Specific Lytic Activity Research Articles

Impact of effluent parameters and vancomycin concentration on vancomycin resistant Escherichia coli and its host specific bacteriophage lytic activity in hospital effluent

Vancomycin resistance in bacteria has been classified under high priority category by World Health Organization (WHO) and its presence in hospital effluent is reported to be increasing owing to excess antibiotics use. Among various strategies, bacteriophage has been recently considered as a promising biological agent for combating such antimicrobial resistant bacteria (ARB). However, the influence of effluent's properties on phage-ARB interaction in actual hospital effluent is not completely understood. The present works intends to study this influence of hospital effluent and its parameters on the interaction between vancomycin resistant E. coli (VRE) and its host specific bacteriophage. The isolated VRE was identified by 16S rRNA sequencing, matrix-assisted laser desorption/ionization–time of flight (MALDI – TOF) and whole genome sequencing. The infectivity of phage onto host bacteria was investigated using electron microscopic techniques, dynamic light scattering (DLS), spectrofluorophotometer and confirmed using double agar overlay method. The monovalency and polyvalency of isolated phage against various bacterial species were determined. The phage morphology was identical to T7 phage belonging to Podoviridae. The phage lysis was maximum at pH 7 (90.2%), 37 °C (91.6%) and vancomycin concentration of 50 μg/mL in both synthetic media (89.13%) and effluent (100%). At a maximum vancomycin concentration of 100 μg/mL, decrease in Ca, K, Mg and P (up to 19.70, 14.18, 28, and 15.82% respectively) concentration in effluent was observed due to phage infectivity when compared to control. The whole genome sequencing was performed and the bioinformatics analysis presented the role of mdfA gene encoding the efflux pump in causing vancomycin resistance in E. coli. It also depicted the presence of multiple genes responsible for mercury, cobalt, zinc and cadmium resistance in VRE. These results clearly indicate that bacteriophage mediated combating of VRE is possible in actual hospital effluent and can be used as one of the treatment methods.

Targeting Superbugs: Efficacy of Bacteriophage Therapy against Antibiotic-Resistant Pseudomonas Aeruginosa in Urinary Tract Infections

The rise of antibiotic-resistant bacterial infections poses a significant challenge to global health, particularly in the treatment of urinary tract infections (UTIs). This study evaluates the potential of bacteriophage therapy as an alternative treatment against antibiotic-resistant Pseudomonas Aeruginosa isolated from UTI samples. We isolated 12 strains of P. aeruginosa from 50 UTI samples and conducted extensive antibiotic susceptibility testing, revealing significant resistance to multiple conventional antibiotics such as Tetracycline, Septran, Ceftazidime, and Cefepime, while showing susceptibility to Imipenem and Meropenem. Concurrently, we isolated and characterized bacteriophages from sewage samples that demonstrated specific lytic activity against the antibiotic-resistant strains. Our findings suggest that bacteriophage therapy provides a high specificity and efficacy in targeting antibiotic-resistant P. aeruginosa, offering a promising alternative to traditional antibiotic therapies. This study underscores the potential of phages in clinical applications, advocating for further clinical trials and the development of a regulatory framework to integrate phage therapy into mainstream medical practice for combating antibioticresistant infections.

Complete genome analysis and biological characterization of a novel phage vB_CP_qdyz_P5 with lytic activity against Clostridium perfringens

Clostridium perfringens, a common foodborne pathogen, exhibit high-stress resistance. The prevailing reliance on antibiotics in the farming industry for its prevention and control has led to increasing concerns over antibiotic residue and bacterial resistance. Bacteriophages that possess specific lytic activity against C. perfringens are of significant interest. Here, a novel C. perfringens phage, named vB_CP_qdyz_P5, was isolated and characterized. The phage displayed high stability at temperatures below 70 °C and pH levels ranging from 4 to 12. Genome analysis revealed that vB_CP_qdyz_P5 has a double-stand DNA of 18,888 bp with a G + C composition of 28.8%. Among the 27 identified opening reading frames (ORFs), eight were found to be functional genes. BLASTn analysis showed that vB_CP_qdyz_P5 is closely related to phage DCp1, with a genome homology coverage of 83%. Phylogenetic analysis indicated that vB_CP_qdyz_P5 may be a novel phage of the family Guelinviridae, Susfortunavirus. This study provides important preliminary information for further research on the potential use of vB_CP_qdyz_P5 in protecting against C. perfringens and maintaining intestinal health.

