Antimicrobial Polypeptides Research Articles

Bacteria-Assisted Activation of Antimicrobial Polypeptides by a Random-Coil to Helix Transition.

The application of antimicrobial peptides (AMPs) is largely hindered by their non-specific toxicity against mammalian cells, which is usually associated with helical structure, hydrophobicity, and charge density. A random coil-to-helix transition mechanism has now been introduced into the design of AMPs, minimizing the toxicity against mammalian cells while maintaining high antimicrobial activity. By incorporating anionic phosphorylated tyrosine into the cationic polypeptide, the helical structure of AMPs was distorted owing to the side-chain charge interaction. Together with the decreased charge density, the AMPs exhibited inhibited toxicity against mammalian cells. At the infectious site, the AMPs can be activated by bacterial phosphatase to restore the helical structure, thus contributing to strong membrane disruptive capability and potent antimicrobial activity. This bacteria-activated system is an effective strategy to enhance the therapeutic selectivity of AMPs.

Proteomic and transcriptomic analysis of saliva components from the hematophagous reduviid Triatoma pallidipennis

Species belonging to the Triatominae subfamily are commonly associated with Chagas disease, as they are potential vectors of the parasite Trypanosoma cruzi. However, their saliva contains a cocktail of diverse anti-hemostatic proteins that prevent blood coagulation, vasodilation and platelet aggregation of blood; components with indisputable therapeutic potential. We performed a transcriptomic and proteomic analyses of salivary glands and protein spots from 2DE gels of milked saliva, respectively, from the Mexican Triatoma pallidipennis. Massive sequencing techniques were used to reveal this protein diversity. A total of 78 out of 233 transcripts were identified as proteins in the saliva, divided among 43 of 55 spots from 2DE gels of saliva, identified by LC-MS/MS analysis. Some of the annotated transcripts putatively code for anti-hemostatic proteins, which share sequence similarities with proteins previously described for South American triatomines. The most abundant as well as diverse transcripts and proteins in the saliva were the anti-hemostatic triabins. For the first time, a transcriptomic analysis uncovered other unrelated but relevant components in triatomines, including antimicrobial and thrombolytic polypeptides. Likewise, unique proteins such as the angiotensin-converting enzyme were identified not just in the salivary gland transcriptome but also at saliva proteome of this North American bloodsucking insect. Biological significanceThis manuscript is the first report of the correlation between proteome and transcriptome of Triatoma pallidipennis, which shows for the first time the presence of proteins in this insect that have not been characterized in other species of this family. This information contributes to a better understanding of the multiple host defense mechanisms that are being affected at the moment of blood ingestion by the insect. Furthermore, this report gives a repertoire of possible therapeutic proteins.

Characterization and activity of antimicrobial polypeptide of Bacillus spp from wastelands of Kathmandu valley

Background: Bacillus genus have been found to produce antimicrobial polypeptides that have a remarkable capability to combat the current increasing problem of antibiotic resistance. The aim of this cross sectional study was to determine the proportion and pattern of distribution of antibiotic producing and non-producing species of Bacillus in soil and also to analyse the activity of the antibiotic producers. Methods: A total of 40 soil samples were collected from the wastelands of Kathmandu Valley and 121 isolates were studied from them. They were identified according to Bergey’s Manual. The antibiotic were extracted and the activity was analysed by agar- well diffusion method against the test organisms Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Result: Bacillus subtilis species were found to be in highest number (17.3%) and Bacillus macerans (0.82%) in lowest number. 18.33% of Bacillus isolates were found to produce antibiotics. The highest number of antibiotic producing isolate was B. subtilis (40.09%) and the least was B. circulans (4.54%). The number of antibiotic producing bacteria were not found to be affected by moisture content of soil and pH change. Among the antibiotic producers, 63.63% produced antibiotic against Gram positive bacteria, 27.27% against Gram negative bacteria and B. alvei (9.09%) had a broad spectrum activity. Some colonies were even found to produce antibiotic with a greater potential than the conventional antibiotics. Conclusion: Based on property of soil, moist-neutral soil had the highest number of bacilli isolates and dry-acidic and moist-alkaline had least number of isolates. DOI: 10.21276/AABS.2017.1316

Synthesis of silver nanoparticles modified with lysozyme and sodium dioctyl sulfosuccinate and comparison of their biological activities

