Wojciech Kamysz

The major surface-metalloprotease of the parasitic protozoan, Leishmania, protects against antimicrobial peptide-induced apoptotic killing

Human infection by the vector‐borne protozoan Leishmania is responsible for substantial worldwide morbidity and mortality. The surface‐metalloprotease (leishmanolysin) of Leishmania is a virulence factor which contributes to a variety of functions including evasion of complement‐mediated parasite‐killing and host intramacrophage survival. We tested the hypothesis that leishmanolysin serves to protect parasites from the cytolytic effects of various antimicrobial peptides (AMPs) which are important components of the innate immune system. We found that members of the α‐ and θ‐defensins, magainins and cathelicidins had substantially higher leishmanicidal activity against leishmanolysin‐knock out mutants of L. major. Using the magainin analogue, pexiganan, as a model peptide we show that AMP evasion is due to rapid and extensive peptide degradation by wild‐type parasites. Pexiganan‐treatment of knock out mutants induced disruption of surface‐membrane permeability and expression of features of apoptosis including smaller cell size, loss of mitochondrial membrane potential, exposure of surface phosphatidyl serine as well as induction of caspase 3/7 activity. These results demonstrate leishmanolysin as a virulence factor preventing AMP‐mediated apoptotic killing. This study serves as a platform for the dissection of the AMP‐mediated death pathways of Leishmania and demonstrates the potential that AMP evasion plays during host infection by this parasite.

Prophylactic efficacy of topical temporin A and RNAIII-inhibiting peptide in a subcutaneous rat Pouch model of graft infection attributable to staphylococci with intermediate resistance to glycopeptides.

Background—Bacteria that adhere to implanted medical devices play an important role in industry and in modern medicine. Staphylococci are among the most common pathogens that cause biomaterial infections. Vascular prosthetic graft infection is one of the most feared complications that the vascular surgeon treats, frequently resulting in prolonged hospitalization, organ failure, amputation, and death. A rat model was used to investigate the topical efficacies of temporin A and the quorum-sensing inhibitor RNAIII-inhibiting protein (RIP) as prophylactic agents of vascular prosthetic graft infections caused by Staphylococcus aureus and Staphylococcus epidermidis with intermediate resistance to glycopeptides. Methods and Results—Graft infections were established in the back subcutaneous tissue of adult male Wistar rats by implantation of Dacron prostheses 1 cm2 followed by topical inoculation with 2×107 colony-forming units of bacterial strains. The study included, for each staphylococcal strain, a control group (no graft contamination), a contaminated group that did not receive antibiotic prophylaxis, and 6 contaminated groups that received grafts soaked with temporin A, RIP, rifampin, temporin A plus RIP, RIP plus rifampin, or temporin A plus RIP. The infection was evaluated by quantitative agar culture. When tested alone, temporin A and RIP showed comparable efficacies, and their efficacies were significantly higher than that of rifampin against both strains. All combinations showed efficacies significantly higher than that of each single compound. The combinations of temporin A and RIP exerted the strongest antistaphylococcal efficacies, eliminating infection by 100%. Conclusions—The results of the present study make these molecules potentially useful for antimicrobial chemoprophylaxis in vascular surgery.

Antimicrobial Peptide-induced Apoptotic Death of Leishmania Results from Calcium-de pend ent, Caspase-independent Mitochondrial Toxicity

α- and θ-defensin-, magainin-, and cathelicidin-type antimicrobial peptides (AMPs) can kill the pathogenic protozoan Leishmania. Comparative studies of a panel of AMPs have defined two distinct groups: those that induce nonapoptotic (Class I) and apoptotic (Class II) parasite killing based on their differential ability to induce phosphatidyl serine exposure, loss of mitochondrial membrane potential and decreased ATP production, induction of caspase-3/7 and -12 activity, and DNA degradation. Class II AMPs cause rapid influx of the vital stain SYTOX and an increase in intracellular Ca2+, whereas Class I AMPs cause a slow accumulation of SYTOX and do not affect intracellular Ca2+ levels. Inhibitors of cysteine or caspase proteases diminished fast influx of SYTOX through the surface membrane and DNA degradation but do not ablate the annexin V staining or the induction of apoptosis by Class II AMPs. This suggests that the changes in surface permeability in AMP-mediated apoptosis are related to the downstream events of intracellular cysteine/caspase activation or the loss of ATP. The activation of caspase-12-like activity was Ca2+-dependent, and inhibitors of voltage-gated and nonspecific Ca2+ channels diminished this activity. Flufenamic acid, a nonspecific Ca2+ inhibitor, completely ablated AMP-induced mitochondrial dysfunction and cell death, indicating the importance of dysregulation of Ca2+ in antimicrobial peptide-induced apoptosis.

