Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells.

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

Exebacase Is Active In Vitro in Pulmonary Surfactant and Is Efficacious Alone and Synergistic with Daptomycin in a Mouse Model of Lethal Staphylococcus aureus Lung Infection.

Exebacase (CF-301) is a novel antistaphylococcal lysin (cell wall hydrolase) in phase 3 of clinical development for the treatment of Staphylococcus aureus bacteremia, including right-sided endocarditis, used in addition to standard-of-care antibiotics. In the current study, the potential for exebacase to treat S. aureus pneumonia was explored in vitro using bovine pulmonary surfactant (Survanta) and in vivo using a lethal murine pneumonia model. Exebacase was active against a set of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains, with an MIC90 of 2 µg/ml (n = 18 strains), in the presence of a surfactant concentration (7.5%) inhibitory to the antistaphylococcal antibiotic daptomycin, which is inactive in pulmonary environments due to specific inhibition by surfactant. In a rigorous test of the ability of exebacase to synergize with antistaphylococcal antibiotics, exebacase synergized with daptomycin in the presence of surfactant in vitro, resulting in daptomycin MIC reductions of up to 64-fold against 9 MRSA and 9 MSSA strains. Exebacase was also observed to facilitate the binding of daptomycin to S. aureus and the elimination of biofilm-like structures formed in the presence of surfactant. Exebacase (5 mg/kg of body weight 1 time every 24 h [q24h], administered intravenously for 3 days) was efficacious in a murine model of staphylococcal pneumonia, resulting in 50% survival, compared to 0% survival with the vehicle control; exebacase in addition to daptomycin (50 mg/kg q24h for 3 days) resulted in 70% survival, compared to 0% survival in the daptomycin-alone control group. Overall, exebacase is active in pulmonary environments and may be appropriate for development as a treatment for staphylococcal pneumonia.

Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression.

BACKGROUND: Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and a cause of necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of drug-resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains. RESULTS: An Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) containing a polyhistidine tag was expressed in E. coli. In addition, The E. coli optimized endolysin gene was engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X (PVX)-based transient expression vector. PlyCP41p accumulated to ~ 1% total soluble protein (100µg/gm f. wt. leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal systemic expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue. CONCLUSION: We demonstrated that functionally active bacteriophage PlyCP41 endolysin can be produced in systemically infected plant tissue with potential for use of crude plant sap as an effective antimicrobial agent against C. perfringens.

Thermophile Lytic Enzyme Fusion Proteins that Target Clostridium perfringens.

Clostridium perfringens is a bacterial pathogen that causes necrotic enteritis in poultry and livestock, and is a source of food poisoning and gas gangrene in humans. As the agriculture industry eliminates the use of antibiotics in animal feed, alternatives to antibiotics will be needed. Bacteriophage endolysins are enzymes used by the virus to burst their bacterial host, releasing bacteriophage particles. This type of enzyme represents a potential replacement for antibiotics controlling C. perfringens. As animal feed is often heat-treated during production of feed pellets, thermostable enzymes would be preferred for use in feed. To create thermostable endolysins that target C. perfringens, thermophile endolysin catalytic domains were fused to cell wall binding domains from different C. perfringens prophage endolysins. Three thermostable catalytic domains were used, two from prophage endolysins from two Geobacillus strains, and a third endolysin from the deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2). These domains harbor predicted L-alanine-amidase, glucosaminidase, and L-alanine-amidase activities, respectively and degrade the peptidoglycan of the bacterial cell wall. The cell wall binding domains were from C. perfringens prophage endolysins (Phage LYtic enzymes; Ply): PlyCP18, PlyCP10, PlyCP33, PlyCP41, and PlyCP26F. The resulting fifteen chimeric proteins were more thermostable than the native C. perfringens endolysins, and killed swine and poultry disease-associated strains of C. perfringens.

Characterization of LysBC17, a Lytic Endopeptidase from Bacillus cereus.

Bacillus cereus, a Gram-positive bacterium, is an agent of food poisoning. B. cereus is closely related to Bacillus anthracis, a deadly pathogen for humans, and Bacillus thuringenesis, an insect pathogen. Due to the growing prevalence of antibiotic resistance in bacteria, alternative antimicrobials are needed. One such alternative is peptidoglycan hydrolase enzymes, which can lyse Gram-positive bacteria when exposed externally. A bioinformatic search for bacteriolytic enzymes led to the discovery of a gene encoding an endolysin-like endopeptidase, LysBC17, which was then cloned from the genome of B. cereus strain Bc17. This gene is also present in the B. cereus ATCC 14579 genome. The gene for LysBC17 encodes a protein of 281 amino acids. Recombinant LysBC17 was expressed and purified from E. coli. Optimal lytic activity against B. cereus occurred between pH 7.0 and 8.0, and in the absence of NaCl. The LysBC17 enzyme had lytic activity against strains of B. cereus, B. anthracis, and other Bacillus species.