Silver nanoparticles have been fabricated by the method of chemical reduction in solution using different types of stabilizers: an organic low-molecular compound—sodium dioctyl sulfosuccinate (AOT)— and a natural biologically active substance—antimicrobial cationic polypeptide lysozyme. According to studies of the produced hydrosols, the average size of the shell-coated particles is 20–25 nm. The biological activity of the obtained bioconjugates toward Gram-negative (Escherichia coli ML35p, Pseudomonas aeruginosa (clinical isolate)) and Gram-positive (Listeria monocytogenes EGD (АТСС ВАА-679), MRSA ATCC 33591 (Staphylococcus aureus resistant to methicillin)) bacteria has been investigated by the methods of radial diffusion in an agarose gel and serial dilution in a liquid nutritional medium. It has been established that the antimicrobial activity of the bioconjugates depends on the nature of the used stabilizer.

Enhancement of photobactericidal activity of chlorin-e6-cellulose nanocrystals by covalent attachment of polymyxin B.

Despite the advances made over the last decade, infections caused by multi-drug resistant bacterial strains are increasingly important societal issues that need to be addressed. New approaches have already been developed in order to overcome this problem. Photodynamic antimicrobial chemotherapy (PACT) could provide an alternative to fight infectious bacteria. This approach has already inspired the development of innovative materials. Interesting results have been obtained against Gram-positive bacteria, but it also appeared that Gram-negative strains, especially Pseudomonas aeruginosa, were less sensitive to PACT. Enhanced efficacy against Gram-negative bacteria had been previously obtained with photosensitizers bound to antimicrobial peptides. In this work, we designed a photobactericidal organic material, CNCsc6-PMB, consisting of cellulose nanocrystals to which the photosensitizer chlorin-e6 and the antimicrobial polypeptide polymyxin B (PMB) were covalently attached. These modified nanocrystals were characterized by IR spectroscopy, zeta potential measurements and elemental analyses, after which antibacterial assays were carried out. Following light irradiation, CNCsc6-PMB demonstrated efficiency against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis) by inhibition of bacterial growth. An amplifying effect of chlorin-e6 has been highlighted against these Gram-negative strains, based on membrane weakening and a potential docking effect from the polymyxin moiety. Such results confirmed the importance of using an antimicrobial peptide in order to broaden the spectrum of PACT.

The effect of Ochratoxin A on antimicrobial polypeptide expression and resistance to water mold infection in channel catfish (Ictalurus punctatus)

Mycotoxin contamination of agricultural commodities poses a serious risk to animal health, including aquaculture species. Ochratoxin A (OA) is the most immunotoxic ochratoxin, yet little is known about its effect on immune function in fish. Antimicrobial polypeptides (AMPPs) are one of the most potent, innate, host defense factors, yet very little is known about what types of chronic stressors affect their expression. Among the most prevalent and potent AMPPs in fish are histone-like proteins (HLPs). In this study, fish were fed 2, 4, or 8 mg OA/kg diet. Skin antibacterial activity and HLP-1 levels were measured on Days 0, 28 and 56. Feeding 2, 4 or 8 mg OA/kg diet resulted in significant growth depression, but higher levels (4 or 8 mg OA/kg diet) resulted in lowering feed intake (FI) and impaired feed conversion ratio. In addition, feeding 8 mg OA/kg diet increased susceptibility to experimental water mold (Saprolegnia) challenge, suggesting that OA toxicity might contribute to some saprolegnosis outbreaks. However, there were no changes in AMPP expression in any treatment group. Our data suggests that the increased disease susceptibility of channel catfish due to OA is probably due to mechanisms other than a direct effect on antimicrobial polypeptide expression.

Stenotrophomonas maltophilia PhoP, a Two-Component Response Regulator, Involved in Antimicrobial Susceptibilities.

