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1.
Viruses ; 13(11)2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34834977

RESUMO

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_ϕR2-01 (in short, ϕR2-01) that infects strains of several Yersinia enterocolitica serotypes. The ϕR2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The ϕR2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical ϕR2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the ϕR2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and ϕR2-01 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 ϕR2-01 as a food biocontrol or phage therapy agent.

2.
Viruses ; 13(10)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34696318

RESUMO

The increase of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) causes a threat to human health. LA-MRSA can be transmitted from animals to animal caretakers, which may further spread MRSA to communities and health care facilities. The objective of this work was to study the efficacy of phage treatment in the eradication of LA-MRSA from healthy carrier pigs. A total of 19 MRSA -positive weanling pigs were assigned to a test (n = 10) and a control group (n = 9). A phage cocktail containing three Staphylococcus phages, or a control buffer was administered to the nares and skin of the pigs three times every two days, after which the phage and MRSA levels in nasal and skin swab samples were monitored for a three-week period. The sensitivity of the strains isolated during the follow-up period to the phage cocktail and each phage individually was analyzed and the pig sera were tested for antibodies against the phages used in the cocktail. The phage treatment did not cause any side effects to the pigs. Phages were found in the skin and nasal samples on the days following the phage applications, but there was no reduction in the MRSA levels in the sampled animals. Phage-resistant strains or phage-specific antibodies were not detected during the experiment. The MRSA load in these healthy carrier animals was only 10-100 CFU/swab or nasal sample, which was likely below the replication threshold of phages. The effectiveness of phage treatment to eradicate MRSA from the pigs could thus not be (reliably) determined.

3.
Viruses ; 13(7)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34372590

RESUMO

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.


Assuntos
Bacteriófagos/genética , Bacteriófagos/fisiologia , Genoma Viral , Proteoma , Yersinia pestis/virologia , Bacteriófagos/ultraestrutura , Finlândia , Especificidade de Hospedeiro , Microscopia Eletrônica de Transmissão , Esgotos , Yersinia pestis/classificação
4.
Viruses ; 13(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068736

RESUMO

Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations may induce mutation or may perturb DNA-protein interactions, free living organisms from bacteria to human contain several pathways to counteract uracilation. These efficient and highly specific repair routes uracil-directed excision repair initiated by representative of uracil-DNA glycosylase families. Interestingly, some bacteriophages exist with thymine-lacking uracil-DNA genome. A detailed understanding of the strategy by which such phages can replicate in bacteria where an efficient repair pathway functions for uracil-excision from DNA is expected to reveal novel inhibitors that can also be used for biotechnological applications. Here, we also review the several potential biotechnological applications already implemented based on inhibitors of uracil-excision repair, such as Crispr-base-editing and detection of nascent uracil distribution pattern in complex genomes.


Assuntos
DNA Viral/química , DNA Viral/genética , Uracila , Vírus/genética , Bacteriófagos/efeitos dos fármacos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Biotecnologia , DNA Viral/metabolismo , Desenvolvimento de Medicamentos , Humanos , Modelos Moleculares , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Uracila/química , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo , Vírus/efeitos dos fármacos , Vírus/metabolismo
5.
Viruses ; 13(5)2021 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923360

RESUMO

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.


Assuntos
Bacteriófagos/genética , Toxinas Biológicas/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Biologia Computacional/métodos , Genoma Viral , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Especificidade de Hospedeiro , Humanos , Modelos Moleculares , Conformação Proteica , Proteômica/métodos , Análise de Sequência de DNA , Relação Estrutura-Atividade , Toxinas Biológicas/química , Proteínas Virais/química
6.
Phage (New Rochelle) ; 2(1): 26-42, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33796863

RESUMO

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.

7.
Viruses ; 13(2)2021 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-33668618

RESUMO

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 ß-helices connecting loops.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/isolamento & purificação , Fezes/virologia , Lipopolissacarídeos/metabolismo , Porinas/metabolismo , Receptores Virais/metabolismo , Yersinia pestis/virologia , Yersinia pseudotuberculosis/virologia , Animais , Proteínas de Bactérias/genética , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Composição de Bases , Genoma Viral , Especificidade de Hospedeiro , Filogenia , Porinas/genética , Receptores Virais/genética , Suínos , Yersinia pestis/genética , Yersinia pestis/metabolismo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo
8.
Curr Opin Biotechnol ; 68: 1-7, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33007632

RESUMO

The deeply intertwined evolutionary history between bacteriophages and bacteria has endowed phages with highly specific mechanisms to hijack bacterial cell metabolism for their propagation. Here, we present a comprehensive, phage-driven strategy to reveal novel antibacterial targets by the exploitation of phage-bacteria interactions. This strategy will enable the design of small molecules, which mimic the inhibitory phage proteins, and allow the subsequent hit-to-lead development of these antimicrobial compounds. This proposed small molecule approach is distinct from phage therapy and phage enzyme-based antimicrobials and may produce a more sustainable generation of new antibiotics that exploit novel bacterial targets and act in a pathogen-specific manner.


Assuntos
Bacteriófagos , Terapia por Fagos , Antibacterianos , Bactérias/genética , Bacteriófagos/genética , Evolução Biológica
9.
J Immunol Res ; 2020: 7439506, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33274243

RESUMO

Yersinia enterocolitica O:3 is mentioned among the most common arthritogenic pathogens. Bacterial components (including lipopolysaccharide (LPS)) may persist in the joint after eradication of infection. Having an adjuvant activity, LPS may enhance production of anticollagen antibodies, involved in the pathogenesis of rheumatoid arthritis. Furthermore, its ability to activate complement contributes to the inflammation. The aim of this work was to investigate whether Yersinia LPS (coinjected with collagen) is associated with arthritis progression or other pathological effects and to elucidate the mechanism of this association. It was demonstrated that murine mannose-binding lectin C (MBL-C) recognizes the inner core heptoses of the Rd1 chemotype LPS of Yersinia. In addition, the Rd1 LPS activates the MBL-associated serine protease 1 (MASP-1) stronger than the S and Ra chemotype LPS and comparable to Klebsiella pneumoniae O:3 LPS. However, in contrast to the latter, Yersinia Rd1 LPS was associated neither with the adjuvancity nor with the enhancement of pathological changes in animal paws/impairment of motility. On the other hand, it seemed to be more hepatotoxic when compared with the other tested endotoxins, while the enlargement of inguinal lymph nodes and drop in hepatic MBL-C expression (at the mRNA level) were independent of LPS chemotype. Our data did not suggest no greater impact Y. enterocolitica O:3 on the development or severity of arthropathy related to anticollagen antibody-induced arthritis in mice, although its interaction with MBL-C and subsequent complement activation may contribute to some adverse effects.


Assuntos
Artrite Reumatoide/etiologia , Lipopolissacarídeos/farmacologia , Yersiniose/complicações , Yersiniose/imunologia , Yersinia enterocolitica/imunologia , Animais , Artrite Experimental , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Autoimunidade , Biomarcadores , Colágeno/efeitos adversos , Colágeno/imunologia , Lectina de Ligação a Manose da Via do Complemento/imunologia , Suscetibilidade a Doenças , Masculino , Lectina de Ligação a Manose/metabolismo , Camundongos , Fenótipo , Ligação Proteica , RNA Mensageiro/genética , Yersiniose/microbiologia
10.
Front Microbiol ; 11: 1356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636826

RESUMO

We report here the complete genome sequence and characterization of Yersinia bacteriophage vB_YenP_ϕ80-18. ϕ80-18 was isolated in 1991 using a Y. enterocolitica serotype O:8 strain 8081 as a host from a sewage sample in Turku, Finland, and based on its morphological and genomic features is classified as a podovirus. The genome is 42 kb in size and has 325 bp direct terminal repeats characteristic for podoviruses. The genome contains 57 predicted genes, all encoded in the forward strand, of which 29 showed no similarity to any known genes. Phage particle proteome analysis identified altogether 24 phage particle-associated proteins (PPAPs) including those identified as structural proteins such as major capsid, scaffolding and tail component proteins. In addition, also the DNA helicase, DNA ligase, DNA polymerase, 5'-exonuclease, and the lytic glycosylase proteins were identified as PPAPs, suggesting that they might be injected together with the phage genome into the host cell to facilitate the take-over of the host metabolism. The phage-encoded RNA-polymerase and DNA-primase were not among the PPAPs. Promoter search predicted the presence of four phage and eleven host RNA polymerase -specific promoters in the genome, suggesting that early transcription of the phage is host RNA-polymerase dependent and that the phage RNA polymerase takes over later. The phage tolerates pH values between 2 and 12, and is stable at 50°C but is inactivated at 60°C. It grows slowly with a 50 min latent period and has apparently a low burst size. Electron microscopy revealed that the phage has a head diameter of about 60 nm, and a short tail of 20 nm. Whole-genome phylogenetic analysis confirmed that ϕ80-18 belongs to the Autographivirinae subfamily of the Podoviridae family, that it is 93.2% identical to Yersinia phage fHe-Yen3-01. Host range analysis showed that ϕ80-18 can infect in addition to Y. enterocolitica serotype O:8 strains also strains of serotypes O:4, O:4,32, O:20 and O:21, the latter ones representing similar to Y. enterocolitica serotype O:8, the American pathogenic biotype 1B strains. In conclusion, the phage ϕ80-18 is a promising candidate for the biocontrol of the American biotype 1B Y. enterocolitica.

11.
Viruses ; 12(6)2020 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-32486497

RESUMO

Acinetobacter baumannii is 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-incurable A. baumannii infections. In search for new A. baumannii specific bacteriophages, 20 clinical A. baumannii strains 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 family Siphoviridae. 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, an attP site and one tRNA gene each. A database search revealed an >99% identical prophage in the genome of A. baumannii strain 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 two A. baumannii strains. In conclusion, we have isolated and characterized three novel temperate Siphoviridae phages that infect A. baumannii.


Assuntos
Acinetobacter baumannii/virologia , Siphoviridae/fisiologia , DNA Viral/genética , DNA Viral/isolamento & purificação , Genoma Viral/genética , Lisogenia , Microscopia Eletrônica de Transmissão , Filogenia , Análise de Sequência de DNA , Siphoviridae/genética , Siphoviridae/isolamento & purificação , Siphoviridae/patogenicidade , Ensaio de Placa Viral , Ativação Viral
12.
Viruses ; 12(6)2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517038

RESUMO

YerA41 is a Myoviridae bacteriophage that was originally isolated due its ability to infect Yersinia ruckeri bacteria, the causative agent of enteric redmouth disease of salmonid fish. Several attempts to determine its genomic DNA sequence using traditional and next generation sequencing technologies failed, indicating that the phage genome is modified in such a way that it is an unsuitable template for PCR amplification and for conventional sequencing. To determine the YerA41 genome sequence, we performed RNA-sequencing from phage-infected Y. ruckeri cells at different time points post-infection. The host-genome specific reads were subtracted and de novo assembly was performed on the remaining unaligned reads. This resulted in nine phage-specific scaffolds with a total length of 143 kb that shared only low level and scattered identity to known sequences deposited in DNA databases. Annotation of the sequences revealed 201 predicted genes, most of which found no homologs in the databases. Proteome studies identified altogether 63 phage particle-associated proteins. The RNA-sequencing data were used to characterize the transcriptional control of YerA41 and to investigate its impact on the bacterial gene expression. Overall, our results indicate that RNA-sequencing can be successfully used to obtain the genomic sequence of non-sequencable phages, providing simultaneous information about the phage-host interactions during the process of infection.


Assuntos
Bacteriófagos/genética , Genoma Viral , Yersinia ruckeri/virologia , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Filogenia , RNA Viral/genética , Análise de Sequência de RNA
13.
Viruses ; 12(5)2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32429141

RESUMO

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.


Assuntos
Bacteriófagos/metabolismo , Klebsiella pneumoniae/virologia , Podoviridae/metabolismo , Proteínas Virais/toxicidade , Sequência de Aminoácidos , Antibacterianos/química , Cápsulas Bacterianas/genética , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Genoma Viral/genética , Especificidade de Hospedeiro , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Filogenia , Podoviridae/classificação , Podoviridae/isolamento & purificação , Podoviridae/fisiologia , Esgotos/virologia , Proteínas Virais/química , Proteínas Virais/genética , Vírion/ultraestrutura , Resistência beta-Lactâmica
14.
Microb Pathog ; 141: 103993, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31988008

RESUMO

Yersinia pestis, a Gram-negative bacterium, is the etiologic agent of plague. A hallmark of Y. pestis infection is the organism's ability to rapidly disseminate through an animal host. Y. pestis expresses the outer membrane protein, Ail (Attachment invasion locus), which is associated with host invasion and serum resistance. However, whether Ail plays a role in host dissemination remains unclear. In this study, C57BL/6J mice were challenged with a defined Y. pestis strain, KimD27, or an isogenic ail-deleted mutant derived from KimD27 via metacarpal paw pad inoculation, nasal drops, orogastric infection, or tail vein injection to mimic bubonic, pneumonic, oral, or septicemic plague, respectively. Our results showed that ail-deleted Y. pestis KimD27 lost the ability to invade host cells, leading to failed host dissemination in the pneumonic and oral plague models but not in the bubonic or septicemic plague models, which do not require invasiveness. Therefore, this study demonstrated that whether Ail plays a role in Y. pestis pathogenesis depends on the infection route.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Peste/microbiologia , Fatores de Virulência/metabolismo , Virulência , Yersinia pestis , Animais , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Boca/microbiologia , Yersinia pestis/metabolismo , Yersinia pestis/patogenicidade
15.
Viruses ; 12(2)2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31979276

RESUMO

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.


Assuntos
Agentes de Controle Biológico/isolamento & purificação , Bioprospecção , Fagos de Staphylococcus/classificação , Fagos de Staphylococcus/fisiologia , Staphylococcus aureus/virologia , Albânia , Clorofórmio/farmacologia , Etanol/farmacologia , Genoma Viral , Genômica , Especificidade de Hospedeiro , Concentração de Íons de Hidrogênio , Terapia por Fagos , Análise de Sequência de DNA , Infecções Estafilocócicas/terapia , Temperatura
16.
Viruses ; 11(12)2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31795231

RESUMO

One of the human- and animal-pathogenic species in genus Yersinia is Yersinia enterocolitica, a food-borne zoonotic pathogen that causes enteric infections, mesenteric lymphadenitis, and sometimes sequelae such as reactive arthritis and erythema nodosum. Y. enterocolitica is able to proliferate at 4 C, making it dangerous if contaminated food products are stored under refrigeration. The most common source of Y. enterocolitica is raw pork meat. Microbiological detection of the bacteria from food products is hampered by its slow growth rate as other bacteria overgrow it. Bacteriophages can be exploited in several ways to increase food safety with regards to contamination by Y. enterocolitica. For example, Yersinia phages could be useful in keeping the contamination of food products under control, or, alternatively, the specificity of the phages could be exploited in developing rapid and sensitive diagnostic tools for the identification of the bacteria in food products. In this review, we will discuss the present state of the research on these topics.


Assuntos
Bacteriófagos/fisiologia , Microbiologia de Alimentos , Inocuidade dos Alimentos , Yersiniose/microbiologia , Yersinia enterocolitica/virologia , Animais , Humanos , Yersinia enterocolitica/isolamento & purificação
17.
Viruses ; 11(11)2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31739448

RESUMO

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.


Assuntos
Bactérias/virologia , Bacteriólise/genética , Bacteriófagos/fisiologia , Proteômica/métodos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Escherichia coli/virologia , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais/química
18.
Microbiol Resour Announc ; 8(38)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537666

RESUMO

We report the genomic sequences of phages KpCHEMY26 and KpGranit, isolated in Israel during a worldwide effort against a multidrug- and phage-resistant strain of Klebsiella pneumoniae from a patient in Finland. These results demonstrate the importance of an efficient worldwide network for collaborating in personalized therapy for infectious diseases.

19.
Front Microbiol ; 10: 1674, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396188

RESUMO

The production of phages for therapeutic purposes demands fast, efficient and scalable purification procedures. Phage lysates have a wide range of impurities, of which endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species are harmful to humans. The highest allowed endotoxin concentration for parenterally applied medicines is 5 EU/kg/h. The aim of this study was to evaluate the feasibility of different purification methods in endotoxin and protein toxin removal in the production of phage preparations for clinical use. In the purification assays, we utilized three phages: Escherichia phage vB_EcoM_fHoEco02, Acinetobacter phage vB_ApiM_fHyAci03, and Staphylococcus phage vB_SauM_fRuSau02. The purification methods tested in the study were precipitation with polyethylene glycol, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, two different endotoxin removal columns, and different combinations thereof. The efficiency of the applied purification protocols was evaluated by measuring phage titer and either endotoxins or staphylococcal enterotoxins A and C (SEA and SEC, respectively) from samples taken from different purification steps. The most efficient procedure in endotoxin removal was the combination of ultrafiltration and EndoTrap HD affinity column, which was able to reduce the endotoxin-to-phage ratio of vB_EcoM_fHoEco02 lysate from 3.5 × 104 Endotoxin Units (EU)/109 plaque forming units (PFU) to 0.09 EU/109 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/109 PFU, and the addition of octanol extraction step into this procedure still reduced this ratio threefold. The other methods tested either resulted to less efficient endotoxin removal or required the use of harmful chemicals that should be avoided when producing phage preparations for medical use. Ultrafiltration with 100,000 MWCO efficiently removed enterotoxins from vB_SauM_fRuSau02 lysate (from 1.3 to 0.06 ng SEA/109 PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay.

20.
PLoS One ; 14(7): e0219422, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31287844

RESUMO

Yersinia pseudotuberculosis is an important foodborne pathogen threatening modern food safety due to its ability to survive and grow at low temperatures. DEAD-box RNA helicase CsdA has been shown to play an important role in the low-temperature growth of psychrotrophic Y. pseudotuberculosis. A total of five DEAD-box RNA helicase genes (rhlB, csdA, rhlE, dbpA, srmB) have been identified in Y. pseudotuberculosis IP32953. However, their role in various stress conditions used in food production is unclear. We studied the involvement of the DEAD-box RNA helicase-encoding genes in the cold tolerance of Y. pseudotuberculosis IP32953 using quantitative real-time reverse transcription (RT-qPCR) and mutational analysis. Quantitative RT-PCR revealed that mRNA transcriptional levels of csdA, rhlE, dbpA and srmB were significantly higher after cold shock at 3°C compared to non-shocked culture at 28°C, suggesting the involvement of these four genes in cold shock response at the transcriptional level. The deletion of csdA ceased growth, while the deletion of dbpA or srmB significantly impaired growth at 3°C, suggesting the requirement of these three genes in Y. pseudotuberculosis at low temperatures. Growth of each DEAD-box RNA helicase mutant was also investigated under pH, osmotic, ethanol and oxidative stress conditions. The five helicase-encoding genes did not play major roles in the growth of Y. pseudotuberculosis IP32953 under pH, osmotic, ethanol or oxidative stress.


Assuntos
Temperatura Baixa , RNA Helicases DEAD-box/genética , Etanol/metabolismo , Concentração de Íons de Hidrogênio , Pressão Osmótica , Estresse Oxidativo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo , Mutação
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