A novel chimeric endolysin with enhanced lytic and binding activity against Clostridium perfringens

Clostridium perfringens is a gram-positive, anaerobic, and spore-forming bacterium that produces toxins causing various diseases in humans and livestock animals. With the growth in antibiotic resistance, finding alternative biocontrol agents against C. perfringens is necessary for food safety and animal health. Here, we isolated a C. perfringens-infecting bacteriophage CPD9 and characterized its endolysin LysCPD9. Although LysCPD9 has specific lytic activity against C. perfringens over a wide range of environmental conditions, its activity was lower than expected. To improve its lytic activity, we generated chimeric endolysins by shuffling the domains of LysCPD9 with those of thermostable C. perfringens endolysin, LysCPS2. Among the chimeras, a novel chimeric endolysin, ClyY, showed higher antimicrobial activity than its parental endolysin LysCPD9. In addition, ClyY significantly reduced C. perfringens cells in artificially contaminated milk and beef by 4-log CFU/ml and 3-log CFU/cm2, respectively, within 2 h. This enhanced lytic activity might be due to the increased cell wall binding because ClyY showed at least 3-fold higher binding activity than LysCPD9. ClyY could be utilized as an effective therapeutic as well as diagnostic tool for C. perfringens.

Isolation and evaluation of the efficacy of bacteriophages against multidrug-resistant (MDR), methicillin-resistant (MRSA) and biofilm-producing strains of Staphylococcus aureus recovered from bovine mastitis

BackgroundStaphylococcus aureus (S. aureus) is one of the major causes of bovine mastitis with significant economic losses around the worldwide. The emergence of multidrug-resistant (MDR), methicillin-resistant (MRSA) and biofilm-producing strains of S. aureus challenges the treatment strategies based on the antibiotic application. Today, alternative or combinational treatment options such as bacteriophage application has received much attention. The goal of the present study was to focus on isolation and evaluation of the efficacy of bacteriophages with specific lytic activity against S. aureus strains with low cure rates (MDR, MRSA and biofilm-producing strains).ResultsIn the present study, two phages belonging to the Podoviridae family with specific lytic activity against S. aureus were isolated from the sewage of dairy farms and designated as Staphylococcus phage M8 and Staphylococcus phage B4. Latent period and burst size for Staphylococcus phage M8 (70 min, 72 PFU/cell) and Staphylococcus phage B4 (30 min, 447 PFU/cell) were also defined. Our results revealed the susceptibility of MDR (4/20; 20%), MRSA (4/13; 30.8%) and biofilm-producing (1/10; 10%) strains to Staphylococcus phage M8. Moreover, one biofilm-producing strain (1/10; 10%) was susceptible to Staphylococcus phage B4. Furthermore, both phages kept their lytic activity in milk. They reduced the S. aureus population by about 3 logs in cultured milk after 8 h of incubation.ConclusionIn conclusion, it seems that both phages had the potential to serve as biological control agents alone or in combination with other agents such as antibiotics against infections induced by S. aureus. However, further studies are needed to investigate the efficacy of these phages in vivo.

Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity.

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15–0.6 μM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 μM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 μg/cm2). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.

Novel Drexlerviridae bacteriophage KMI8 with specific lytic activity against Klebsiella michiganensis and its biofilms.

The bacterial genus Klebsiella includes the closely related species K. michiganensis, K. oxytoca and K. pneumoniae, which are capable of causing severe disease in humans. In this report we describe the isolation, genomic and functional characterisation of the lytic bacteriophage KMI8 specific for K. michiganensis. KMI8 belongs to the family Drexlerviridae, and has a novel genome which shares very little homology (71.89% identity over a query cover of only 8%) with that of its closest related bacteriophages (Klebsiella bacteriophage LF20 (MW417503.1); Klebsiella bacteriophage 066039 (MW042802.1). KMI8, which possess a putative endosialidase (depolymerase) enzyme, was shown to be capable of degrading mono-biofilms of a strain of K. michiganensis that carried the polysaccharide capsule KL70 locus. This is the first report of a lytic bacteriophage for K. michiganensis, which is capable of breaking down a biofilm of this species.

Characterization of a Broad-Host-Range Lytic Phage SHWT1 Against Multidrug-Resistant Salmonella and Evaluation of Its Therapeutic Efficacy in vitro and in vivo.