Stenotrophomonas maltophilia, a gram-negative bacterium, has increasingly emerged as an important nosocomial pathogen. It is well-known for resistance to a variety of antimicrobial agents including cationic antimicrobial polypeptides (CAPs). Resistance to polymyxin B, a kind of CAPs, is known to be controlled by the two-component system PhoPQ. To unravel the role of PhoPQ in polymyxin B resistance of S. maltophilia, a phoP mutant was constructed. We found MICs of polymyxin B, chloramphenicol, ampicillin, gentamicin, kanamycin, streptomycin and spectinomycin decreased 2–64 fold in the phoP mutant. Complementation of the phoP mutant by the wild-type phoP gene restored all of the MICs to the wild type levels. Expression of PhoP was shown to be autoregulated and responsive to Mg2+ levels. The polymyxin B and gentamicin killing tests indicated that pretreatment of low Mg2+ can protect the wild-type S. maltophilia from killing but not phoP mutant. Interestingly, we found phoP mutant had a decrease in expression of SmeZ, an efflux transporter protein for aminoglycosides in S. maltophilia. Moreover, phoP mutant showed increased permeability in the cell membrane relative to the wild-type. In summary, we demonstrated the two-component regulator PhoP of S. maltophilia is involved in antimicrobial susceptibilities and low Mg2+ serves as a signal for triggering the pathway. Both the alteration in membrane permeability and downregulation of SmeZ efflux transporter in the phoP mutant contributed to the increased drug susceptibilities of S. maltophilia, in particular for aminoglycosides. This is the first report to describe the role of the Mg2+-sensing PhoP signaling pathway of S. maltophilia in regulation of the SmeZ efflux transporter and in antimicrobial susceptibilities. This study suggests PhoPQ TCS may serve as a target for development of antimicrobial agents against multidrug-resistant S. maltophilia.

Development of an antimicrobial endotracheal tube

Event Abstract Back to Event Development of an antimicrobial endotracheal tube Jun Li1, Matthew Kuc1, Fanni Torok1, Michael Bouchard1, Gregory Brotske1, Ashley Banks1 and Zheng Zhang1 1 Teleflex, United States Introduction: Endotracheal tubes (ETTs) provide a route for nosocomial organisms to colonize the lower respiratory tract and cause ventilator-associated pneumonia (VAP), which is the most common nosocomial infection in the intensive care unit. The antimicrobial ETTs are developed by the combination of the release of an antimicrobial polypeptide (AMP) with non-fouling surface modification technology. Materials and Methods: The unmodified ETTs were made of PVC by Teleflex. The AMP (KSL-W) was developed and provided by the USAISR-DTRD[1]. The formulated ETT had three layers: the methacrylate copolymer reservoir layer containing the KSL-W AMP, then a methacrylate copolymer capping layer, and finally a non-fouling, sulfobetaine methacrylate (SBMA) copolymer as the top coat layer (Figure 1). Automatic dip coating machine was used to make these coatings on full ETT devices. Results and Discussion: Zwitterionic SBMA modified ETTs: The non-fouling SBMA modified ETT was compared with ETTs modified by hydrophilic polymers including non-ionic, cationic, and anionic hydrophilic polymers, hydrophobic polymers, and superhydrophobic coatings made of boehmite[2] or commercial NeverWet (from Rust-OleumÒ). From the I-125 labelled fibrinogen and mucin analysis, SBMA modified ETT showed super fibrinogen resistance and better to similar mucin resistance to other modified surfaces. In vivo test was 5hrs of intubation in sheep. After extubated and stained with periodic acid, the non-fouling SBMA modified ETT showed significant reduction in the mucus/sputum adsorption to 58% and 81% compared to 20% and 35% for the control (Figure 2). Antimicrobial ETTs: When non-fouling SBMA was combined with KSL-W AMP to make three layers coating on ETTs, they demonstrated moderate resistance to mucin attachment in the presence of the AMP (36-55%).Antimicrobial testing against S. aureus and P. aeruginosa, showed substantially reduce the attachment of the test organisms by 2-4 log10 compared to unmodified ETTs for a period of 72 hours (Figure 3). The AMP release showed the AMP was rapid released within the first 72h and followed by a slower release up to 5d. A sheep model was developed based on the previously published model by Berra et al[3]. to study the antimicrobial activity of this AMP-releasing ETT. The result demonstrated that there were substantially fewer culturable microbial cells (2 to 4 log10) for all of the trachea, bronchial, and lung cultures of the antimicrobial ETTs. Conclusion: The antimicrobial ETT with AMP-releasing developed by Teleflex has the ability to substantially reduce microbial colonization of the lower trachea, bronchi, and lungs in sheep. Studies was funded by a grant from USAMRAA (award no. W81XWH-12-2-0084)

