26 research outputs found
Screening and in silico characterization of prophages in Helicobacter pylori genomes
Temperate bacterio(phages) play an important role on the evolution of pathogenic bacteria. Nevertheless, information on their role in Helicobacter pylori (an important gastric pathogen bacterium) is scarce.
The present study developed a workflow for the identification of prophages in Portuguese H. pylori clinical strains, proposing the use of a new PCR-based screening method. The genome of strains with different PCR profiles were then sequenced.
In the fourteen genomes analysed, nine intact prophages were identified by PHASTER. These prophages were annotated by analogy with other identified phages, where seven contained the integrase gene, corroborating the results obtained in the PCR screening, with only one exception. Still, in PCR screening, the holin gene was identified in 75 % of the strains containing intact phages, but BLASTp homologies only recognized this gene in one of the prophages. Fifty-six percent are podovirus, while in 44 % it was not possible to assign any family, according to the VirFam tool. Using the Resistance Gene Identifier of CARD it was identified the Acinetobacter mutant Lpx gene conferring resistance to colistin in two intact prophages. The BLASTp search identified a putative ABC binding cassette transporter in one of the intact prophages. On the bacterial genomes, 71 % have the CRISPR-Cas system classified as evidence level 1 by CRISPRCasFinder, which typically indicate potentially invalid CRISPR arrays.
The use of an initial PCR screening method increased the identification of intact prophage-containing strains from 20 % to 57 %. Furthermore, the few virulence factors identified in prophages, and the possible inactivity of CRISPR-Cas in the bacterial genomes, allow the choice of strains for the isolation of phages for future studies. Overall, our results represent a significant contribution to the knowledge of prophages in H. pylori, and provide valuable insights into their potential use in phage therapy.info:eu-repo/semantics/publishedVersio
Helicobacter pylori prophages: screening, detection, induction and potential therapeutic use
Helicobacter pylori is a microaerophilic bacterium that chronically infects the human gastric
mucosa. Infections caused by this pathogen are difficult to treat, mainly due to the increased
resistance of this species to conventional antibiotics. Therefore, it is important to develop
antibiotic alternative or complementary approaches to tackle H. pylori infections.
Bacterio(phages) have proven to be efficient antibacterial agents, however it is very difficult to
isolate strictly lytic phages infecting H. pylori. Nevertheless, this bacterial species presents
prophages in their genomes and although strictly lytic phages have been consensually
preferred for phage therapy purposes, temperate prophages holds a great but an exploited
potential.
In the present work, we developed a new PCR-based screening method to detect the presence
of prophages genes in a set of H. pylori Portuguese clinical strains. The genomes of selected
strains were then sequenced using a combined Illumina platform and MinION nanopore-based
sequencing strategy. Prophages content was then analysed using the PHASTER tool. After
sequencing analysis, UV light was used to induce phages, from which one was further
characterized in terms of morphology, host range, stability on an in vitro gastric model, genome
analysis and efficacy against a H. pylori culture.
The complementarity between Illumina and Nanopore results, allowed us to identify a total of
10 intact, 7 questionable and 47 incomplete prophages on the 14 sequenced strains. One
predicted intact prophage was induced successfully, and presents a genome length of 31 162
bp with 37.1 % G+C content. Interestingly, this new podovirus infects five H. pylori strains, and
in the gastric in vitro model only a small loss of phage titer was observed in the gastric phase,
suggesting that this phage could be adapted to the stomach environment. Farther, this phage
demonstrated to be capable of maintaining the H. pylori population at low levels for up to 24 h post-infection with MOIs of 0.01, 0.1 and 1.