Potential Therapeutic Role of Cationic Peptides in Three Experimental Models of Septic Shock

ABSTRACT The therapeutic efficacies of buforin II, indolicidin, and KFFKFFKFF were investigated in three rat models of septic shock: (i) rats injected intraperitoneally with 10 μg of Escherichia coli O111:B4 lipopolysaccharide, (ii) rats given an intraperitoneal injection of 2 × 1010 CFU of Escherichia coli ATCC 25922, and (iii) rats in which intra-abdominal sepsis was induced via cecal ligation and single puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1 mg of buforin II per kg of body weight, 1 mg of indolicidin per kg, 1 mg of KFFKFFKFF per kg, and 20 mg of imipenem per kg. The main outcome measures were bacterial growth in abdominal exudate and plasma, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. Treatment with all peptides resulted in significant reductions in plasma endotoxin and TNF-α concentrations compared with those resulting from the imipenem and saline treatments. On the other hand, imipenem treatment significantly reduced the levels of bacterial growth compared with the reductions achieved with the peptide and saline treatments. All compounds reduced the rates of death compared to that for the controls. Although the peptides demonstrated lower levels of antimicrobial activity than imipenem, they exhibited the dual properties of antimicrobial and antiendotoxin agents.

Comparative activities of cecropin A, melittin, and cecropin A–melittin peptide CA(1–7)M(2–9)NH2 against multidrug-resistant nosocomial isolates of Acinetobacter baumannii

The in vitro activity of three polycationic peptides, cecropin A, melittin, and cecropin A-melittin hybrid peptide CA(1-7)M(2-9)NH2, alone and in combination with various clinically used antimicrobial agents, was investigated against 32 nosocomial isolates of Acinetobacter baumannii. Antimicrobial activities were measured by MIC, MBC and bacterial killing assay. The peptides demonstrated different ranges of inhibitory values: overall, the organisms were more susceptible to CA(1-7)M(2-9)NH2 (MIC range, 0.25-16 mg/l) than to cecropin A (0.50-32 mg/l) and melittin (0.50-32 mg/l). Synergy was observed when CA(1-7)M(2-9)NH2 and melittin were combined with beta-lactam antibiotics.

The hypoxia-inducible miR-429 regulates hypoxia-inducible factor- 1α expression in human endothelial cells through a negative feedback loop

Hypoxia‐inducible factors (HIFs) 1 and 2 are dimeric α/β transcription factors that regulate cellular responses to low oxygen. HIF‐1 is induced first, whereas HIF‐2 is associated with chronic hypoxia. To determine how HIF1A mRNA, the inducible subunit of HIF‐1, is regulated during hypoxia, we followed HIF1A mRNA levels in primary HUVECs over 24 hours using quantitative PCR. HIF1A and VEGF A (VEGFA) mRNA, a transcriptional target of HIF‐1, increased ~2.5‐ and 8‐fold at 2‐4 hours, respectively. To determine how the mRNAs were regulated, we identified a microRNA (miRNA), miR‐429, that destabilized HIF1A message and decreased VEGFA mRNA by inhibiting HIF1A. Target protector analysis, which interferes with miRNA‐mRNA complex formation, confirmed that miR‐429 targeted HIF1A message. Desferoxamine treatment, which inhibits the hydroxylases that promote HIF‐1α protein degradation, stabilized HIF‐1 activity during normoxic conditions and elevated miR‐429 levels, demonstrating that HIF‐1 promotes miR‐429 expression. RNA‐sequencing‐based transcriptome analysis indicated that inhibition of miRNA‐429 in HUVECs up‐regulated 209 mRNAs, a number of which regulate angiogenesis. The results demonstrate that HIF‐1 is in a negative regulatory loop with miR‐429, that miR‐429 attenuates HIF‐1 activity by decreasing HIF1A message during the early stages of hypoxia before HIF‐2 is activated, and this regulatory network helps explain the HIF‐1 transition to HIF‐2 during chronic hypoxia in endothelial cells.—Bartoszewska, S., Kochan, K., Piotrowski, A., Kamysz, W., Ochocka, R. J., Collawn, J. F., Bartoszewski, R. The hypoxia‐inducible miR‐429 regulates hypoxia hypoxia‐inducible factor‐1α expression in human endothelial cells through a negative feedback loop. FASEB J. 29, 1467‐1479 (2015). www.fasebj.org

Composition and antimicrobial activity of fatty acids detected in the hygroscopic secretion collected from the secretory setae of larvae of the biting midge Forcipomyia nigra (Diptera: Ceratopogonidae)