Helical antimicrobial polypeptides with radial amphiphilicity

α-Helical antimicrobial peptides (AMPs) generally have facially amphiphilic structures that may lead to undesired peptide interactions with blood proteins and self-aggregation due to exposed hydrophobic surfaces. Here we report the design of a class of cationic, helical homo-polypeptide antimicrobials with a hydrophobic internal helical core and a charged exterior shell, possessing unprecedented radial amphiphilicity. The radially amphiphilic structure enables the polypeptide to bind effectively to the negatively charged bacterial surface and exhibit high antimicrobial activity against both gram-positive and gram-negative bacteria. Moreover, the shielding of the hydrophobic core by the charged exterior shell decreases nonspecific interactions with eukaryotic cells, as evidenced by low hemolytic activity, and protects the polypeptide backbone from proteolytic degradation. The radially amphiphilic polypeptides can also be used as effective adjuvants, allowing improved permeation of commercial antibiotics in bacteria and enhanced antimicrobial activity by one to two orders of magnitude. Designing AMPs bearing this unprecedented, unique radially amphiphilic structure represents an alternative direction of AMP development; radially amphiphilic polypeptides may become a general platform for developing AMPs to treat drug-resistant bacteria.

Glucocorticoid dexamethasone down-regulates basal and vitamin D3 induced cathelicidin expression in human monocytes and bronchial epithelial cell line

Glucocorticoids (GCs) have been extensively used as the mainstream treatment for chronic inflammatory disorders. The persistent use of steroids in the past decades and the association with secondary infections warrants for detailed investigation into their effects on the innate immune system and the therapeutic outcome. In this study, we analyse the effect of GCs on antimicrobial polypeptide (AMP) expression. We hypothesize that GC related side effects, including secondary infections are a result of compromised innate immune responses. Here, we show that treatment with dexamethasone (Dex) inhibits basal mRNA expression of the following AMPs; human cathelicidin, human beta defensin 1, lysozyme and secretory leukocyte peptidase 1 in the THP-1 monocytic cell-line (THP-1 monocytes). Furthermore, pre-treatment with Dex inhibits vitamin D3 induced cathelicidin expression in THP-1 monocytes, primary monocytes and in the human bronchial epithelial cell line BCi NS 1.1. We also demonstrate that treatment with the glucocorticoid receptor (GR) inhibitor RU486 counteracts Dex mediated down-regulation of basal and vitamin D3 induced cathelicidin expression in THP-1 monocytes. Moreover, we confirmed the anti-inflammatory effect of Dex. Pre-treatment with Dex inhibits dsRNA mimic poly IC induction of the inflammatory chemokine IP10 (CXCL10) and cytokine IL1B mRNA expression in THP-1 monocytes. These results suggest that GCs inhibit innate immune responses, in addition to exerting beneficial anti-inflammatory effects.

Crystal structures of a polypeptide processing and secretion transporter.

Bacteria secrete peptides and proteins to communicate, to poison competitors, and to manipulate host cells. Among the various protein-translocation machineries, the peptidase-containing ATP-binding cassette transporters (PCATs) are appealingly simple. Each PCAT contains two peptidase domains that cleave the secretion signal from the substrate, two transmembrane domains that form a translocation pathway, and two nucleotide-binding domains that hydrolyse ATP. In Gram-positive bacteria, PCATs function both as maturation proteases and exporters for quorum-sensing or antimicrobial polypeptides. In Gram-negative bacteria, PCATs interact with two other membrane proteins to form the type 1 secretion system. Here we present crystal structures of PCAT1 from Clostridium thermocellum in two different conformations. These structures, accompanied by biochemical data, show that the translocation pathway is a large α-helical barrel sufficient to accommodate small folded proteins. ATP binding alternates access to the transmembrane pathway and also regulates the protease activity, thereby coupling substrate processing to translocation.

Evaluation of biodegradable film packaging to improve the shelf-life of Boletus edulis wild edible mushrooms

Abstract The objective of this work was to evaluate the effect of poly(lactic acid) (PLA) based biodegradable film packaging combining 0.5% nisin antimicrobial polypeptide on the physicochemical and microbial quality of Boletus edulis wild edible mushrooms stored at 4 ± 1 °C. The experiment was set up by packaging mushrooms with extruded PLA films containing 0, 7.5, and 15 wt.% triethyl citrate plasticizer. The low-density polyethylene (LDPE) film was used as the control. Mushrooms stored in PLA films containing 7.5 and 15 wt.% plasticizer provided better retention of quality characteristics and received higher sensory ratings compared to mushrooms stored in pure PLA film and LDPE film. Samples with these two treatments underwent minimal changes in texture, PPO activity, total bacteria count, and sensory attributes. Results suggest that nisin in combination with plasticized PLA film has the potential to maintain B. edulis wild edible mushroom quality and extend its postharvest life to 18 days. Industrial relevance B. edulis is one of the most commercialized mushrooms worldwide. However, as with all fresh mushrooms, there are severe preservation problems. Extruded PLA films containing triethyl citrate plasticizer plus antimicrobial agent nisin proved to be a suitable technology for mushroom conservation. This material exhibits an environmental-friendliness potential and a high versatility in food packaging.

