Enhancing thermal stability and lytic activity of phage lysin PlyAB1 from Acinetobacter baumannii.

With the increasingly serious drug resistance of Acinetobacter baumannii, there is an increasingly urgent need for new antibacterial drugs. Phage lysin PlyAB1 has a bactericidal effect on drug-resistant A. baumannii, which has the potential to replace antibiotics to fight infection caused by A. baumannii. However, its application is limited by its thermal stability and lytic activity. To solve these problems, molecular dynamics (MD) simulations combined with Hotspot wizard 3.0 were used to identify key residue sites affecting thermal stability, and evolutionary analysis combined with multiple sequence alignment was used to identify key residue sites affecting lytic activity. Four single-point variants with significantly increased thermal stability and four single-point variants with significantly lytic activity were obtained, respectively. Furthermore, by superimposing mutations, we obtained three double-point variants, G100Q/K69R, G100R/K69R, and G100K/K69R, with significantly improved thermal stability and improved lytic activity. At 45°C, the lytic activity and half-life of the optimal variant G100Q/K69R were 1.51- and 24-fold higher than those of the wild PlyAB1, respectively. These results deepen our understanding of the structure and function of phage lysin and contribute to the application of phage lysin in antibiotic substitution.

Bronchodilatory effect of higenamine as antiallergic asthma treatment.

Asthma is a complex airway disease that affects more than 350 million humans worldwide. Allergic asthma symptoms are induced by Th2 immune response with the release of cytokines and allegro-inflammatory mediators that amplify the inflammatory response, airway hyper-responsiveness (AHR) and hyperproduction of mucus. Higenamine, as a chemical compound, is a ß2 adrenoreceptor agonist and can be used as bronchodilator in allergic asthma.BALB/c mice were allocated in four groups and then allergic asthma was induced in three groups. One of the asthmatic groups was treated with albuterol and other one was treated with higenamine. At least, methacholine challenge to determine the AHR, measurement of cytokines, total immunoglobulin E (IgE), LTB4 and LTC4 levels, evaluation of gene expression of Muc5ac, Muc5b, Agr2 and Arg1, and histopathological study were done.Higenamine treatment reduced AHR, interleukin (IL)-4, IL-13 levels, mRNA expression of MUC5ac, MUC5b, Arg1 and Agr2, goblet cell hyperplasia and mucus hypersecretion. Higenamine had no significant effect on IL-5, interferon-γ (INF-γ), IgE, LTB4, LTC4 levels and eosinophilic inflammation in lung tissue.Higenamine treatment controls asthma acute attack and breathlessness and can be used as asthma treatment with control of AHR and decrease of airflow obstruction and mucus hypersecretion and had allegro-immune-regulatory effect. But higenamine treatment had no notable effect on the inflammation and inflammatory factors.

Plant-derived saccharides and their inhibitory potential on metastasis associated cellular processes of pancreatic ductal adenocarcinoma cells.

This study intends to investigate the inhibitory potential of different plant derived saccharides on cell migration and adhesion of pancreatic ductal adenocarcinoma (PDAC) cells to microvascular liver endothelium, particularly considering the role of transmembranous galectin-3. PDAC cell lines PancTu1 and Panc1 were characterized by considerable (transmembranous) galectin-3 (Gal3) expression. SiRNA mediated Gal3 knockdown as well as treatment with differentially processed pectins and arabinogalactan-proteins (AGPs) did not impact on cell migration of either PDAC cell line. In contrast, Gal3 knockdown reduced adhesion of PDAC cells to the liver endothelial cell line TMNK-1 being more pronounced in Panc1 cells. Similarly, plant derived substances did not impact cell adhesion of PancTu1 cells while partially hydrolyzed citrus pectin (MCP), pectinase-treated MCP (MCPPec) and partially hydrolized AGP (AGPTFA) clearly diminished adhesive properties of Panc1 cells. MCPPec or AGPTFA could not further intensify the adhesion reducing effect of galectin-3 knockdown, indicating that these plant derived polysaccharides are able to inhibit PDAC cell adhesion to liver endothelial cells in a galectin-3 dependent manner. Overall, these data suggest an inhibitory potential of plant derived processed saccharides which have undergone chemical modification in impairing PDAC cell adhesion to liver endothelium.

