Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules

Discovery of Three Toxic Proteins of Klebsiella Phage fHe-Kpn01

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic

A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts

Characterization of Anti-ECA Antibodies in Rabbit Antiserum Against Rough Yersinia enterocolitica O:3

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Bioprospecting Staphylococcus Phages with Therapeutic and Bio-Control Potential

Emergence of antibiotic-resistant bacteria is a serious threat to the public health. This is also true for Staphylococcus aureus and other staphylococci. Staphylococcus phages Stab20, Stab21, Stab22, and Stab23, were isolated in Albania. Based on genomic and phylogenetic analysis, they were classified to genus Kayvirus of the subfamily Twortvirinae. In this work, we describe the in-depth characterization of the phages that electron microscopy confirmed to be myoviruses. These phages showed tolerance to pH range of 5.4 to 9.4, to maximum UV radiation energy of 25 µJ/cm2, to temperatures up to 45 °C, and to ethanol concentrations up to 25%, and complete resistance to chloroform. The adsorption rate constants of the phages ranged between 1.0 × 10−9 mL/min and 4.7 × 10−9 mL/min, and the burst size was from 42 to 130 plaque-forming units. The phages Stab20, 21, 22, and 23, originally isolated using Staphylococcus xylosus as a host, demonstrated varied host ranges among different Staphylococcus strains suggesting that they could be included in cocktail formulations for therapeutic or bio-control purpose. Phage particle proteomes, consisting on average of ca 60–70 gene products, revealed, in addition to straight-forward structural proteins, also the presence of enzymes such DNA polymerase, helicases, recombinases, exonucleases, and RNA ligase polymer. They are likely to be injected into the bacteria along with the genomic DNA to take over the host metabolism as soon as possible after infection

Bioprospecting Staphylococcus Phages with Therapeutic and Bio-Control Potential

Emergence of antibiotic-resistant bacteria is a serious threat to the public health. This is also true for Staphylococcus aureus and other staphylococci. Staphylococcus phages Stab20, Stab21, Stab22, and Stab23, were isolated in Albania. Based on genomic and phylogenetic analysis, they were classified to genus Kayvirus of the subfamily Twortvirinae. In this work, we describe the in-depth characterization of the phages that electron microscopy confirmed to be myoviruses. These phages showed tolerance to pH range of 5.4 to 9.4, to maximum UV radiation energy of 25 µJ/cm2, to temperatures up to 45 °C, and to ethanol concentrations up to 25%, and complete resistance to chloroform. The adsorption rate constants of the phages ranged between 1.0 × 10−9 mL/min and 4.7 × 10−9 mL/min, and the burst size was from 42 to 130 plaque-forming units. The phages Stab20, 21, 22, and 23, originally isolated using Staphylococcus xylosus as a host, demonstrated varied host ranges among different Staphylococcus strains suggesting that they could be included in cocktail formulations for therapeutic or bio-control purpose. Phage particle proteomes, consisting on average of ca 60–70 gene products, revealed, in addition to straight-forward structural proteins, also the presence of enzymes such DNA polymerase, helicases, recombinases, exonucleases, and RNA ligase polymer. They are likely to be injected into the bacteria along with the genomic DNA to take over the host metabolism as soon as possible after infection

The effect of waaL genes deletion from Yersinia enterocolitica O:3 genome on bacteria LPS’ phenotype