Complete Genome Sequence of Bacillus amyloliquefaciens subsp. plantarum CC178, a Phyllosphere Bacterium Antagonistic to Plant Pathogenic Fungi.

Bacillus amyloliquefaciens subsp. plantarum strain CC178 is a phyllosphere bacterium with antagonistic activity against a wide range of plant fungal pathogens. The genome of strain CC178 is 3,916,828 bp in size and harbors 3,972 genes. Six giant gene clusters are dedicated to the nonribosomal synthesis of antimicrobial polypeptides and polyketides.

Effect of Histidine Rich Antimicrobial Peptides, Gad-1 and Gad-2, On Model Membranes at Different pH Values

Antimicrobial polypeptides (AMPs) are an important component of the innate immune system of many different organisms and are generally amphipathic and cationic in nature. Electrostatic interactions between positively charged residues of the AMP and negatively charged lipids in pathogen membranes are an important component of AMP mechanism and specificity. AMPs such as Gad-1 and Gad-2 that are rich in histidine, an amino acid that is uncharged at neutral pH and positively charged at slightly acidic pH, can thus exhibit pH dependent activity. 2H solid state NMR spectroscopy was used to study the effect of Gad-1 and Gad-2 on model membranes at different pH values. The addition of peptides to the model membrane system POPE/POPG-d31 (3:1) results in a change in the splitting of spectrum both at pH 7 and pH 5. Further investigation of peptide effects on lipid acyl chain motion were determined through calculations of orientational order parameter values (SCD). The order parameter profile demonstrated that Gad-1 causes more lipid acyl chain disordering than Gad-2, at both pH 7 and pH 5. These results are consistent with Gad-1's greater activity in assays for bacterial growth inhibition. On the other hand, while Gad-2 is more active at inhibiting bacterial growth at low pH than at neutral pH, its effect on lipid acyl chain disorder is the same at pH 7 and pH 5.

Preliminary study on expression of antimicrobial peptides in European sea bass (Dicentrarchus labrax) following in vivo infection with Vibrio anguillarum. A time course experiment

Antimicrobial polypeptides (AMPPs) are humoral components of the vertebrates and invertebrates innate immune system. Their potent broad spectrum antimicrobial activities have drawn the attention of the scientific community to their potential use not only as an alternative to antibiotics but also as functional targets for immunostimulants in order to enhance the host immunity. Fish synthesize a great number of these peptides but in European sea bass, an important fish species in the Mediterranean aquaculture, only a few AMPPs have been studied and these surveys have highlighted their functional role as predictive markers of stressful conditions. Many aspects concerning AMPP mode of action in the host during bacterial infections are still unknown. In this work a 72 h time course experiment, performed on juvenile sea bass intraperitoneally (i.p.) injected with a sub-lethal dose of Vibrio anguillarum, was aimed to investigate the mRNA expression of four specific AMPP genes and interleukin-1β (IL-1β) in skin, gills, spleen, and head kidney. AMPP genes were: dicentracin (DIC), histone-like protein 1 (HLP-1), histone-like protein 2 (HLP-2) and hemoglobin-like protein (Hb-LP). The delta–delta CT method in real-time RT-PCR allowed to gain more knowledge about temporal dynamics, preferential sites of expression as well as immunological and physiological role of these molecular markers. DIC was significantly up-regulated mainly in head kidney at 1.5–3 h post-infection (p.i.). HLP-1 showed an extended-time overexpression in gills and a significant up-regulation in spleen. HLP-2 was interestingly overexpressed in gills at 24 h p.i., while Hb-LP showed a significant up-regulation in skin for all the 72 h trial as well as lower but always significant values either in gills or in spleen. Different was the response of IL-1β that showed a dramatic up-regulation in spleen and head kidney at 8 h p.i. whilst in gills it displayed a severe inhibition. During this survey the i.p. stimulus surely conditioned the AMPP expression in skin and gills, especially as regards the DIC that as piscidin-related gene has an important defensive role in the mucosal tissues. However, two unconventional AMPP genes such as HLP-2 and Hb-LP, strictly related to the physiological mechanisms of fish, were less affected in terms of expression by the route of infection, being more evident in peripheral loci. These findings might suggest them as potential markers to be analyzed within plans of health survey in fish farms.