Chemical characterization and complement modulating activities of an arabinogalactan-protein-rich fraction from an aqueous extract of avocado leaves.

The aim of this study was to chemically characterize an arabinogalactan-protein-rich fraction (FRAGP) obtained from an aqueous extract of avocado leaves and investigate its effects on the classical pathway of the complement system. The FRAGP contained 4.6% ±â€¯1.8%, 22.5% ±â€¯4.9%, and 76.7% ±â€¯8.8% of total protein, arabinogalactan-protein, and carbohydrates, respectively. Arabinose and galactose were the main monosaccharide constituents. FT-IR and NMR data, together with linkage analyses, indicated the presence of a structure that included a (1 → 3)-linked ß-D-Galp main chain, mainly substituted at O-6 by Gal and Ara residues, which was characteristic of a type II arabinogalactan. The effect of FRAGP on the classical pathway of complement system was examined by a hemolytic fixation test and comparing with heparin, which was used as a control for inhibition. With pre-incubation, the IC50 of FRAGP was 1.90 ±â€¯1.1 µg/mL, which was similar to that of heparin (IC50 = 2.90 ±â€¯0.3 µg/mL). Without pre-incubation, the IC50 values were 18.6 ±â€¯3.7 and 8.0 ±â€¯4.1 µg/mL for FRAGP and heparin, respectively. Collectively, these results suggested that FRAGP has an inhibitory effect on the classical pathway of the complement system.

Phage-derived lysins as potential agents for eradicating biofilms and persisters.

Bacterial biofilms are highly resistant to the action of antibiotics. Presence of persisters, phenotypically resistant populations of bacterial cells, is thought to contribute toward recalcitrance of biofilms. The phage-derived lysins, by virtue of their ability to cleave the peptidoglycan of bacterial cells in an enzymatic manner, have the unique ability to kill dormant cells. Several lysins have shown potent antibiofilm activity in vitro. The fact that lysins have shown better efficacy than conventional drugs in animal models of endocarditis and other infections involving biofilms suggests that the lysins can potentially be developed against difficult-to-treat bacterial infections.

Arabinogalactan Proteins From Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes In Vitro.

Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However, little is currently known regarding their potential activity toward skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability. Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. J. Cell. Physiol. 232: 2558-2568, 2017. © 2016 Wiley Periodicals, Inc.

LysGH15 kills Staphylococcus aureus without being affected by the humoral immune response or inducing inflammation.

The lysin LysGH15, derived from the staphylococcal phage GH15, exhibits a wide lytic spectrum and highly efficient lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we found that LysGH15 did not induce resistance in MRSA or methicillin-sensitive S. aureus (MSSA) strains after repeated treatment. Although LysGH15 triggered the generation of LysGH15-specific antibodies in mice, these antibodies did not block lytic activity in vitro (nor the binding capacity of LysGH15). More importantly, when the antibody titre was highest in mice immunized with LysGH15, a single intravenous injection of LysGH15 was sufficient to protect mice against lethal infection with MRSA. These results indicated that LysGH15-specific antibodies did not affect the killing efficiency of LysGH15 against MRSA in vitro or in vivo. LysGH15 also reduced pro-inflammatory cytokines in mice with lethal infections. Furthermore, a high-dose LysGH15 injection did not cause significant adverse effects or pathological changes in the main organs of treated animals. These results provide further evidence for the administration of LysGH15 as an alternative strategy for the treatment of infections caused by MRSA.

Post-Synthetic Defucosylation of AGP by Aspergillus nidulans α-1,2-Fucosidase Expressed in Arabidopsis Apoplast Induces Compensatory Upregulation of α-1,2-Fucosyltransferases.