Aim. To estimate WaaL ligase contribution in the lipopolysaccharide (LPS) phenotype profile formation of Y. enterocolitica O:3 (YeO3) bacteria. Methods. The waaL-knock-out mutants were created by an allelic exchange strategy. The LPS phenotypes of created mutants were visualized by silver-stained DOC-PAGE and immunoblotting with specific outer core (core oligosaccharide, hexasaccharide, OC) and O-polysaccharide (OPS or O-Ag) monoclonal antibodies. Results. Deletion of waaLOS gene from YeO3 genome has a marked effect on OC ligation in either single or double mutants. The waaLPS deletion has an opposite effect on the OPS ligation – barely detected increasing of OPS bands. Conclusions. The LPS ligases of YeO3 exhibit relaxed donor substrate specificity. Under given conditions the effect of WaaLOS ligase is more significant for OC and OPS ligation onto lipid A than that of WaaLPS.Мета. Дослідити участь лігаз WaaL у формуванні фенотипу ліпополісахариду (LPS) серед бактерій Y. enterocolitica O:3 (YeO3). Методи. Нокаутні мутанти по генах лігаз waaL створено внаслідок обміну алелями. Фенотипи LPS отриманих мутантів візуалізували, забарвлюючи сріблом гель DOC-PAGE, а також використовували імуноблот зі специфічними моноклональними антитілами до кору (корового олігосахариду, гексасахариду, ОC) та О-полісахариду (OPS, O-Ag). Результати. Делеція гена лігази waaLOS з геному бактерій YeO3 чинить помітний вплив на лігування ОC як в одиночних, так і в подвійних мутантах. Проте маніпуляції з геном лігази waaLPS призводять до ледь помітної стимуляції утворення OPS. Висновки. Лігази LPS бактерій YeO3 демонструють низьку субстратну специфічність. Участь лігази WaaLOS у формуванні повноцінної структури LPS є суттєвішою, аніж WaaLPS, за даних умов.Цель. Исследовать участие лигаз WaaL в формировании фенотипа липополисахарида (LPS) среди бактерий Y. enterocolitica O:3 (YeO3). Методы. Нокаутные мутанты по генам лигаз waaL созданы вследствие обмена аллелями. Фенотипы LPS полученных мутантов визуализировали, окрашивая серебром гель DOC-PAGE, а также с использованием иммуноблота со специфическими моноклональными антителами к кору (коровому олигосахариду, гексасахариду, OC) и О-полисахариду (OPS, O-Ag). Результаты. Делеция гена лигазы waaLOS из генома бактерий YeO3 оказывает заметное влияние на лигирование OC как в одиночных, так и двойных мутантах. Однако манипуляции с геном лигаз waaLPS приводят к едва заметной стимуляции образования OPS. Выводы. Лигазы LPS бактерий YeO3 демонстрируют низкую субстратную специфичность. Участие лигазы WaaLOS в образовании полноценной структуры LPS является более существенным, чем WaaLPS, при данных условиях

Biological and molecular characterization of fEg-Eco19, a lytic bacteriophage active against an antibiotic-resistant clinical Escherichia coli isolate

Characterization of bacteriophages facilitates better understanding of their biology, host specificity, genomic diversity, and adaptation to their bacterial hosts. This, in turn, is important for the exploitation of phages for therapeutic purposes, as the use of uncharacterized phages may lead to treatment failure. The present study describes the isolation and characterization of a bacteriophage effective against the important clinical pathogen Escherichia coli, which shows increasing accumulation of antibiotic resistance. Phage fEg-Eco19, which is specific for a clinical E. coli strain, was isolated from an Egyptian sewage sample. Phage fEg-Eco19 formed clear, sharp-edged, round plaques. Electron microscopy showed that the isolated phage is tailed and therefore belongs to the order Caudovirales, and morphologically, it resembles siphoviruses. The diameter of the icosahedral head of fEg-Eco19 is 68 +/- 2 nm, and the non-contractile tail length and diameter are 118 +/- 0.2 and 13 +/- 0.6 nm, respectively. The host range of the phage was found to be narrow, as it infected only two out of 137 clinical E. coli strains tested. The phage genome is 45,805 bp in length with a GC content of 50.3% and contains 76 predicted genes. Comparison of predicted and experimental restriction digestion patterns allowed rough mapping of the physical ends of the phage genome, which was confirmed using the PhageTerm tool. Annotation of the predicted genes revealed gene products belonging to several functional groups, including regulatory proteins, DNA packaging and phage structural proteins, host lysis proteins, and proteins involved in DNA/RNA metabolism and replication.Peer reviewe