Determination of the Microbicidal Capacity of Platelets By a Biomonitoring System

Introduction: Various antimicrobial polypeptides are contained in platelet α-granules and collectively termed platelet microbicidal proteins. Remarkably, these constituents identified by purification and structural determination of platelet-derived antimicrobial functions were found to be identical with or derived from proteins previously known for other functions, among them thymosin β-4 (Tβ-4), a ubiquitous actin-binding protein. Others are the fibrinopeptides A and B (FPA and FPB), which are released upon proteolytic activation of fibrinogen by thrombin. Two other platelet microbicidal proteins are CXC chemokines, namely platelet factor 4 (PF4; new designation CXCL4) and platelet basic protein (PBP/CXCL7). Another platelet microbicidal proteins was found to be identical with the CC chemokine RANTES (renamed CCL5). Two proteolytic products of PBP known as connective tissue-activating peptide-3 (CTAP-3) and neutrophil-activating peptide-2 (NAP-2), were also isolated as platelet microbicidal proteins. Apparently, the most potent antimicrobial activity is associated with C-terminal truncation products of NAP-2 and CTAP-3, both lacking two amino acids at the N-terminus. These products were are designated thrombocidin-1 (TC-1) and thrombocidin-2 (TC-2) (Virulence 2010; 1:5; 440-464).The antimicrobial functions of platelets are donor-dependent, agonist-induced, and influenced by target microorganisms. It is cost intensive and time consuming to determine the microbicidal capacity of platelets in vitro. We therefore developed a biomonitoring system to assess the microbicidal capacityof platelets in response to pathogenic gram-negative strains.Methods: Platelets, obtained by apheresis from healthy volunteers, were washed repeatedly in HEPES-buffered (pH 7.3) Tyrode solution (Cold Spring Harb Protoc 2007). For specific experiments, platelet-poor plasma or serum, incubated after preparation at 37°C for 1 hour, was used. Washed platelets (5x108/ml) were activated by 10 U/ml thrombin and incubated at 37°C for 1 hour. After adding 1 % agarose, blood-derived samples were casted onto wedge-shaped low concentrated lysogeny broth (low LB) agar (1 g/l tryptone, 0.5 g/l yeast extract, 1 g/l NaCl, and 1.8 % agar) into square dishes (12 cm x 12 cm) to obtain a linear agar gradient. Mid-log cultures of acinetobacter baumanni, aeromonas caviae, citrobacter freundii, klebsiella oxytoca, pseudomonas aeroginosa, pseudomonas fluorescence, stenotrophomonas maltophilia grown in LB-Miller medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, and 1.8 % agar) were washed and the OD was adjusted to 0.4. To incubate the bacteria over night at 37°C, lines of diluted bacterial cultures were distributed along to the agar gradients. Photographs were taken to measure the growth inhibition (n=7) longitudinal to the increasing concentration of a blood-derived sample agar gradient in comparison to the negative control (plasma agar gradient) or in comparison to the agar gradient without blood-derived samples.Results: Apart from S. maltophilia, overall growth of the bacterial strains was inhibited by (mean+SD) 25.9+10,5 % (p<0.05) on the serum agar gradient compared to the plasma control agar gradient, specifically 25.0% (A.baumanni), 38.5 % (A. caviae), 37.5 % (C. freundii), 25.0 % (K. oxytoca), 17.0 % (P. aeroginosa), and 12.5 % (P. fluorescence). Testing of thrombin-activated platelets revealed an inhibition of S. maltophilia by 15.0 % and of A. caviae by 8.8 % compared to the HEPES-buffered control agar (mean 11.9 + 4.4 % inhibition of growth, p>0.05). Other bacterial strains were not inhibited by thrombin-activated platelets.Conclusion: Inhibition of bacterial growth is measurable longitudinal a linear agar gradient containing microbicidal proteins released by activated platelets. Due to their strain-specific biochemistry not all gram-negative strains tested are sensitive to platelet microbicidal proteins. Serum and thrombin-activated platelet convey different antimicrobial effects. Thus, distinct bacterial strains can resist to platelet microbicidal proteins to different extents.Biomonitoring allows monitoring of inhibition of bacterial growth caused by platelet microbicidal proteins. The bacterial strains evaluated in this study may be used as bioindicators to determine the microbicidal capacity of platelets. DisclosuresNo relevant conflicts of interest to declare.