Inappropriate use of antibiotics has accelerated to the emergence of multidrug-resistant bacteria, becoming a major health threat. Moreover, bacterial biofilms contribute to antibiotic resistance and prolonged infections. Bacteriophage (phage) therapy may provide an alternative strategy for controlling multidrug-resistant bacterial infections. In this study, a broad-host-range phage, SHWT1, with lytic activity against multidrug-resistant Salmonella was isolated, characterized and evaluated for the therapeutic efficacy in vitro and in vivo. Phage SHWT1 exhibited specific lytic activity against the prevalent Salmonella serovars, such as Salmonella Pullorum, Salmonella Gallinarum, Salmonella Enteritidis, and Salmonella Typhimurium. Morphological analysis showed that phage SHWT1 was a member of the family Siphoviridae and the order Caudovirales. Phage SHWT1 had a latent period of 5 min and burst size of ~150 plaque-forming units (PFUs)/cell. The phage was stable from pH 3-12 and 4–65°C. Phage SHWT1 also showed capacity to lyse Salmonella planktonic cells and inhibit the biofilm formation at optimal multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 100, respectively. In addition, phage SHWT1 was able to lyse intracellular Salmonella within macrophages. Genome sequencing and phylogenetic analyses revealed that SHWT1 was a lytic phage without toxin genes, virulence genes, antibiotic resistance genes, or significant genomic rearrangements. We found that phage SHWT1 could successfully protect mice against S. enteritidis and S. typhimurium infection. Elucidation of the characteristics and genome sequence of phage SHWT1 demonstrates that this phage is a potential therapeutic agent against the salmonellosis caused by multidrug-resistant Salmonella.

A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm.

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

Lysis of cells of diverse bacteria by l,d-peptidases of Escherichia coli bacteriophages RB43, RB49 and T5.

The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance. Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4β) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts. Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures. This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology.

Anti-PSMA CAR-engineered NK-92 Cells: An Off-the-shelf Cell Therapy for Prostate Cancer.

Prostate cancer (PCa) has become the most common cancer among males in Europe and the USA. Adoptive immunotherapy appears a promising strategy to control the advanced stages of the disease by specifically targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the treatment of hematological malignancies, solid tumors still represent a challenge. To overcome current limits, other cellular effectors than T lymphocytes are under study as possible candidates for CAR-engineered cancer immunotherapy. A novel approach involves the NK-92 cell line, which mediates strong cytotoxic responses against a variety of tumor cells but has no effect on non-malignant healthy counterparts. Here, we report a novel therapeutic approach against PCa based on engineering of NK-92 cells with a CAR recognizing the human prostate-specific membrane antigen (PSMA), which is overexpressed in prostatic neoplastic cells. More importantly, the potential utility of NK-92/CAR cells to treat PCa has not yet been explored. Upon CAR transduction, NK-92/CAR cells acquired high and specific lytic activity against PSMA-expressing prostate cancer cells in vitro, and also underwent degranulation and produced high levels of IFN-γ in response to antigen recognition. Lethal irradiation of the effectors, a safety measure requested for the clinical application of retargeted NK-92 cells, fully abrogated replication but did not impact on phenotype and short-term functionality. PSMA-specific recognition and antitumor activity were retained in vivo, as adoptive transfer of irradiated NK-92/CAR cells in prostate cancer-bearing mice restrained tumor growth and improved survival. Anti-PSMA CAR-modified NK-92 cells represent a universal, off-the-shelf, renewable, and cost-effective product endowed with relevant potentialities as a therapeutic approach for PCa immunotherapy.

Isolation and purification of Escherichia coli bacteriophage from Tigris River, Baghdad, Iraq

Hospitals and industrial wastes are discharged directly to Tigris River in Baghdad city, Iraq without any specific treatment. They are a potential hot spot of selection of antibiotic resistance bacteria and increases pressure on environmental microorganisms. However, bacteriophages are the most promising biological alternative for antimicrobial therapy in recent years is also found in the same environment. The aim of this study was to isolate Escherichia coli and identify their antibiotic resistance determinants from Tigris River in Baghdad city, Iraq. The isolation of the potential bacteriophages present in the same samples collected from river water and to test inhibitory effect of bacteriophages against Escherichia coli isolates. Seven water samples were obtained from different locations in Tigris River near Baghdad Medical city; Water samples were cultured on selective media, morphological characteristics, and biochemical methods were used to characterize Escherichia coli strains. Antibiotic sensitivity was measured by disc diffusion method. Bacteriophages isolated from site 1 showed specific lytic activity against Escherichia coli strain isolated from the same water sample by using double agar overlay method. Further identification was carried out with the VITEK system for Escherichia coli isolate and were found to be a phenotypic extended-spectrum beta-lactamases (ESBLs) producer.

Characterization of a novel phage infecting the pathogenic multidrug-resistant Bacillus cereus and functional analysis of its endolysin.

Bacillus cereus is widely distributed food-borne pathogenic bacterium. Due to the harmness to human hearth and the generation of multidrug-resistant B. cereus, it is urgent to develop novel antimicrobial agents. Phage and phage endolysin were taken as novel antimicrobial substance for their specific lytic activity against pathogenic bacteria. In this study, a Myoviridae family phage, designated as vB_BceM-HSE3, infecting the pathogenic multidrug-resistant B. cereus strain was isolated and characterized along with its endolysin. Phage vB_BceM-HSE3 can specially infect the B. cereus group strains, including B. cereus, B. anthracis, and B. thuringiensis, and exhibits high temperature and pH tolerance, which endow it with high potential for been used in controlling pathogenic B. cereus group strains. Genomic analysis reveals that vB_BceM-HSE3 is a novel phage and only shows extremely low genome similarity with available phage genome. Functional analysis of endolysin PlyHSE3 encoding by vB_BceM-HSE3 shows that PlyHSE3 exhibits broader lytic spectrum than the phage and can lyse all the tested B. cereus group strains as well as the tested pathogenic strain of P. aeruginosa. PlyHSE3 also shows broad temperature and pH tolerance, and can efficiently lyse B. cereus strain at temperature at 4°C and higher than 45°C, which indicating that PlyHSE3 might can be used in controlling food-borne B. cereus during both the cold storage of food and the stage after the heat treatment of food. The findings of this study enrich our understanding of phage diversity as well as providing resources for developing phage therapy.

Characterization of a newly isolated phage infecting pathogenic Escherichia coli and analysis of its mosaic structural genes

The outbreak of multidrug-resistant pathogenic bacteria made the discovery of novel control strategies necessary. Phages have regained attention for their specific lytic activity against pathogenic bacterium. A newly isolated phage infecting the clinical Escherichia coli isolates, including several multidrug-resistant strains, was isolated, and this phage showed high control effects against the tested pathogenic E. coli strains. Host range analysis revealed that although the phage exhibited broad lytic spectrum against the tested E. coli strains, it could not lyse strains from the other species. Comparative genomic analysis showed that phages had undergone at least three genome recombination events during the evolutionary process at the position of the three phage tail genes, which was reported to be associated with the host range determination of the phage. The recombinant tail proteins contained functional domains that were highly similar with genes of the Salmonella phage and genes of Pseudomonas and Neisseria. The findings of this study not only provide resources for developing phage therapy against E. coli, but also showed the highly variable genome structure of the phage.

ХАРАКТЕРИСТИКА ЧУВСТВИТЕЛЬНОСТИ К БАКТЕРИОФАГАМ ШТАММОВ STAPHYLOCOCCUS AUREUS, ВЫДЕЛЕННЫХ ИЗ МИКРОБИОЦЕНОЗА РОТОГЛОТКИ У ДЕТЕЙ Г. ИРКУТСКА

Dysbiotic disorders of the oropharynx takes one of the first places in the structure of morbidity worldwide. The aim
 of the study was to determine the peculiarities of microbiocenosis of the oropharynx in children at different ages
 and to assess the level of sensitivity of S. aureus strains to specific bacteriophages. Material for the study consisted of
 84 smears from the oropharynx of children with inflammatory diseases of the upper respiratory tract (nasopharyngi-
 tis, pharyngitis, tonsillitis, laryngitis (J00, J02, J03, J04 according to ICD-10). The biological material was distributed
 depending on the age of the surveyed: the first group – children 3–6 years (n = 61) and the second age group – children
 7–12 years (n = 23). Studies of the microflora of the oropharynx were performed according to standard methods. Isolated
 microorganisms were identified by morphological, tinctorial, cultural and biochemical properties. The results were
 evaluated in accordance with the Order of USSR Ministry of Health N 535 as of April 22, 1985. Specific lytic activity of
 bacteriophages against S. aureus was determined using preparations of staphylococcal bacteriophage and polyvalent
 pyobacteriophage Sexta. The sensitivity of strains of S. aureus to specific bacteriophages was assessed by the method
 of «crosses», according to the guidelines. Statistical analysis was performed with the use of licensed applications Office
 MS Excel 2007 for Windows 7 BioStat v. 5.0.1. 2008. Correlation analysis (Pearson method) was used. It has been shown
 that with increasing age children in the oropharynx microbial density is increasing for indicators of both normoflora
 (S. oralis), and of opportunistic microflora (S. ß-hemolytic, S. pyogenes, S. pneumoniae, bacteria Enterobacteriaceae),
 especially of S. aureus (up to 74 %). Registration of sensitive strains of S. aureus in more than 70 % of cases in preschool
 and school-aged children is evidence of the correctness of correction of the dysbiotic disorders of the oropharynx with
 the use of bacteriophages of staphylococcus and Sexta.

Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform.

Chimeric antigen receptor (CAR)-modified T-cell adoptive immunotherapy is a remarkable therapeutic option proven effective in the treatment of hematological malignancies. In order to optimize cell manufacturing, we sought to develop a novel clinical-grade protocol to obtain CAR-modified cytokine-induced killer cells (CIKs) using the Sleeping Beauty (SB) transposon system. Administration of irradiated PBMCs overcame cell death of stimulating cells induced by non-viral transfection, enabling robust gene transfer together with efficient T-cell expansion. Upon single stimulation, we reached an average of 60% expression of CD123- and CD19- specific 3rd generation CARs (CD28/OX40/TCRzeta). Furthermore, modified cells displayed persistence of cell subsets with memory phenotype, specific and effective lytic activity against leukemic cell lines and primary blasts, cytokine secretion, and proliferation. Adoptive transfer of CD123.CAR or CD19.CAR lymphocytes led to a significant anti-tumor response against acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) disseminated diseases in NSG mice. Notably, we found no evidence of integration enrichment near cancer genes and transposase expression at the end of the differentiation. Taken all together, our findings describe a novel donor-derived non-viral CAR approach that may widen the repertoire of available methods for T cell-based immunotherapy.

Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component.

Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall.

Elucidating the contribution of IRF3 to effector and memory cytotoxic T cell responses after influenza virus infection

Abstract The role of interferon regulatory factor (IRF) 3 in initiating the type I interferon response in innate immune cells has been well studied in the context of viral infection. However, it is less clear what impact IRF3 has in shaping and maintaining adaptive immune responses. Previous reports have demonstrated that when restimulated, both CD4 and CD8 T cells from IRF3 knockout (KO) mice have significantly reduced expression of Granzyme B 30 days after infection or following secondary influenza A virus (IAV) challenge. To determine whether deficiencies in Granzyme B correlate with CTL function, we are examining the cytotoxic potential and specific antiviral lytic activity after primary and secondary challenge with IAV in CD4 and CD8 cells. Additionally, as early signals derived from antigen presenting cells (APCs) influence effector and memory T cell development, the innate immune response following IAV infection was examined. Our results demonstrate the innate response is not compromised in IRF3 KO mice at day 1 or 3 post IAV infection in the draining lymph node. Interestingly, macrophages and dendritic cells had decreased expression of MHCII and CD86 at both time points in the lung. These results suggest that while initial priming is sufficient in the draining lymph nodes, secondary factors in the lung could provide T cells with the signals needed for effective memory development. Together, this work provides insight into the molecular regulation of T cell responses to viral infections and highlights the role IRFs play in the development of high quality effector and memory T cells.

Characterization of LysPBC4, a novel Bacillus cereus-specific endolysin of bacteriophage PBC4.

Bacillus cereus is a spore-forming, Gram-positive bacterium and is a major food-borne pathogen. A B. cereus-specific bacteriophage PBC4 was isolated from the soil of a stock farm, and its genome was analyzed. PBC4 belongs to the Siphoviridae family and has a genome consisting of 80647-bp-long double-stranded DNA, including 123 genes and two tRNAs. LysPBC4, the endolysin of PBC4, has an enzymatically active domain (EAD) on its N-terminal region and a putative cell wall-binding domain (CBD) on its C-terminal region, respectively. Although the phage PBC4 showed a very limited host range, LysPBC4 could lyse all of the B. cereus strains tested. However, LysPBC4 did not kill other bacteria such as B. subtilis or Listeria, indicating that the endolysin has specific lytic activity against the B. cereus group species. Furthermore, LysPBC4_CBD fused with enhanced green fluorescent protein (EGFP) could decorate limited strains of B. cereus group, suggesting that the LysPBC4_CBD may be a promising material for specific detection of B. cereus.

Genetic characterization of ØVC8 lytic phage for Vibrio cholerae O1.

BackgroundEpidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholerae serogroups O1 and O139. In recent years, specific lytic phages of V. cholerae have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholerae O1 strains.MethodsSixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry.ResultsSixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the “single-stranded cohesive ends” packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function.ConclusionsØVC8 is a lytic phage with specific activity against V. cholerae O1 strains and is grouped as a member of the VP2-like phage subfamily. The encoding of an Ig domain by ØVC8 makes this phage a good candidate for use in phage therapy and an alternative tool for monitoring V. cholerae populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0490-x) contains supplementary material, which is available to authorized users.