The Human Cathelicidin Antimicrobial Peptide LL-37 Promotes the Growth of the Pulmonary Pathogen Aspergillus fumigatus.

The pulmonary mucus of cystic fibrosis (CF) patients displays elevated levels of the cathelicidin antimicrobial peptide LL-37, and the aim of this work was to assess the effect of LL-37 on the growth of Aspergillus fumigatus, a common pathogen of CF patients. Exposure of A. fumigatus to LL-37 and its derived fragment RK-31 (1.95 µg/ml) for 24 h had a positive effect on growth (199.94% ± 6.172% [P < 0.05] and 218.20% ± 4.63% [P < 0.05], respectively), whereas scrambled LL-37 peptide did not (85.12% ± 2.92%). Exposure of mycelium (preformed for 24 h) to 5 µg/ml intact LL-37 for 48 h increased hyphal wet weight (4.37 ± 0.23 g, P < 0.001) compared to the control (2.67 ± 0.05 g) and scrambled LL-37 (2.23 ± 0.09 g) treatments. Gliotoxin secretion from LL-37 exposed hyphae (169.1 ± 6.36 ng/mg hyphae, P < 0.05) was increased at 24 h compared to the results seen with the control treatment (102 ± 18.81 ng/mg hyphae) and the scrambled LL-37 treatment (96.09 ± 15.15 ng/mg hyphae). Shotgun proteomic analysis of 24-h LL-37-treated hyphae revealed an increase in the abundance of proteins associated with growth (eukaryotic translation initiation factor 5A [eIF-5A] [16.3-fold increased]), tissue degradation (aspartic endopeptidase [4.7-fold increased]), and allergic reactions (Asp F13 [10-fold increased]). By 48 h, there was an increase in protein levels indicative of cellular stress (glutathione peroxidase [9-fold increased]), growth (eIF-5A [6-fold increased]), and virulence (RNase mitogillin [3.7-fold increased]). These results indicate that LL-37 stimulates A. fumigatus growth and that this stimulation can result in increased fungal growth and secretion of toxins in the lungs of CF patients.

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance.

Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However, the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.

The effect of Aspergillus fumigatus infection on vitamin D receptor expression in cystic fibrosis.

RATIONALE: Aspergillus fumigatus (A. fumigatus) in cystic fibrosis (CF) is increasingly recognized. Although allergic bronchopulmonary aspergillosis (ABPA) leads to deterioration of pulmonary function, the effect of A. fumigatus colonization in the absence of ABPA remains unclear. OBJECTIVES: To address this, we examined individuals with CF with A. fumigatus who were ABPA negative to identify the effects of itraconazole therapy on Aspergillus-induced lung inflammation. METHODS: The effect of A. fumigatus on nuclear vitamin D receptor (VDR) expression was investigated using qRT-PCR and Western blotting. IL-5 and IL-13 levels were quantified by ELISA. The effect of itraconazole was assessed by a combination of high-resolution computed tomography, lung function test, and microbiological analysis. MEASUREMENTS AND MAIN RESULTS: We demonstrate that A. fumigatus down-regulates VDR in macrophages and airway epithelial cells and that the fungal metabolite gliotoxin (Gt) is the main causative agent. Gt overcame the positive effect of 1,25-OH vitamin D(3) on VDR expression in vitro, resulting in increased IL-5 and IL-13 production. In vivo, A. fumigatus positivity was associated with increased Gt in CF bronchoalveolar lavage fluid and increased bronchoalveolar lavage fluid levels of IL-5 and IL-13. After airway eradication of A. fumigatus with itraconazole, we observed decreased Gt, IL-5 and IL-13, improved respiratory symptoms, and diminished high-resolution computed tomography mosaic pattern consistent with sustained pulmonary function. CONCLUSIONS: This study provides a rationale for the therapeutic effect of itraconazole and implied that the therapeutic potential of vitamin D supplementation in preventing ABPA is only feasible with concurrent elimination of A. fumigatus to permit VDR expression and its positive functional consequences.

Pharmacodynamic synergy strictly dependent on the co-operative aggregation of enantiomers of cyclic peptides in the bacterial cell membrane.

The antimicrobial activity of the peptide enantiomers cyclo[D-Tle-D-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala] and cyclo[L-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] against Bacillus megaterium was investigated. Both these peptides showed very low activity in both an agar diffusion assay and a broth microdilution assay. However, when both peptides were present during the experiments a potent inhibition with an IC(50) value of 2 µM was observed. Furthermore, the peptides also showed low hemolytic activity. Neither peptide had any hemolytic activity in concentrations up to 1mM but when erythrocytes were exposed to both peptides a weak hemolytic activity could be observed with a HC(50) value of 316 µM.

Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases.

Elafin is a 6-kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases [neutrophil elastase (NE) and proteinase 3] with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P. aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), are able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaves elafin at the amino-terminal Lys6-Gly7 peptide bond, resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidence that elafin is susceptible to proteolytic cleavage at alternative sites by P. aeruginosa metalloproteinases, which can affect different biological functions of elafin.

LL-37 complexation with glycosaminoglycans in cystic fibrosis lungs inhibits antimicrobial activity, which can be restored by hypertonic saline.