Detection of antimicrobial (poly)peptides with acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.

Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887-895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620-631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483-6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210-219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313-1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host-pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and protein transfer to the membrane, and conducting the immunodetection using an alkaline phosphatase/NBT/BCIP system. A standard SDS-PAGE in comparison with AU-PAGE and the corresponding Western immunoblot are depicted in Fig. 1.

Short Communication: Genital Tumor Growth Factor-β1 Levels in HIV-Infected Indian Women Are Associated with Reduced Levels of Innate Antimicrobial Products and Increased HIV Shedding

Tumor growth factor (TGF)-β1 is a cytokine with potent immunoinhibitory functions and is known to be secreted by vaginal epithelial cells. The present study was designed to determine the association of cervicovaginal levels of TGF- β1 with various innate immune secretions such as cytokines and antimicrobial polypeptides [Trappin-2/Elafin and secretory leukocyte protease inhibitor (SLPI)] and cervical HIV shedding in HIV-infected Indian women. TGF- β1, antimicrobial polypeptides, and cytokine levels were estimated in the cervicovaginal lavages (CVLs) of 36 age-matched HIV-infected and 31 HIV-uninfected asymptomatic Indian women using an ELISA and Bio-Plex Assay, respectively. The nonparametric Mann-Whitney test and Spearman's test were used to compare the levels from both the groups and to determine the association of the TGF-β1 levels with cervical viral shedding and antimicrobial peptides. The levels of Trappin-2/Elafin and SLPI were similar in the CVLs of HIV-infected and HIV-uninfected women, but were significantly associated with a low cervical viral load (r=-0.501, p=0.005 for Trappin-2/Elafin and r=-0.488, p=0.007 for SLPI). Eleven (30.5%) of the 36 HIV-infected women showed 5- to 30-fold higher levels of TGF-β1 as compared to the levels in uninfected women. The TGF-β1 levels were significantly associated with higher cervical viral load (r=0.425, p=0.03) and with lower levels of Trappin-2/Elafin (r=-0.407, p=0.03) and SLPI (r=-0.405, p=0.04). The findings indicate a possible interdependent mechanism driving the identified higher TGF-β1 and lower antimicrobial peptide (Trappin-2/Elafin and SLPI) levels at the genital mucosa surface in HIV-infected women. We postulate that a combination of increased TGF-β1 secretion and altered levels of Trappin-2/Elafin and SLPI contributes to increased HIV shedding. The observation warrants further studies to identify the underlying mechanisms linking increased mucosal TGF-β1 levels and genital HIV shedding. Considering the known association of HIV and cervical cancers, it will also be important to assess the predictive capacity of TGF-β1 levels in HIV-associated cervical malignancies.

Differential expression of antimicrobial polypeptides in cord blood samples of preterm and term infants

To determine levels of antimicrobial polypeptides (AMP) in cord blood of term and preterm neonates and to investigate influencing factors. In a single-centre study, n = 139 preterm infants and n = 36 term infants were included. AMP levels were analysed in supernatants of whole cord blood cultures with a standardised concentration of 5 × 10(6) white blood cells/mL via enzyme-linked immunosorbent assay (ELISA). Lactoferrin, human neutrophil peptides (HNP) 1-3 and bacterial permeability-increasing protein (BPI) expression in cord blood of preterm infants were influenced by the cause of preterm delivery, that is increased levels in infants with clinical amniotic infection. AMP levels also weakly correlated with white blood cell and neutrophil count at birth. In the whole cohort, no association between gestational age or birthweight with AMP levels was found. In the subgroup of infants without clinical amniotic infection (n = 77 preterm infants, n = 36 healthy term infants), we noted a weak correlation between gestational age and lactoferrin, calprotectin and HNP1-3 levels. In addition to that, we observed higher levels of lactoferrin and HNP1-3 in large-for-gestational-age infants. Our study confirms that several factors influence cord blood AMP levels which underlines the difficulties of using AMP levels as biomarkers of immunological response.

Studies on the role of insect hemolymph polypeptides: Galleria mellonella anionic peptide 2 and lysozyme

The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella.