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Biochemical characterization and evaluation of cytotoxicity of antistaphylococcal chimeric protein P128
Shilpa E George, Ravisha Chikkamadaiah, Murali Durgaiah, Amruta A Joshi, Ullas P Thankappan, Shampur N Madhusudhana, Bharathi Sriram
BMC Research Notes , 2012, DOI: 10.1186/1756-0500-5-280
Abstract: We evaluated the requirement or effect of divalent cations and the metal ion chelator, EDTA upon biological activity of P128. As the protein is intended for therapeutic use, we tested its activity in presence of body fluids and antibodies specific to P128. For the same reason, we used standard human cell lines to evaluate cytotoxic effects, if any.The divalent cations, calcium and magnesium at upto 25?mM and Zinc upto 2.5?mM neither inhibited nor enhanced P128 activity. Incubation of this protein with EDTA, human serum, plasma and blood also did not alter the antibacterial properties of the molecule. No inhibitory effect was observed in presence of hyper-immune sera raised against the protein. Finally, P128 did not show any cytotoxic effect on HEp2 and Vero cells at the highest concentration (5?mg/mL) tested.The results presented here throw light on several properties of protein P128. Taken together, these substantiate the potential of P128 for therapeutic use against S. aureus. Further development of the protein and conduct of preclinical safety studies in animals is warranted.Current treatment for staphylococcal infections face multiple challenges including spread of highly virulent strains in the hospital and community environments. Novel therapeutic agents and modalities are becoming a necessity to combat the often invasive infections by these strains [1,2].In S. aureus and other Gram-positive bacteria, where the outer membrane is absent, the multilayered peptidoglycan or murein, commonly referred to as the cell wall, represents the limit of the cell. Peptidoglycan is an important component in these bacteria, conferring strength and rigidity to the cell, allowing growth and division, maintaining cell shape, and protecting against osmotic lysis [3]. The peptidoglycan therefore presents an excellent target for antimicrobial action. Murein degrading enzymes that are capable of hydrolyzing the various carbohydrate or protein linkages of the peptidoglycan layer bring
A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein
Vivek Paul, Sanjeev Rajagopalan, Sudarson Sundarrajan, Shilpa E George, Jiya Y Asrani, Renjith Pillai, Ravisha Chikkamadaiah, Murali Durgaiah, Bharathi Sriram, Sriram Padmanabhan
BMC Microbiology , 2011, DOI: 10.1186/1471-2180-11-226
Abstract: We identified a phage K gene, designated orf56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to S. aureus cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of S. aureus including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of S. aureus USA300 in an experimental model.We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient Staphylococcus-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of S. aureus and against the genus Staphylococcus in general. In vivo, P128 was efficacious against methicillin-resistant S. aureus in a rat nasal colonization model.Peptidoglycan-degrading enzymes or murein hydrolases have the ability to digest bacterial cell walls. Such enzymes from bacteriophages represent a unique class of antibacterial agents because of their ability to cleave bacterial peptidoglycan in a species-specific or genus-specific manner. Thus, they provide a means to selectively target pathogens [1-3].At the end of the bacteriophage infection process, progeny are released from the host cell by lysis, which is mediated by two phage-encoded gene products, endolysins
A Bacteriophage-Related Chimeric Marine Virus Infecting Abalone  [PDF]
Jun Zhuang,Guiqin Cai,Qiying Lin,Zujian Wu,Lianhui Xie
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0013850
Abstract: Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV) can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin). The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs), eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB) protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria.
Bacteriophage-Derived Vectors for Targeted Cancer Gene Therapy  [PDF]
Md Zahidul Islam Pranjol,Amin Hajitou
Viruses , 2015, DOI: 10.3390/v7010268
Abstract: Cancer gene therapy expanded and reached its pinnacle in research in the last decade. Both viral and non-viral vectors have entered clinical trials, and significant successes have been achieved. However, a systemic administration of a vector, illustrating safe, efficient, and targeted gene delivery to solid tumors has proven to be a major challenge. In this review, we summarize the current progress and challenges in the targeted gene therapy of cancer. Moreover, we highlight the recent developments of bacteriophage-derived vectors and their contributions in targeting cancer with therapeutic genes following systemic administration.
Bacteriophage-Derived Peptidase Eliminates and Prevents Staphylococcal Biofilms  [PDF]
Mark Fenton,Ruth Keary,Olivia McAuliffe,R. Paul Ross,Jim O'Mahony,Aidan Coffey
International Journal of Microbiology , 2013, DOI: 10.1155/2013/625341
Abstract: New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, , as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4?h. In addition, was able to prevent biofilm formation by this strain. The lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors. 1. Introduction Staphylococcal species commonly colonise the skin and mucosal membranes of both humans and animals. They are a significant causative agent of bovine mastitis in dairy herds [1] and are also associated with a number of diseases in humans, ranging from a variety of skin conditions to more serious infections such as septicemia [2]. Staphylococcal food poisoning is among the most common food-borne microbial diseases [3] and contamination of food industrial surfaces with staphylococcal species has been demonstrated to be a considerable risk factor [4–6]. Along with the urgent requirement for novel antibacterials to combat the prevalence of antibiotic/disinfectant resistant staphylococci in food processing, veterinary and healthcare settings, there is an increasing need for effective antimicrobial agents which can prevent and treat staphylococcal biofilm-associated infections [7–11]. Biofilms are multilayered communities of sessile cells protected by an extracellular matrix, which often adhere to food contact surfaces, damaged tissue and indwelling medical devices [12–14]. Once formed, biofilms may be up to 1,000 times more resistant to antimicrobial agents than planktonic cells alone making them particularly difficult to eliminate [15]. This can ultimately lead to increased risk of persistent infections, as is commonly the case with bovine mastitis [16]. In addition, because of their increased levels of resistance, biofilm-associated infections can result in a need for explantation of medical devices in human healthcare settings [17, 18]. Although the precise mechanisms of biofilm antibiotic resistance have yet to be fully resolved, failure to
Expression of chimeric HCV peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plant-derived vaccine against HCV
AK El Attar, AM Shamloul, AA Shalaby, BY Riad, A Saad, HM Mazyad, JM Keith
African Journal of Biotechnology , 2004,
Abstract: Hepatitis C virus (HCV) is the major etiologic agent of blood transfusion–associated and sporadic non-A non-B hepatitis affecting more than 180 million worldwide. Vaccine development for HCV has been difficult and there is no vaccine or effective therapy against this virus. In this paper, we describe the development of an experimental plant-derived subunit vaccine against HCV. Our subunit vaccine originates from a consensus HCV-HVR1 epitope (R9) that antigenically mimics many natural HVR1 variants. This HVR1 sequence was cloned into the open reading frame of a plant virus, Alfalfa Mosaic Virus (ALMV) coat protein (CP). The chimeric ALMV RNA4 containing sequence-encoding R9 epitope was introduced into full-length infectious ALMV-RNA3 that was utilized as an expression vector. The recombinant chimeric protein is expressed in transgenic tobacco plants (P12) expressing ALMV RNA1 and 2. Plant–derived HVR1/ALMV-CP reacted with HVR1 and/or ALMV-CP specific monoclonal antibodies and immune sera from individuals infected with HCV. Using plant-virus based transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccine. Key Words: Hepatitis C virus (HCV), transgenic tobacco plants (P12), consensus HCV HVR1 epitope (R9), and chimeric ALMV-RNA4. African Journal of Biotechnology Vol.3(11) 2004: 588-594
Chimeric Newcastle Disease Virus Protects Chickens against Avian Influenza in the Presence of Maternally Derived NDV Immunity  [PDF]
Constanze Steglich, Christian Grund, Kristina Ramp, Angele Breithaupt, Dirk H?per, Günther Keil, Jutta Veits, Mario Ziller, Harald Granzow, Thomas C. Mettenleiter, Angela R?mer-Oberd?rfer
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0072530
Abstract: Newcastle disease virus (NDV), an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV). However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of avian paramyxovirus type 8 (APMV-8) instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination.
Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus
Choonghee Lee, Hyung-Hwan Kim, Kyung Mi Choi, Kyung Won Chung, Yien Choi, Mi Jang, Tong-Soo Kim, Nam-Jun Chung, Ho-Gun Rhie, Ho-Sa Lee, Youngjoo Sohn, Hyuck Kim, Sung-Jae Lee, Hyeong-Woo Lee
Malaria Journal , 2011, DOI: 10.1186/1475-2875-10-106
Abstract: A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot.The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38) and a clinical specificity of 100% (n = 24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice.The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.Plasmodium vivax, a causative agent of relapsing benign tertian malaria, is the second most important malaria-causing species; it afflicts several hundred million people annually [1,2]. Malaria constitutes a major health problem and is closely associated with socioeconomic burden in many temperate and most tropical countries. The malaria situation of Korea peninsula is also not different from other countries. It reemerged in the early 1990s after two decade-long absence. Following government intervention, reported cases of malaria decreased over the course of several years. However, it is unlikely that malaria has been completely eradicated from Korea; not only is there a steady influx of travelers and workers from countries where malaria is endemic
APPLICATION OF FACTORIAL DESIGN FOR THE OPTIMIZED PRODUCTION OF ANTISTAPHYLOCOCCAL METABOLITE BY AUREOBASIDIUM PULLULANS  [cached]
E Kalantar,R Deopurkar
Jundishapur Journal of Natural Pharmaceutical Products , 2007,
Abstract: Background: Antimicrobial substances are mainly produced by bacteria and lower fungi, and have great roles in the treatment of most infectious diseases..Purpose: Production of antistaphylococcal metabolite from Aureobasidium pullulans by development of a cultural medium using response surface methodology. Methods: Production of antistaphylococcal metabolite from Aureobasidium pullulans was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, glucose, peptone, KH2PO4 as well as initial pH and temperature were statistically combined using a 2 level, 4 factor experimental design and tested for their influence on maximal antistaphylococcal metabolite production. Results were analyzed using response surface methodology (RSM) software. Results: Optimum production of antistaphylococcal metabolite occurred at glucose 2.0%, peptone 2.5%, KH2PO4 0.15%, pH 4.0 and temperature 30°C. The maximum amount of antistaphylococcal metabolite 900 U/flask from about 0.85 g of dry weight biomass was extracted. Conclusion: The antistaphylococcal activity of Aureobasidium pullulans seemed to be associated with primary metabolite rather than secondary metabolite. However, this conclusion should be taken with caution because both secondary metabolites as well as antibiotics are heterogeneous group and our knowledge regarding the exact definitions and of secondary metabolite / antibiotics are far from the perfection.
Molecular weight dependent antistaphylococcal activities of oligomers/polymers synthesized from 3-aminopyridi
CAHIT AKGUL,MEHMET YILDIRIM
Journal of the Serbian Chemical Society , 2010,
Abstract: The main aim of this study was to investigate the relationship between molecular weight and the antistaphylococcal activity of oligomers/polymers synthesized from 3-aminopyridine. Different oligomers/polymers were synthesized from 3-aminopyridine by changing the oxidative polycondensation reaction conditions. They were characterized by size exclusion chromatography and their antibacterial activities were compared by employing standardized susceptibility assays. The obtained experimental results demonstrated that 3-aminopyridine had no antistaphylococcal activity. However, as a result of polymerization, strong antistaphylococcal activity was obtained. Oligomers/polymers synthesized from 3-aminopyridine had varying degrees of antistaphylococcal activity and the maximum activity was obtained from relatively very short oligomers. It was therefore concluded that polymerization of 3-aminopyridine is required for antistaphylococcal activity and strength of this activity depends on the molecular weights of the synthesized molecules.
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