Microbial-derived products as potential new antimicrobials.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

Characterization of two glycosyl hydrolases, putative prophage endolysins, that target Clostridium perfringens.

Clostridium perfringens, a spore-forming anaerobic bacterium, causes food poisoning and gas gangrene in humans and is an agent of necrotizing enteritis in poultry, swine and cattle. Endolysins are peptidoglycan hydrolases from bacteriophage that degrade the bacterial host cell wall causing lysis and thus harbor antimicrobial therapy potential. The genes for the PlyCP10 and PlyCP41 endolysins were found in prophage regions of the genomes from C. perfringens strains Cp10 and Cp41, respectively. The gene for PlyCP10 encodes a protein of 351 amino acids, while the gene for PlyCP41 encodes a protein of 335 amino acids. Both proteins harbor predicted glycosyl hydrolase domains. Recombinant PlyCP10 and PlyCP41 were expressed in E. coli with C-terminal His-tags, purified by nickel chromatography and characterized in vitro. PlyCP10 activity was greatest at pH 6.0, and between 50 and 100 mM NaCl. PlyCP41 activity was greatest between pH 6.5 and 7.0, and at 50 mM NaCl, with retention of activity as high as 600 mM NaCl. PlyCP10 lost most of its activity above 42°C, whereas PlyCP41 survived at 50°C for 30 min and still retained >60% activity. Both enzymes had lytic activity against 75 C. perfringens strains (isolates from poultry, swine and cattle) suggesting therapeutic potential.

Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs.

The domestic pig is an attractive model for biomedical research because of similarities in anatomy and physiology to humans. However, key gaps remain in our understanding of the role of developmental genes in pig, limiting its full potential. In this publication, the role of NEUROGENIN 3 (NGN3), a transcription factor involved in endocrine pancreas development has been investigated by CRISPR/Cas9 gene ablation. Precomplexed Cas9 ribonucleoproteins targeting NGN3 were injected into in vivo derived porcine embryos, and transferred into surrogate females. On day 60 of pregnancy, nine fetuses were collected for genotypic and phenotypic analysis. One of the piglets was identified as an in-frame biallelic knockout (Δ2/Δ2), which showed a loss of putative NGN3-downstream target genes: NEUROD1 and PAX4, as well as insulin, glucagon, somatostatin and pancreatic polypeptide-Y. Fibroblasts from this fetus were used in somatic cell nuclear transfer to generate clonal animals to qualify the effect of mutation on embryonic lethality. Three live piglets were born, received colostrum and suckled normally, but experienced extreme weight loss over a 24 to 36-hour period requiring humane euthanasia. Expression of pancreatic endocrine hormones: insulin, glucagon, and somatostatin were lost. The data support a critical role of NGN3 in porcine endocrine pancreas development.

The endolysin from the Enterococcus faecalis bacteriophage VD13 and conditions stimulating its lytic activity.

Bacteriophages produce endolysins (peptidoglycan hydrolases) to lyse the host cell from within and release nascent bacteriophage particles. Recombinant endolysins can lyse Gram-positive bacteria when added exogenously. As a potential alternative antimicrobial, we cloned and expressed the enterococcal VD13 bacteriophage endolysin. VD13 endolysin has a CHAP catalytic domain with 92% identity with the bacteriophage IME-EF1 endolysin. The predicted size of VD13 endolysin is ∼27 kDa as verified by SDS-PAGE. The VD13 endolysin lyses Enterococcus faecalis strains, but not E. faecium or other non-enterococci. VD13 endolysin has activity from pH 4 to pH 8, with peak activity at pH 5, and exhibits greater activity in the presence of calcium. Optimum activity at pH 5 occurs in the absence of NaCl. VD13 endolysin, in ammonium acetate (C2H3O2NH4) calcium chloride (CaCl2) buffer pH 5, is stimulated to higher activity upon heating at temperatures up to 65°C for 30 min, whereas activity is lost upon heating to 42°C, in pH 7 buffer.

A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability.

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A.

Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.

Complete Genome Sequence of Staphylococcus aureus Phage GRCS.

The Staphylococcus aureus phage GRCS was isolated from a sewage treatment facility in India and has shown potential for phage therapy in a mouse model of bacteremia. Here, we report the complete genome sequence of this bacteriophage.

Differential coupling of Arg- and Gly389 polymorphic forms of the beta1-adrenergic receptor leads to pathogenic cardiac gene regulatory programs.