Cell walls are essential components of plant cells which perform a variety of important functions for the different cell types, tissues and organs of a plant. Besides mechanical function providing cell shape, cell walls participate in intercellular communication, defense during plant-microbe interactions, and plant growth. The plant cell wall consists predominantly of polysaccharides with the addition of structural glycoproteins, phenolic esters, minerals, lignin, and associated enzymes. Alterations in the cell wall composition created through either changes in biosynthesis of specific constituents or their post-synthetic modifications in the apoplast compromise cell wall integrity and frequently induce plant compensatory responses as a result of these alterations. Here we report that post-synthetic removal of fucose residues specifically from arabinogalactan proteins in the Arabidopsis plant cell wall induces differential expression of fucosyltransferases and leads to the root and hypocotyl elongation changes. These results demonstrate that the post-synthetic modification of cell wall components presents a valuable approach to investigate the potential signaling pathways induced during plant responses to such modifications that usually occur during plant development and stress responses.

Effect of the native polysaccharide of cashew-nut tree gum exudate on murine peritoneal macrophage modulatory activities.

The native polysaccharide of cashew-nut tree gum exudate (CNTG) and its arabinogalactan-protein component (CNTG-AGP) were tested by using immuno-stimulant and anti-inflammatory in vitro assays of murine peritoneal macrophage activities. In the assay for immuno-stimulant activity (without previous treatment with lipopolysaccharide; LPS), CNTG increased the production of interleukin (IL)-10 and both CNTG and CNTG-AGP decreased the concentrations of IL6. When the macrophages were incubated in the presence of LPS and CNTG a decrease in the levels of nitric oxide (NO(·)) and IFN-γ was observed. The results could explain the popular use of CNTG as an anti-inflammatory. In addition, CNTG is the main component of the cashew-nut tree gum exudate, which has been considered a versatile polymer with potential pharmaceutical and food industry applications. These data may contribute to the study of the immunomodulation activity of plant polysaccharides, as well as encourage future experiments in the field of cashew-nut tree gum exudate applications.

EC300: a phage-based, bacteriolysin-like protein with enhanced antibacterial activity against Enterococcus faecalis.

Bacteriophage lytic enzymes, either endolysins or virion-associated lysins, have been receiving considerable attention as potential antibacterial agents, particularly for the combat of antibiotic-resistant Gram-positive pathogens. A conclusion that easily emerges from the careful analysis of a great number of reports on the field is that the activity of phage lytic enzymes is rarely studied in conditions that support robust growth of the target bacteria. Here, we report the construction and study of a chimerical lysin, EC300, which was designed to target and kill Enterococcus faecalis in conditions supporting vigorous bacterial growth. EC300 resulted from the fusion of a predicted M23 endopeptidase domain of a virion-associated lysin to the putative cell wall binding domain of a previously characterized amidase endolysin, both produced by the E. faecalis phage F170/08. This bacteriolysin-like protein exhibited a clear enhanced lytic activity over the parental endolysin when both were assayed in a rich bacterial growth medium. We demonstrate the killing efficacy of EC300 against growing cells of a panel of typed E. faecalis clinical strains with high level of antibiotic resistance. The possible reasons for the marked difference between the lytic performance of EC300 and that of the amidase are discussed.

A novel chimeric phage lysin with high in vitro and in vivo bactericidal activity against Streptococcus pneumoniae.

OBJECTIVES: Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide and new antimicrobials are urgently needed. Our aim was new chimeric phage endolysins, or lysins, with improved bactericidal activity by swapping the structural components of two pneumococcal phage lysozymes: Cpl-1 (the best lysin tested to date) and Cpl-7S. METHODS: The bactericidal effects of four new chimeric lysins were checked against several bacteria. The purified enzymes were added at different concentrations to resuspended bacteria and viable cells were measured after 1 h. Killing capacity of the most active lysin, Cpl-711, was tested in a mouse bacteraemia model, following mouse survival after injecting different amounts (25-500 µg) of enzyme. The capacity of Cpl-711 to reduce pneumococcal biofilm formation was also studied. RESULTS: The chimera Cpl-711 substantially improved the killing activity of the parental phage lysozymes, Cpl-1 and Cpl-7S, against pneumococcal bacteria, including multiresistant strains. Specifically, 5 µg/mL Cpl-711 killed ≥7.5 log of pneumococcal R6 strain. Cpl-711 also reduced pneumococcal biofilm formation and killed 4 log of the bacterial population at 1 µg/mL. Mice challenged intraperitoneally with D39_IU pneumococcal strain were protected by treatment with a single intraperitoneal injection of Cpl-711 1 h later, resulting in about 50% greater protection than with Cpl-1. CONCLUSIONS: Domain swapping among phage lysins allows the construction of new chimeric enzymes with high bactericidal activity and a different substrate range. Cpl-711, the most powerful endolysin against pneumococci, offers a promising therapeutic perspective for the treatment of multiresistant pneumococcal infections.

