery on solid media, where (unless in the presence of K+ 150 to 250 mM) CAP18(106-137) at 12 microM fulfilled the MBC criteria (99.9% killing). We conclude that CAP18(106-137) exerts a rapid and profound action on E. coli cytoplasmic membranes and viability as measured by colony formation. The results suggest, however, that CAP18(106-137) may exert its action at sites additional to the cell membrane and that its activity profile is unique among cationic antimicrobial proteins.

Human antimicrobial peptide CAP18/LL37 (hCAP18/LL37) was expressed in Pichia pastoris and its antibacterial activity was tested against pathogenic bacteria. The full length ORF of hCAP18/LL37 was cloned into the pPICZaA vector followed by integration into the genomic AOX1 gene of P. pastoris. Agar diffusion assay demonstrated that the different hCAP18/LL37 transformants showed various antibacterial activities against Staphylococcus aureus, Micrococcus luteus, and Salmonella gastroenteritis. The secreted form of hCAP18/LL37 exhibited its maximum activity after 72 h incubation with 2% methanol in MM media, not in BMM. This result suggests that the yeast secreted expression system can be used as a production tool of antimicrobial peptides for industrial or pharmaceutical application.

In recent years, several endogenous mammalian antibacterial peptides have been described. An amphipathic cationicalpha-helix is a common feature in many cases; therefore, other peptides with this characteristic might also possess antibiotic activity. In fact, a 30-mer peptide of apoprotein E 133-162 (LRVRLASHLRKLRKRLLRDADDLQKRLAVY) was found to have antibiotic activity comparable to those of a classic antibiotic (Gentamicin) and a neutrophil-derived antibiotic peptide (CAP18). Calculation of cationicity, hydrophobicity, and hydrophobic moment and the helical wheel diagram of apoprotein E 133-162 revealed close similarities to CAP18.

CAP18 (cationic antimicrobial protein of 18 kDa) was originally isolated from rabbit granulocytes using as an assay the agglutination of Re-lipopolysaccharide coated erythrocytes. The C-terminal 37 amino acids of CAP18 comprise the LPS-binding domain called RNIP, reactive nitrogen inhibitory peptide. Synthetic RNIP has broad antimicrobial activity versus both gram positive [IC50 = 130-200 nM] and gram negative bacteria [IC50 = 20-100 nM). Susceptible strains include Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes. Antimicrobial activity is highly dependent upon peptide structure. Although a 32 amino peptide resulting from truncation of five amino acids from the C terminus of RNIP is highly active, other fragments of RNIP including truncation of its N-terminus do not exhibit antimicrobial activity. Unlike previously characterized antimicrobial peptides derived from granulocyte proteins RNIP is active in serum. RNIP or a derivative peptide may have therapeutic potential for bacterial sepsis.

Antimicrobial peptides (AMPs) are multifunctional factors with an important role in the innate immune system. Our previous studies revealed that the human cathelicidin LL‑37 and its analog, FF/CAP18, limit the proliferation of colon cancer cell lines. In the present study, the exosomes released by HCT116 cells treated with FF/CAP18 were analyzed. After the treatment, exosomes were isolated from the culture supernatant by ultrafiltration and using the miRCURY™ Exosome Isolation Kit. Membrane vesicles 40‑100‑nm expressing CD63 and CD81 were identified before and after FF/CAP18 treatment. Exosome concentration in the culture supernatant was increased after treatment with FF/CAP18. Exosomes formed in HCT116 cells treated with FF/CAP18 induced growth suppression of the cells in a dose‑dependent manner. By contrast, the exosomes formed in non‑treated HCT116 cells did not affect cell viability. Microarray analysis of miRNA expression indicated that FF/CAP18 treatment induced increases in the expression of three miRNAs (miR‑584‑5p, miR‑1202 and miR‑3162‑5p) in both HCT116 cells and exosomes. These results suggest that FF/CAP18 treatment increases exosome formation, and that exosome‑encapsulated miRNAs suppress HCT116 cell proliferation. Exosomal miRNAs are considered to be involved in the dissemination of cell signals to control local cellular microenvironments. The present findings suggest that FF/CAP18 regulates cancer growth by modulating cell‑to‑cell communication. AMPs localize in the cytoplasm of cancer cells and enhance the expression of growth‑suppressing miRNAs. These miRNAs are also transported to other cancer cells via exosomes. Therefore, transportation of these miRNAs has the potential to suppress cancer growth. AMPs exert their effects directly by targeting cancer cells and indirectly via exosomes.