There is an abundance of antimicrobial peptides in cystic fibrosis (CF) lungs. Despite this, individuals with CF are susceptible to microbial colonization and infection. In this study, we investigated the antimicrobial response within the CF lung, focusing on the human cathelicidin LL-37. We demonstrate the presence of the LL-37 precursor, human cathelicidin precursor protein designated 18-kDa cationic antimicrobial protein, in the CF lung along with evidence that it is processed to active LL-37 by proteinase-3. We demonstrate that despite supranormal levels of LL-37, the lung fluid from CF patients exhibits no demonstrable antimicrobial activity. Furthermore Pseudomonas killing by physiological concentrations of exogenous LL-37 is inhibited by CF bronchoalveolar lavage (BAL) fluid due to proteolytic degradation of LL-37 by neutrophil elastase and cathepsin D. The endogenous LL-37 in CF BAL fluid is protected from this proteolysis by interactions with glycosaminoglycans, but while this protects LL-37 from proteolysis it results in inactivation of LL-37 antimicrobial activity. By digesting glycosaminoglycans in CF BAL fluid, endogenous LL-37 is liberated and the antimicrobial properties of CF BAL fluid restored. High sodium concentrations also liberate LL-37 in CF BAL fluid in vitro. This is also seen in vivo in CF sputum where LL-37 is complexed to glycosaminoglycans but is liberated following nebulized hypertonic saline resulting in increased antimicrobial effect. These data suggest glycosaminoglycan-LL-37 complexes to be potential therapeutic targets. Factors that disrupt glycosaminoglycan-LL-37 aggregates promote the antimicrobial effects of LL-37 with the caveat that concomitant administration of antiproteases may be needed to protect the now liberated LL-37 from proteolytic cleavage.

Antimicrobial components of the neonatal gut affected upon colonization.

Antimicrobial peptides (AMP) produced throughout our body are important effectors in the defense barrier of innate immunity. Here, we have analyzed antimicrobial activity and polypeptide composition of meconium versus neonatal feces to address the development of antimicrobial defense of the neonatal gut. Extracts of meconium exhibited antimicrobial activity against Bacillus megaterium, Escherichia coli, and group B streptococci (GBS) but not against the yeast Candida albicans. Extracts of neonatal feces were found to possess low activity against E. coli, GBS, and C. albicans. However, the anti-B. megaterium activity was higher in the fecal extracts than in meconium. All activities were reduced or abolished when salt was added to the antimicrobial assay. The AMP cathelicidin LL-37, alpha-defensin HNP-1-2, alpha-defensin HD 5, and lysozyme were identified in both meconium and fecal extracts. In addition, HNP-3 and a fragment of azurocidin were found in meconium, whereas the holoprotein azurocidin was detected in feces. In meconium, histones H2A and H4 were isolated and identified by their antimicrobial activity. Notably, LL-37 and lysozyme were found at significantly higher levels in feces than in meconium. Our findings reveal that meconium and feces contain AMP, acting in the defense of the neonatal gut, and may be implicated in the control of the initial colonization.

Stable concentrated emulsions of the 1-monoglyceride of capric acid (monocaprin) with microbicidal activities against the food-borne bacteria Campylobacter jejuni, Salmonella spp., and Escherichia coli.

Of 11 fatty acids and monoglycerides tested against Campylobacter jejuni, the 1-monoglyceride of capric acid (monocaprin) was the most active in killing the bacterium. Various monocaprin-in-water emulsions were prepared which were stable after storage at room temperature for many months and which retained their microbicidal activity. A procedure was developed to manufacture up to 500 ml of 200 mM preconcentrated emulsions of monocaprin in tap water. The concentrates were clear and remained stable for at least 12 months. They were active against C. jejuni upon 160- to 200-fold dilution in tap water and caused a >6- to 7-log(10) reduction in viable bacterial count in 1 min at room temperature. The addition of 0.8% Tween 40 to the concentrates as an emulsifying agent did not change the microbicidal activity. Emulsions of monocaprin killed a variety of Campylobacter isolates from humans and poultry and also killed strains of Campylobacter coli and Campylobacter lari, indicating a broad anticampylobacter activity. Emulsions of 1.25 mM monocaprin in citrate-lactate buffer at pH 4 to 5 caused a >6- to 7-log(10) reduction in viable bacterial counts of Salmonella spp. and Escherichia coli in 10 min. C. jejuni was also more susceptible to monocaprin emulsions at low pH. The addition of 5 and 10 mM monocaprin emulsions to Campylobacter-spiked chicken feed significantly reduced the bacterial contamination. These results are discussed in view of the possible utilization of monocaprin emulsions in controlling the spread of food-borne bacteria from poultry to humans.

Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua).

The epidermal mucus of fish species has been found to contain antimicrobial proteins and peptides, which is of interest in regard to fish immunity. An acidic extract from the epidermal mucus of the Atlantic cod (Gadus morhua) was found to exhibit antimicrobial activity against Bacillus megaterium, Escherichia coli and Candida albicans. This activity varied significantly when salt was added to the antimicrobial assay, and was eliminated by pepsin digestion. No lysozyme activity was detected in the extract. By using weak cationic exchange chromatography together with reversed-phase chromatography, and monitoring the antimicrobial activity, we have isolated four cationic proteins from the mucus extract. Using N-terminal and C-terminal amino acid sequence analysis, together with MS, the antimicrobial proteins were identified as histone H2B (13 565 Da), ribosomal protein L40 (6397 Da), ribosomal protein L36A (12 340 Da) and ribosomal protein L35 (14 215 Da). The broad spectra of antimicrobial activities in the cod mucus and the characterization of four antimicrobial polypeptides suggest that mucus compounds contribute to the innate host defence of cod.

Bactericidal effects of fatty acids and monoglycerides on Helicobacter pylori.

The susceptibility of Salmonella spp., Escherichia coli and Helicobacter pylori to fatty acids and monoglycerides was studied. None of the lipids showed significant antibacterial activity against Salmonella spp. and E. coli but eight of 12 lipids tested showed high activity against H. pylori, monocaprin and monolaurin being the most active. The high activity of monoglycerides against H. pylori suggests that they may be useful as active ingredients in pharmaceutical formulations.