Tobacco arabinogalactan protein NtEPc can promote banana (Musa AAA) somatic embryogenesis.

Banana is an important tropical fruit worldwide. Parthenocarpy and female sterility made it impossible to improve banana varieties through common hybridization. Genetic transformation for banana improvement is imperative. But the low rate that banana embryogenic callus was induced made the transformation cannot be performed in many laboratories. Finding ways to promote banana somatic embryogenesis is critical for banana genetic transformation. After tobacco arabinogalactan protein gene NtEPc was transformed into Escherichia coli (DE3), the recombinant protein was purified and filter-sterilized. A series of the sterilized protein was added into tissue culture medium. It was found that the number of banana immature male flowers developing embryogenic calli increased significantly in the presence of NtEPc protein compared with the effect of the control medium. Among the treatments, explants cultured on medium containing 10 mg/l of NtEPc protein had the highest chance to develop embryogenic calli. The percentage of lines that developed embryogenic calli on this medium was about 12.5 %. These demonstrated that NtEPc protein can be used to promote banana embryogenesis. This is the first paper that reported that foreign arabinogalactan protein (AGP) could be used to improve banana somatic embryogenesis.

Structural and biochemical characterization reveals LysGH15 as an unprecedented "EF-hand-like" calcium-binding phage lysin.

The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an "EF-hand-like" calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.

Degradation of methicillin-resistant Staphylococcus aureus biofilms using a chimeric lysin.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for a large number of chronic infections due to its ability to form robust biofilms. Herein, the authors evaluated the anti-biofilm activity of a Staphylococcus specific chimeric lysin ClyH on MRSA biofilms. ClyH is known to be active against planktonic MRSA cells in vitro and in vivo. The minimum concentrations for biofilm eradication (MCBE) of ClyH were 6.2-50 mg l(-1), much lower than those of antibiotics. Scanning electron microscope (SEM) analysis revealed that ClyH eliminated MRSA biofilms through cell lytic activity in a time-dependent manner. Viable plate counts and kinetic analysis demonstrated that biofilms of different ages displayed varying susceptibility to ClyH. Together with previously demonstrated in vivo efficacy of ClyH against MRSA, the degradation efficacy against biofilms of different ages indicates that ClyH could be used to remove MRSA biofilms in vivo.

Antiadhesive properties of arabinogalactan protein from ribes nigrum seeds against bacterial adhesion of Helicobacter pylori.

Fruit extracts from black currants (Ribes nigrum L.) are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2) was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with ß-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. ¹²5I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects.

Combination therapy with lysin CF-301 and antibiotic is superior to antibiotic alone for treating methicillin-resistant Staphylococcus aureus-induced murine bacteremia.

Lysins are bacteriophage-derived enzymes that degrade bacterial peptidoglycans. Lysin CF-301 is being developed to treat Staphylococcus aureus because of its potent, specific, and rapid bacteriolytic effects. It also demonstrates activity on drug-resistant strains, has a low resistance profile, eradicates biofilms, and acts synergistically with antibiotics. CF-301 was bacteriolytic against 250 S. aureus strains tested including 120 methicillin-resistant S. aureus (MRSA) isolates. In time-kill studies with 62 strains, CF-301 reduced S. aureus by 3-log10 within 30 minutes compared to 6-12 hours required by antibiotics. In bacteremia, CF-301 increased survival by reducing blood MRSA 100-fold within 1 hour. Combinations of CF-301 with vancomycin or daptomycin synergized in vitro and increased survival significantly in staphylococcal-induced bacteremia compared to treatment with antibiotics alone (P < .0001). Superiority of CF-301 combinations with antibiotics was confirmed in 26 independent bacteremia studies. Combinations including CF-301 and antibiotics represent an attractive alternative to antibiotic monotherapies currently used to treat S. aureus bacteremia.