Naturally present antibacterial proteins play an important role in innate host defense. A synthetic peptide mimicking the C-terminal lipopolysaccharide (LPS)-binding domain of rabbit cathelicidin CAP18 was coupled to immunoglobulin (Ig) G to create CAP18(106-138)-IgG, a construct that, in concentrations equimolar to those of peptide alone, binds and neutralizes LPS and kills multiple gram-negative bacterial strains. The protective efficacy of CAP18(106-138)-IgG was evaluated in a model of cecal ligation and puncture in mice. A single intravenous administration of 20 mg/kg CAP18(106-138)-IgG protected against mortality, compared with sham-coupled IgG (P<.03). There was no protection offered by administration of equimolar peptide alone (P=.96). There was a trend toward protection in C3H/HeJ mice that are minimally sensitive to LPS (P=.06), suggesting that direct detoxification of LPS was not the only mechanism of protection. Chemical or genetic coupling of antimicrobial peptides to IgG may be a means of using these peptides to treat infections.

OBJECTIVE

There is increasing evidence that the innate immune system plays an important role in the pathogenesis of COPD. The objective of this study was to quantify several innate immune biomarkers in serum and induced sputum of COPD patients, and healthy non-smokers and smokers.

METHODS

Serum and induced sputum levels of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator inhibitor (PAI-1) and human cationic antimicrobial protein 18 (CAP18) were measured by ELISA, in 13 patients with stage I or stage II COPD (COPD I + II), 15 patients with stage III or stage IV COPD (COPD III + IV), 18 healthy non-smokers and 14 healthy smokers. In addition, membrane-bound uPAR in peripheral blood and induced sputum was assessed by flow cytometry.

RESULTS

Levels of uPAR, PAI-1 and CAP18 were elevated in induced sputum of COPD I + II and COPD III + IV patients, compared with healthy non-smokers (P < 0.05) and healthy smokers (P < 0.05). uPAR, PAI-1 and CAP18 levels were significantly higher in COPD III + IV patients compared with COPD I + II patients (P < 0.05). The expression of uPAR on induced sputum neutrophils and macrophages was significantly higher in COPD patients compared with healthy non-smokers (P < 0.05) and healthy smokers (P < 0.05). Sputum uPAR and CAP18 levels showed significant inverse correlations with FEV(1)% and 6MWD, and significant positive correlations with St. George's Respiratory Questionnaire scores.

CONCLUSIONS

In COPD patients, increased induced sputum levels of uPAR, PAI-1 and CAP18 were associated with airflow limitation, health status and exercise tolerance, suggesting that these biomarkers may be implicated in the pathogenesis of COPD.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic pathogen. An emerging problem in treating S. aureus infections is the increasing resistance against antibiotics. A possible way to overcome this issue is to boost the host immune system and one target are antimicrobial peptides (AMPs), especially cathelicidins. The aim of this study was to characterize the antimicrobial activity of cathelicidins from different animal species against LA-MRSA and to reveal whether major antimicrobial resistance mechanisms influence the bactericidal activity of these peptides. The MICs of 153 LA-MRSA field isolates for different cathelicidins (LL-37, mCRAMP, CAP18, BMAP-27 and BMAP-28) were analysed. The cathelicidin MICs of S. aureus RN4220 and isogenic transformants, that carried 14 functionally active antimicrobial resistance genes, were determined. These resistance genes have been identified in LA-MRSA and specify the resistance mechanisms active efflux, enzymatic inactivation and modification/protection/replacement of target sites. The data showed that mode MIC values for the cathelicidins did not differ among the LA-MRSA isolates of different animal origin. However, distinct differences were detected between the MIC values for the different cathelicidins. MIC values were lowest for bovine cathelicidins (BMAP-27 and BMAP-28) and highest for the human and mouse cathelicidins (LL-37 and mCRAMP). None of the tested antimicrobial resistance genes affected the antimicrobial activity of the cathelicidins. The findings obtained in this study support the hypothesis that cathelicidins might be a promising target to support the host defense against LA-MRSA, especially since the antimicrobial activity of these peptides is not affected by common staphylococcal antimicrobial resistance genes.

Antimicrobial peptides (AMPs) represent an important part of the innate host immune system. Although they are active against a broad range of pathogens, bacteria have evolved different resistance mechanisms to avoid killing by AMPs. Since not much is known about the impact of efflux pumps on the susceptibility of Gram-positive bacteria to AMPs, especially to the cathelicidins, the aim of this study was to analyze whether Staphylococcus aureus can use efflux pumps to resist the antimicrobial effects of cathelicidins derived from different animal species (human, mouse, rabbit or cattle). For this purpose the minimal inhibitory concentrations (MICs) of S. aureus field isolates for the cathelicidins LL-37, mCRAMP, CAP18, BMAP-27 and BMAP-28 in the presence and absence of different efflux pump inhibitors were determined. Furthermore, the MICs of mutants lacking SecDF, a member of the RND efflux pump family, were determined and compared to the MICs of their respective wildtype and complemented strains. The data demonstrated that after blocking RND-type efflux pumps with 1-(1-naphthylmethyl)-piperazine, the MICs for CAP18, but not those for the other cathelicidins tested, were significantly decreased. In good correlation with these data, significantly decreased MICs for CAP18 and also BMAP-27 have been observed for SecDF knockout mutants as compared to their isogenic wildtype strains. In addition, the MIC values increased again after re-introducing a cloned secDF via plasmid complementation. These results indicated an involvement of SecDF in a reduced efficacy of species-specific cathelicidins against S. aureus.

Lipopolysaccharide (LPS) is a toxic and immunogenic agent for human. Additionally, LPS is a good target for some antimicrobial compounds, including antimicrobial peptides (AMPs). LPS-binding peptides (LBPs) can recognize and neutralize LPS. Rabbit and human cathelicidins are AMPs with LPS-binding activity. In this study, we designed and synthesized two new truncated LBPs from rabbit and human CAP18 peptides by in silico methods. After synthesis of peptides, the antimicrobial properties and LPS-binding activity of these peptides were evaluated. The parental rabbit and human CAP18 peptides were selected as positive controls. Next, the changes in the secondary structure of these peptides before and after treatment with LPS were measured by circular dichroism (CD). Human cytotoxicity of the peptides was evaluated by MTT and red blood cells (RBCs) hemolysis assays. Finally, field emission scanning electron microscopy (FE-SEM), confocal microscopy, and flow cytometry were performed to study the action mechanism of these peptides. Results indicated that the hCap18 and rCap18 had antibacterial activity (at a MIC of 4-128 μg/mL). The results of the quantitative LAL test demonstrated that LPS-binding activity of hCap18 peptide was better than rCap18, while rCap18 peptide had better antimicrobial properties. Furthermore, rCap18 had less cytotoxicity than hCap18. However, both peptides were nontoxic for normal human skin fibroblast cell in MIC range. In conclusion, rCap18 has good antibacterial properties, while hCap18 can be tested as a diagnostic molecule in our future studies.

Keywords: Antimicrobial peptides; Cap18; In silico drug design; LPS-binding peptide; Lipopolysaccharide.