Browsing by Subject "bacteriophage"

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  • Skurnik, Mikael; Jaakkola, Salla; Mattinen, Laura; von Ossowski, Lotta; Nawaz, Ayesha; Pajunen, Maria; Happonen, Lotta J. (2021)
    Bacteriophages vB_YpeM_fEV-1 (fEV-1) and vB_YpeM_fD1 (fD1) were isolated from incoming sewage water samples in Turku, Finland, using Yersinia pestis strains EV76 and KIM D27 as enrichment hosts, respectively. Genomic analysis and transmission electron microscopy established that fEV-1 is a novel type of dwarf myovirus, while fD1 is a T4-like myovirus. The genome sizes are 38 and 167 kb, respectively. To date, the morphology and genome sequences of some dwarf myoviruses have been described; however, a proteome characterization such as the one presented here, has currently been lacking for this group of viruses. Notably, fEV-1 is the first dwarf myovirus described for Y. pestis. The host range of fEV-1 was restricted strictly to Y. pestis strains, while that of fD1 also included other members of Enterobacterales such as Escherichia coli and Yersinia pseudotuberculosis. In this study, we present the life cycles, genomes, and proteomes of two Yersinia myoviruses, fEV-1 and fD1.
  • Oduor, Joseph M. Ochieng; Kadija, Ermir; Nyachieo, Atunga; Mureithi, Marianne W.; Skurnik, Mikael (2020)
    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 mu J/cm(2), to temperatures up to 45 degrees C, and to ethanol concentrations up to 25%, and complete resistance to chloroform. The adsorption rate constants of the phages ranged between 1.0 x 10(-9) mL/min and 4.7 x 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.
  • Mäntynen, Sari; Laanto, Elina; Oksanen, Hanna M.; Poranen, Minna M.; Diaz-Munoz, Samuel L. (2021)
    The canonical lytic-lysogenic binary has been challenged in recent years, as more evidence has emerged on alternative bacteriophage infection strategies. These infection modes are little studied, and yet they appear to be more abundant and ubiquitous in nature than previously recognized, and can play a significant role in the ecology and evolution of their bacterial hosts. In this review, we discuss the extent, causes and consequences of alternative phage lifestyles, and clarify conceptual and terminological confusion to facilitate research progress. We propose distinct definitions for the terms 'pseudolysogeny' and 'productive or non-productive chronic infection', and distinguish them from the carrier state life cycle, which describes a population-level phenomenon. Our review also finds that phages may change their infection modes in response to environmental conditions or the physiological state of the host cell. We outline known molecular mechanisms underlying the alternative phage-host interactions, including specific genetic pathways and their considerable biotechnological potential. Moreover, we discuss potential implications of the alternative phage lifestyles for microbial biology and ecosystem functioning, as well as applied topics such as phage therapy.
  • Happonen, Lotta J.; Pajunen, Maria I.; Jun, Jin Woo; Skurnik, Mikael (2021)
    Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_phi R2-01 (in short, phi R2-01) that infects strains of several Yersinia enterocolitica serotypes. The phi R2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The phi R2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical phi R2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the phi R2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and phi R2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of phi R2-01 as a food biocontrol or phage therapy agent.
  • Skurnik, Mikael (2022)
    Increasing antibiotic resistance numbers force both scientists and politicians to tackle the problem, and preferably without any delay. The application of bacteriophages as precision therapy to treat bacterial infections, phage therapy, has received increasing attention during the last two decades. While it looks like phage therapy is here to stay, there is still a lot to do. Medicine regulatory authorities are working to deliver clear instructions to carry out phage therapy. Physicians need to get more practical experience on treatments with phages. In this opinion article I try to place phage therapy in the context of the health care system and state that the use phages for precision treatments will require a seamless chain of events from the patient to the phage therapy laboratory to allow for the immediate application of phages therapeutically. It is not likely that phages will replace antibiotics, however, they will be valuable in the treatment of infections caused by multidrug resistant bacteria. Antibiotics will nevertheless remain the main treatment for a majority of infections.
  • Assimakopoulou, Irini Jr (Helsingin yliopisto, 2022)
    The genetic and morphological diversity of viruses and more specifically membrane-containing bacteriophages (phages) with single-stranded DNA (ssDNA) genomes is largely unexplored. It can be difficult to detect evolutionary relationships of viruses using solely sequence-based methods due to their rapid sequence evolution. However, more distant evolutionary connections of viruses have been observed based on structure data. Here we introduce an icosahedral tailless ssDNA phage, Cellulophaga phage phi48:2, isolated from the Baltic Sea that has not been assigned to any virus family or taxa. Phage phi48:2 has been previously linked to the family Finnlakeviridae whose members are icosahedral, internal membrane-containing phages with circular ssDNA genomes. However, the presence of lipids in phi48:2 virion has not been studied. In this study, different buffer conditions were tested for infectivity and stability of phi48:2 allowing us to optimize the purification of the phage particles by rate zonal and equilibrium ultracentrifugation in sucrose. Solvent tests in chloroform and ether, as well as low buoyant density of the virion suggested the presence of lipids in the phi48:2 virion. Analysis of the phi48:2 lipids extracted from highly purified virions by thin-layer chromatography revealed that phi48:2 is a membrane-containing phage and acquires its lipids unselectively from its host bacterium Cellulophaga baltica. Lastly, cryogenic electron microscopy of the purified virions also proposed that lipids form a membrane vesicle under the capsid. Altogether our results show that phi48:2 is an icosahedral membrane-containing phage, thus connecting it further with FLiP, which is the sole member of family Finnlakeviridae. Moreover, FLiP and phi48:2 virions are both ~60 nm in diameter and showed some similarity in their major capsid protein sequences (~21% amino acid identity). To conclude, even though phi48:2 and FLiP share various similarities they cannot be placed within the same family due to the low similarity in their genome sequences. However, for now we can assume they are possible distant relatives. The diversity and abundancy of membrane-containing ssDNA phages is gradually starting to uncover and through their characterization and classification we might consequently understand better their significance in microbial ecology.
  • Hietikko, Alli (Helsingin yliopisto, 2019)
    Antibiotic-resistant bacteria are an increasing threat to global health, caused by the excessive use of antibiotics and the lack of new antimicrobial agents being introduced to the market. New approaches to prevent and cure bacterial infections are needed to halt the growing crisis. One of the most promising alternatives is phage therapy which utilizes bacteriophages to target and kill pathogens with specificity. Pseudomonas aeruginosa is a common opportunistic pathogen that is intrinsically resistant to antibiotics, making it one of the most heavily studied targets of phage therapy. In this study, I characterized four P. aeruginosa phages, fHo-Pae01, PA1P1, PA8P1 and PA11P1, and evaluate their potency in therapeutic applications. Bioinformatic analysis of the genomes revealed the phages to be genetically highly similar and belonging to the Pbunavirus genus of the Myoviridae family. No genes encoding harmful toxins, antibiotic-resistance, or lysogeny were predicted. On the other hand, many of the predicted genes had unknown functions. The host ranges of the phages were assessed using 47 clinical P. aeruginosa strains and predicted host receptor binding tail proteins were compared. Some correlation between the host ranges and mutations in the tail proteins were observed but this alone was not sufficient to explain the differences in the host ranges. The recently isolated vB_PaeM_fHoPae01 (fHo-Pae01) phage was further characterized by a one-step growth curve and imaged with a promising atomic force microscopy method that had not been used before in the Skurnik group. Though the imaging results failed to provide any further knowledge of the phage, the 70-minute-long latent period of infection could be determined from the growth curve. Anion- exchange chromatography was found inefficient in purifying the fHo-Pae01 phage, so alternative methods such as endotoxin columns should be used when purifying these phages for patient use. In conclusion, all four phages appeared to be safe for therapeutic use based on current knowledge, and PA1P1 and PA11P1 were the most promising candidates due to their broad host ranges.
  • Leskinen, Katarzyna; Tuomala, Henni; Wicklund, Anu; Horsma-Heikkinen, Jenni; Kuusela, Pentti; Skurnik, Mikael; Kiljunen, Saija (2017)
    Staphylococcus aureus is a commensal and pathogenic bacterium that causes infections in humans and animals. It is a major cause of nosocomial infections worldwide. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the pathogen are necessary. In this respect, polyvalent staphylococcal myoviruses have been demonstrated to be excellent candidates for phage therapy. Here we present the characterization of the bacteriophage vB_SauM-fRuSau02 (fRuSau02) that was isolated from a commercial Staphylococcus bacteriophage cocktail produced by Microgen (Moscow, Russia). The genomic analysis revealed that fRuSau02 is very closely related to the phage MSA6, and possesses a large genome (148,464 bp), with typical modular organization and a low G+ C (30.22%) content. It can therefore be classified as a new virus among the genus Twortlikevirus. The genome contains 236 predicted genes, 4 of which were interrupted by insertion sequences. Altogether, 78 different structural and virion-associated proteins were identified from purified phage particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The host range of fRuSau02 was tested with 135 strains, including 51 and 54 Staphylococcus aureus isolates from humans and pigs, respectively, and 30 coagulase-negative Staphylococcus strains of human origin. All clinical S. aureus strains were at least moderately sensitive to the phage, while only 39% of the pig strains were infected. Also, some strains of Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus and Staphylococcus pseudointer were sensitive. We conclude that fRuSau02, a phage therapy agent in Russia, can serve as an alternative to antibiotic therapy against S. aureus.
  • Ravantti, Janne; Laanto, Elina; Papponen, Petri; Sundberg, Lotta-Riina (2019)
  • Eskelin, Katri Johanna; Poranen, Minna Marjetta (2018)
    Viruses protect their genomes by enclosing them into protein capsids that sometimes contain lipid bilayers that either reside above or below the protein layer. Controlled dissociation of virions provides important information on virion composition, interactions, and stoichiometry of virion components, as well as their possible role in virus life cycles. Dissociation of viruses can be achieved by using various chemicals, enzymatic treatments, and incubation conditions. Asymmetrical flow field-flow fractionation (AF4) is a gentle method where the separation is based on size. Here, we applied AF4 for controlled dissociation of enveloped bacteriophage phi 6. Our results indicate that AF4 can be used to assay the efficiency of the dissociation process and to purify functional subviral particles.
  • Spruit, Cindy; Wicklund, Anu; Wan, Xing; Skurnik, Mikael; Pajunen, Maria (2020)
    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.
  • Badawy, Shimaa; Pajunen, Maria I.; Haiko, Johanna; Baka, Zakaria A. M.; Abou-Dobara, Mohamed; El-Sayed, Ahmed K. A.; Skurnik, Mikael (2020)
    Acinetobacter baumanniiis an opportunistic pathogen that presents a serious clinical challenge due to its increasing resistance to all available antibiotics. Phage therapy has been introduced recently to treat antibiotic-incurableA. baumanniiinfections. In search for newA. baumanniispecific bacteriophages, 20 clinicalA. baumanniistrains were used in two pools in an attempt to enrich phages from sewage. The enrichment resulted in induction of resident prophage(s) and three temperate bacteriophages, named vB_AbaS_fEg-Aba01, vB_AbaS_fLi-Aba02 and vB_AbaS_fLi-Aba03, all able to infect only one strain (#6597) of the 20 clinical strains, were isolated. Morphological characteristics obtained by transmission electron microscopy together with the genomic information revealed that the phages belong to the familySiphoviridae. The ca. 35 kb genomic sequences of the phages were >99% identical to each other. The linear ds DNA genomes of the phages contained 10 nt cohesive end termini, 52-54 predicted genes, anattPsite and one tRNA gene each. A database search revealed an >99% identical prophage in the genome ofA. baumanniistrain AbPK1 (acc. no. CP024576.1). Over 99% identical prophages were also identified from two of the original 20 clinical strains (#5707 and #5920) and both were shown to be spontaneously inducible, thus very likely being the origins of the isolated phages. The phage vB_AbaS_fEg-Aba01 was also able to lysogenize the susceptible strain #6597 demonstrating that it was fully functional. The phages showed a very narrow host range infecting only twoA. baumanniistrains. In conclusion, we have isolated and characterized three novel temperateSiphoviridaephages that infectA.baumannii.
  • Malmgren, Rasmus Albert (Helsingin yliopisto, 2021)
    The COVID-19 pandemic of 2019 has had a huge impact on the hospitality industry, decreasing production by 35.4% in Q4 of 2020. To keep the industry functional, new safety solutions have to be studied and developed for mitigation of the pandemic. In this study, airborne transmission of viruses in an indoor space was studied, and air purifiers and space dividers were tested as potential intervention methods against SARS-CoV-2 by using a non-pathogenic model virus phi 6. Filtered air purifiers were found to work as a possible solution for the mitigation of viruses spreading through aerosols in public spaces such as restaurants, however, the positioning of the devices is crucial, as the air flow to them may increase the concentration of viruses locally. Space dividers were found to increase the possibility of infection via aerosols. Other types of air purifiers were also tested: an ionizer prototype and a hydroxyl radical emitting unit, of which the ionizer prototype proved to be efficient in reducing the virus concentrations in the air. Most importantly, it was confirmed that enveloped viruses resembling coronaviruses are capable of spreading via aerosol transmission indoors.
  • Townsend, Eleanor M; Kelly, Lucy; Gannon, Lucy; Muscatt, George; Dunstan, Rhys; Michniewski, Slawomir; Sapkota, Hari; Kiljunen, Saija J; Kolsi, Anna; Skurnik, Mikael; Lithgow, Trevor; Millard, Andrew D; Jameson, Eleanor (2021)
    Introduction: Klebsiella is a clinically important pathogen causing a variety of antimicrobial resistant infections in both community and nosocomial settings, particularly pneumonia, urinary tract infection, and sepsis. Bacteriophage (phage) therapy is being considered a primary option for the treatment of drug-resistant infections of these types. Methods: We report the successful isolation and characterization of 30 novel, genetically diverse Klebsiella phages. Results: The isolated phages span six different phage families and nine genera, representing both lysogenic and lytic lifestyles. Individual Klebsiella phage isolates infected up to 11 of the 18 Klebsiella capsule types tested, and all 18 capsule-types were infected by at least one of the phages. Conclusions: Of the Klebsiella-infecting phages presented in this study, the lytic phages are most suitable for phage therapy, based on their broad host range, high virulence, short lysis period and given that they encode no known toxin or antimicrobial resistance genes. Phage isolates belonging to the Sugarlandvirus and Slopekvirus genera were deemed most suitable for phage therapy based on our characterization. Importantly, when applied alone, none of the characterized phages were able to suppress the growth of Klebsiella for more than 12 h, likely due to the inherent ease of Klebsiella to generate spontaneous phage-resistant mutants. This indicates that for successful phage therapy, a cocktail of multiple phages would be necessary to treat Klebsiella infections.
  • Marttila, Heli (Helsingin yliopisto, 2021)
    Global warming affects permafrost in the Arctic regions, where melting organic carbon storages will increasingly contribute to the emission of greenhouse gases. Little is known about tundra soil microbial communities, but Acidobacteria and viruses seem to have important roles there. Here, for the first time, we isolated five Acidobacteria infecting viruses from Kilpisjärvi tundra soils using host strains previously isolated from the same area. Three viruses were isolated on Edaphobacter sp. X5P2, one on Edaphobacter sp. M8UP27, and one on Granulicella sp. X4BP1. The viruses had circular double-stranded DNA genomes 63,196–308,711 bp in length and 51–58% GC content. From 108 to 348 putative ORFs were predicted, 54–72% of which were sequences unique to each virus. Annotations indicated that all five phages most likely have tailed virions. The diversity of viruses present in the studied soils was estimated with the metagenome analysis. Only 0.1% (627) of all assembled metagenomic contigs were phage-positive. The gene-sharing network analysis showed approximately genus-level clustering between the virus isolates and a few metagenomic viral contigs, but overall, all (except one) viral contigs clustered only with each other, not with any known viruses from the NCBI database. No taxonomical assignments could be done for the metagenomic viral contigs, highlighting overall undersampling of soil viruses. Further detailed studies on virus-host interactions are needed to understand the impact of viruses on host abundance and metabolism in Arctic soils, as well as the microbial input into biogeochemical cycles.
  • Heinonen, Minna-Maria (Helsingin yliopisto, 2022)
    The tRNA-derived fragments (tsRNAs) are known to play a role in protein translation and post-transcriptional regulation. Viruses exploit the cellular machinery of the host for their replication and therefore the formation of tRNA-derived fragments could be one mechanism utilized by the virus for completing the infection cycle. Virus-induced tRNA-derived fragments have so far been found to suppress the antiviral responses of the host or to favor viral protein translation. However, the biogenesis of tsRNAs, their virus specificity, as well as their putative regulatory roles during infection are still mainly unknown. Research into the roles of tsRNAs in viral infection has enormous potential to reveal novel regulatory functions of tsRNAs and shed light on the mechanisms which viruses utilize to hijack the cellular translation machinery. This Master’s thesis project aimed to investigate the possible regulatory role and the origin of infection-induced tRNA-fragments in Shewanella glacialimarina TZS-4T. S. glacialimarina was infected with Shewanella phage isolate 1/4 and total RNA was isolated from culture samples collected at different timepoints after infection. Additionally, to assess the specificity of the phenomena, S. frigidimarina and S. baltica, two evolutionary close relatives of S. glacialimarina, were also infected with Shewanella phage isolate 1/4. The formation of fragments was found to be dynamic and specific to S. glacialimarina. The observed fragments were further purified from the total RNA and sequenced using an adapted protocol for sequencing library preparation to identify the origin of the fragments. As a result of this thesis, the adapted protocol was further optimized for the fragment isolation, yet the full identification of the sequences was not achieved within the timeframe of this project.
  • Leskinen, Katarzyna; Blasdel, Bob G.; Lavigne, Rob; Skurnik, Mikael (2016)
    Despite the expanding interest in bacterial viruses (bacteriophages), insights into the intracellular development of bacteriophage and its impact on bacterial physiology are still scarce. Here we investigate during lytic infection the whole-genome transcription of the giant phage vB_YecM_phi R1-37 (phi R1-37) and its host, the gastroenteritis causing bacterium Yersinia enterocolitica. RNA sequencing reveals that the gene expression of phi R1-37 does not follow a pattern typical observed in other lytic bacteriophages, as only selected genes could be classified as typically early, middle or late genes. The majority of the genes appear to be expressed constitutively throughout infection. Additionally, our study demonstrates that transcription occurs mainly from the positive strand, while the negative strand encodes only genes with low to medium expression levels. Interestingly, we also detected the presence of antisense RNA species, as well as one non-coding intragenic RNA species. Gene expression in the phage-infected cell is characterized by the broad replacement of host transcripts with phage transcripts. However, the host response in the late phase of infection was also characterized by up-regulation of several specific bacterial gene products known to be involved in stress response and membrane stability, including the Cpx pathway regulators, ATP-binding cassette (ABC) transporters, phage- and cold-shock proteins.
  • Kasurinen, Jutta; Spruit, Cindy M.; Wicklund, Anu; Pajunen, Maria I.; Skurnik, Mikael (2021)
    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.
  • Salem, Mabruka; Pajunen, Maria; Jun, Jin Woo; Skurnik, Mikael (2021)
    The Yersinia bacteriophages fPS-2, fPS-65, and fPS-90, isolated from pig stools, have long contractile tails and elongated heads, and they belong to genus Tequatroviruses in the order Caudovirales. The phages exhibited relatively wide host ranges among Yersinia pseudotuberculosis and related species. One-step growth curve experiments revealed that the phages have latent periods of 50-80 min with burst sizes of 44-65 virions per infected cell. The phage genomes consist of circularly permuted dsDNA of 169,060, 167,058, and 167,132 bp in size, respectively, with a G + C content 35.3%. The number of predicted genes range from 267 to 271. The phage genomes are 84-92% identical to each other and ca 85% identical to phage T4. The phage receptors were identified by whole genome sequencing of spontaneous phage-resistant mutants. The phage-resistant strains had mutations in the ompF, galU, hldD, or hldE genes. OmpF is a porin, and the other genes encode lipopolysaccharide (LPS) biosynthetic enzymes. The ompF, galU, and hldE mutants were successfully complemented in trans with respective wild-type genes. The host recognition was assigned to long tail fiber tip protein Gp38, analogous to that of T-even phages such as Salmonella phage S16, specifically to the distal beta-helices connecting loops.