Novel chimeric lysin with high-level antimicrobial activity against methicillin-resistant Staphylococcus aureus in vitro and in vivo.

The treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is a challenge worldwide. In our search for novel antimicrobial agents against MRSA, we constructed a chimeric lysin (named as ClyH) by fusing the catalytic domain of Ply187 (Pc) with the non-SH3b-like cell wall binding domain of phiNM3 lysin. Herein, the antimicrobial activity of ClyH against MRSA strains in vitro and in vivo was studied. Our results showed that ClyH could kill all of the tested clinical isolates of MRSA with higher efficacy than lysostaphin as well as its parental enzyme. The MICs of ClyH against clinical S. aureus strains were found to be as low as 0.05 to 1.61 mg/liter. In a mouse model, a single intraperitoneal administration of ClyH protected mice from death caused by MRSA, without obvious harmful effects. The present data suggest that ClyH has the potential to be an alternative therapeutic agent for the treatment of infections caused by MRSA.

Lysins: the arrival of pathogen-directed anti-infectives.

Lysins represent a novel class of anti-infectives derived from bacteriophage. Lysins are bacterial cell-wall hydrolytic enzymes that selectively and rapidly kill (≥3 log c.f.u. in 30 min) specific Gram-positive bacteria providing a targeted therapeutic approach with minimal impact on unrelated commensal flora. The potential for bacterial resistance to lysins is considered low due to targeting of highly conserved peptidoglycan components. Through cutting-edge genetic engineering, lysins can be assembled into large libraries of anti-infective agents tailored to any bacterium of interest including drug-resistant Gram-positive pathogens such as meticillin- and vancomycin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, and penicillin-resistant Streptococcus pneumoniae. Lysins can eliminate bacteria systemically and topically from mucosal surfaces and biofilms, as evidenced by experimental models of sepsis, endocarditis, pneumonia, meningitis, and nasopharyngeal, skin and vaginal decolonization. Furthermore, lysins can act synergistically with antibiotics and, in the process, resensitize bacteria to non-susceptible antibiotics. Clinical trials are being prepared to assess the safety and pharmacokinetic properties of lysins in humans.

phiBIOTICS: catalogue of therapeutic enzybiotics, relevant research studies and practical applications.

BACKGROUND: The incidence of bacterial infections in humans along with the growing problem of antibiotic resistance is a major public health concern worldwide. Therefore it is necessary to develop novel therapeutic agents to control microbial pathogens. In this regard, enzybiotics, lytic enzymes endowed with the capacity to degrade bacterial cell wall, are a very promising group of alternative antimicrobials. DESCRIPTION: Numerous experimental studies have confirmed unique therapeutic capabilities of enzybiotics and hence they are worth of wider attention of the medical community. In order to summarize the state of current knowledge of enzybiotics, we have developed phiBIOTICS, an information portal about known and studied therapeutic enzybiotics. phiBIOTICS contains information on chemical and biological properties of enzybiotics together with compendium of facts retrieved from research studies, where enzybiotics were applied. Our auxiliary phiBiScan program utility is dedicated for prediction of novel potential enzybiotics. CONCLUSIONS: phiBIOTICS presents a solid body of knowledge about all studied therapeutic enzybiotics to date. The database brings high-value information on outcomes of applied research and pre-clinical trials of these prospective antimicrobial agents. This information which was scattered in research papers with heterogeneous quality and relevance is now available in the form of manually curated database. phiBIOTICS and phiBiScan are freely accessible at http://www.phibiotics.org/.