The beta(1)-adrenergic receptor (beta(1)AR; ADRB1) polymorphism Arg389Gly is located in an intracellular loop and is associated with distinct human and mouse cardiovascular phenotypes. To test the hypothesis that beta(1)-Arg389 and beta(1)-Gly389 alleles could differentially couple to pathways beyond that of classic G(s)-adenylyl cyclase (AC)/cAMP signaling, we performed comparative gene expression profile analyses on hearts from wild-type and transgenic mice that expressed either human beta(1)-Arg389 or beta(1)-Gly389 receptors, or AC5, sampling at an early age prior to the onset of pathological features. All three models upregulated the expression of genes associated with RNA metabolism and translation and downregulated genes associated with mitochondria and energy metabolism, consistent with shared cAMP-driven increase in cardiac contractility, protein synthesis, and compensatory downregulation of mitochondrial energy production. Both beta(1)AR alleles activated additional genes associated with other pathways. Uniquely, beta(1)-Arg389 hearts exhibited upregulated expression of genes associated with inflammation, programmed cell death, and extracellular matrix. These observations expand the scope of 7-transmembrane domain receptor signaling propagation beyond known cognate G protein couplings. Moreover, they implicate alterations of a repertoire of processes evoked by a single amino acid variation in the cardiac beta(1)AR that might be exploited for genotype-specific heart failure diagnostics and therapeutics.

Variable-length poly-C tract polymorphisms of the beta2-adrenergic receptor 3'-UTR alter expression and agonist regulation.

Beta(2)-adrenergic receptors (beta(2)-AR) expressed on airway epithelial and smooth muscle cells regulate mucociliary clearance and relaxation and are the targets for beta-agonists in the treatment of obstructive lung disease. However, the clinical responses display extensive interindividual variability, which is not adequately explained by genetic variability in the 5'-flanking or coding region of the intronless beta(2)-AR gene. The nonsynonymous coding polymorphism most often associated with a bronchodilator phenotype (Arg16) is found within three haplotypes that differ by the number of Cs (11, 12, or 13) within a 3'-untranslated region (UTR) poly-C tract. To examine potential effects of this variability on receptor expression, BEAS-2B cells were transfected with constructs containing the beta(2)-AR (Arg16) coding sequence followed by its 3'-UTR with the various polymorphic poly-C tracts. beta(2)Arg16-11C had 25% lower mRNA expression and 33% lower beta(2)-AR protein expression compared with the other two haplotypes. Consistent with this lower steady-state expression, beta(2)Arg16-11C mRNA displayed more rapid and extensive degradation after actinomycin D treatment compared with beta(2)Arg16-12C and -13C. However, beta(2)Arg16-12C underwent 50% less downregulation of receptor expression during beta-agonist exposure compared with the other two haplotypes. Thus these haplotypes direct a potential low-response phenotype due to decreased steady-state receptor expression combined with wild-type agonist-promoted downregulation (beta(2)Arg16-11C) and a high-response phenotype due to increased baseline expression combined with decreased agonist-promoted downregulation (beta(2)Arg16-12C). This heterogeneity may contribute to the variability of clinical responses to beta-agonist, and genotyping to identify these 3'-UTR polymorphisms may improve predictive power within the context of beta(2)-AR haplotypes in pharmacogenetic studies.

Pleiotropic beta-agonist-promoted receptor conformations and signals independent of intrinsic activity.

Beta-agonists used for treatment of obstructive lung disease have a variety of different structures but are typically classified by their intrinsic activities for stimulation of cAMP, and predictions are made concerning other downstream signals based on such a classification. We generated modified beta(2)-adrenergic receptors with insertions of energy donor and acceptor moieties to monitor agonist-promoted conformational changes of the receptor using intramolecular bioluminescence resonance energy transfer in live cells. These studies suggested unique conformations stabilized by various agonists that were not based on their classic intrinsic activities. To address the cellular consequences of these differences, G(s)-coupling, G(i)-coupling (p44/p42 activation), G protein-coupled receptor kinase-mediated receptor phosphorylation, internalization, and down-regulation were assessed in response to isoproterenol, albuterol, terbutaline, metaproterenol, salmeterol, formoterol, and fenoterol. In virtually every case, agonists did not maintain the classic rank order, indicating that distinct signaling is evoked by beta-agonists of different structures, which is unrelated to intrinsic activity. The extensive pleiotropy of agonist responses shown here suggests that classification of agonists by cAMP-based intrinsic activity is inadequate as it pertains to other intracellular events and that it may be possible to engineer a beta-agonist that stabilizes conformations that evoke an ideal portfolio of signals for therapeutic purposes.