The hygroscopic secretion produced by the secretory setae of terrestrial larvae of the biting midge Forcipomyia nigra (Winnertz) was analysed using gas chromatography coupled with mass spectrometry (GC-MS). The viscous secretion is stored at the top of each seta and absorbs water from moist air. GC-MS analyses (four independent tests) showed that the secretion contained 12 free fatty acids, the most abundant of which were oleic (18:1), palmitic (16:0), palmitoleic (16:1) and linoleic (18:2). Other acids identified were valeric (5:0), enanthic (7:0), caprylic (8:0), pelargonic (9:0), capric (10:0), lauric (12:0), myristic (14:0) and stearic (18:0). Two other compounds, glycerol and pyroglutamic acid, were also found. The antibacterial activity of the fatty acids and pyroglutamic acid was tested using the agar disc diffusion method and targeted Gram positive (Bacillus cereus, Bacillus subtilis, Enterococcus faecalis) and Gram negative bacterial strains (Citrobacter freundii, Pseudomonas aeruginosa, Pseudomonas fluorescens). The antifungal activity was tested by determining minimal inhibitory concentration (MIC) of examined compounds. Fatty acids were tested against enthomopathogenic fungi (Paecilomyces lilacinus, Paecilomyces fumosoroseus, Lecanicillium lecanii, Metarhizium anisopliae, Beauveria bassiana (Tve-N39), Beauveria bassiana (Dv-1/07)). The most effective acids against bacterial and fungal growth were C(9:0), C(10:0) and C(16:1), whereas C(14:0), C(16:0,) C(18:0) and C(18:1) demonstrated rather poor antifungal activity and did not inhibit the growth of bacteria. The antimicrobial assay investigated mixtures of fatty and pyroglutamic acids (corresponding to the results of each GC-MS test): they were found to be active against almost all the bacteria except P. fluorescens and also demonstrated certain fungistatic activity against enthomopathogenic fungi. The hygroscopic secretion facilitates cuticular respiration and plays an important role in the antimicrobial protection of F. nigra larvae living in moist terrestrial habitats.

Evidence for potential functionality of nuclearly-encoded humanin isoforms

Humanin (HN) is a recently identified neuroprotective and antiapoptotic peptide derived from a portion of the mitochondrial MT-RNR2 gene. We provide bioinformatic and expression data suggesting the existence of 13 MT-RNR2-like nuclear loci predicted to maintain the open reading frames of 15 distinct full-length HN-like peptides. At least ten of these nuclear genes are expressed in human tissues, and respond to staurosporine (STS) and beta-carotene. Sequence comparisons of the nuclear HN isoforms and their homologues in other species reveal two consensus motifs, encompassing residues 5-11 (GFS/NCLLL), and 14-19 (SEIDLP/S). Proline vs serine in position 19 may determine whether the peptide is secreted or not, while threonine in position 13 may be important for cell surface receptor binding. Cytoprotection against the STS-induced apoptosis conferred by the polymorphic HN5 variant, in which threonine in position 13 is replaced with isoleucine, is reduced compared to the wild type HN5 peptide.

Citropin 1.1-treated central venous catheters improve the efficacy of hydrophobic antibiotics in the treatment of experimental staphylococcal catheter-related infection.

An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of citropin 1.1, rifampin and minocycline. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with citropin 1.1 (10 microg/mL). Thirty minutes later the rats were challenged via the CVC with 1.0 x 10(6) CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC (the antibiotic lock technique) began 24 h later. The study included: one control group (no CVC infection), one contaminated group that did not receive any antibiotic prophylaxis, one contaminated group that received citropin 1.1-treated CVC, two contaminated groups that received citropin 1.1-treated CVC plus rifampin and minocycline at concentrations equal to MBCs for adherent cells and 1024 microg/mL in a volume of 0.1 mL that filled the CVC and two contaminated groups that received rifampin or minocycline at the same concentrations. All catheters were explanted 7 days after implantation. Main outcome measures were: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), synergy studies, quantitative culture of the biofilm formed on the catheters and surrounding venous tissues, and quantitative peripheral blood cultures. MICs of conventional antibiotics against the bacteria in a biofilm were at least four-fold higher than against the freely growing planktonic cells. In contrast, when antibiotics were used on citropin 1.1 pre-treated cells they showed comparable activity against both biofilm and planktonic organisms. The in vivo studies show that when CVCs were pre-treated with citropin 1.1 or with a high dose of antibiotics, biofilm bacterial load was reduced from 10(7) to 10(3) CFU/mL and bacteremia reduced from 10(3) to 10(1) CFU/mL. When CVCs were treated both with citropin 1.1 and antibiotics, biofilm bacterial load was further reduced to 10(1) CFU/mL and bacteremia was not detected, suggesting 100% elimination of bacteremia and a log 6 reduction in biofilm load. Citropin 1.1 significantly reduces bacterial load and enhances the effect of hydrophobic antibiotics in the treatment of CVC-associated S. aureus infections.

Efficacy of Tachyplesin III, Colistin, and Imipenem against a Multiresistant Pseudomonas aeruginosa Strain

ABSTRACT An experimental study has been performed to compare the in vitro activity and the in vivo efficacy of tachyplesin III, colistin, and imipenem against a multiresistant Pseudomonas aeruginosa strain. In vitro experiments included MIC determination, time-kill, and synergy studies. For in vivo studies, a mouse model of sepsis has been used. The main outcome measures were bacterial lethality, quantitative blood cultures, and plasma levels of lipopolysaccharide, tumor necrosis factor alpha, and interleukin-6. The combination of tachyplesin III or colistin with imipenem showed in vitro synergistic interaction. A significant increase in efficacy was also observed in vivo: combination-treated groups had significantly lower levels of bacteremia than did groups treated with a single agent. Tachyplesin III combined with imipenem exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of these combinations and provide therapeutic alternatives for serious infections caused by gram-negative bacteria in the coming years.