Overall, a new PCR screening method was developed to detect prophages on H. pylori and positive correlations with sequencing results were observed. Moreover, this new isolated phage seems to have therapeutic potential to treat H. pylori gastric infections.This work was supported by the Portuguese Foundation for Science and Technology – Fundação para a Ciência e
Tecnologia (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and Project PTDC/SAU-PUB/29182/2017
[POCI-01-0145-FEDER-029182]. Rute Ferreira is recipient of a FCT PhD grant with the reference SFRH/BD/146496/2019info:eu-repo/semantics/publishedVersio
Comparison of bioinformatics tools to predict the presence of prophages in Helicobacter pylori genomes
Bacterio(phages) are specific viruses for bacteria, being their natural enemies. When the genome of a phage is integrated into the host bacterial genome, it is named prophage. These are a latent form of phages, in which the viral genes can increase the virulence and/or fitness characteristics of the host. This life cycle - lysogenic - does not cause the bacterial cell to rupture. Prophages have already been identified in most pathogenic bacteria, providing them better chances of survival. In the case of Helicobacter pylori, a human gastric pathogen that causes, among others, chronic gastritis, peptic ulcers, and adenocarcinoma, the presence of important prophage genes in their genomes has already been identified. In our work, a total of 109 complete genomes of human isolates of H. pylori and plasmids, deposited in NCBI archives, between November 5, 2015, and February 21, 2020, and 19 complete genomes of Portuguese clinical isolates, were screened, regarding the presence of prophages. For that, two of the most widely used web servers for identifying putative prophages in bacterial genomes were used: Phaster and Prophage Hunter.
With the use of Phaster, 78 prophage sequences were identified, 6 of which were intact (7.7 %).
Regarding Prophage Hunter, 199 prophages were identified, in a total of 17 active (8.5 %). The differences observed in the number of prophages identified by each tool is probably due to variances in the identification methods that each tool uses, as already reported. However, the intact sequences identified in Phaster were also predicted, in the same strains, in Prophage Hunter. These results suggest a high probability of these strains having inducible sequences of prophages in their genomes. The use of web servers for the rapid identification and annotation of prophage sequences in bacterial genomes and plasmids has been growing, helping to direct laboratory experiments more easily. In this work, we observed some differences in the results between the two tools used, concluding that new prophage prediction tools using Machine-Learning are required to predict more accurately this important viral sequences.info:eu-repo/semantics/publishedVersio
Characterization and genomic analysis of a new phage infecting Helicobacter pylori
Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.This study was supported by the Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of the UIDB/04469/2020 unit, and Project Helicophage PTDC/SAU-PUB/29182/2017 (POCI-01-0145-FEDER-029182). R.F. and R.F.S.G. acknowledge the FCT grants SFRH/BD/146496/2019 and SFRH/BD/140182/2018, respectivelyinfo:eu-repo/semantics/publishedVersio
H. pylori phages: from genome release to hope for use as therapy
The increasing antibiotic-resistant Helicobacter pylori infections worldwide and the ineffectiveness of treatments led the World Health Organization to designate clarithromycin-resistant H. pylori as a high-priority bacterium for antibiotic research and development. (Bacterio)phages, viruses that infect bacteria, showing effectiveness in the treatment of pathogenic bacteria, could be a promising alternative strategy in the fight against H. pylori infections.
Material and methods
In this work, a collection of 74 Portuguese H. pylori-clinical strains was used to screen for the presence of phage genes, using a new PCR-based method. Selected strains were subsequently sequenced and prophage isolation was attempted using UV radiation. Three phages were isolated, one of which was further characterized genetically and biologically.
Results
PCR-based detection indicated the presence of target phage sequences in 14 strains, and the induction strategies resulted in the release of a new phage. It presents a genome length of 31,162 bp with a G+C content of 37.1 %. This podovirus showed capability to form phage plaques in five strains, was stable under an in vitro gastric digestion model, and was able to maintain a H. pylori population at low levels for up to 24h post-infection.
Conclusion
The new PCR screening method proved to be very effective in the selection of strains carrying prophages, resulting in the isolation of a new H. pylori phage. This phage presented very promising characteristics in terms of stability and efficacy, being therefore a small step towards the future use of phage therapy in the fight against H. pylori infections.info:eu-repo/semantics/publishedVersio
Síntese de amidas e sulfonamidas de beta-D-galactopiranosilamina e beta-lactosilamina e avaliação de suas interações com lectinas de Erythrina cristagalli e de Ricinus communis
Síntese de glicodendrímeros e avaliação de sua interação com lectinas
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Previous issue date: 30A superfície celular é composta de proteínas, lipídios e carboidratos. Os carboidratos presentes na superfície celular estão associados a proteínas ou lipídios, e esses glicoconjugados macromoleculares são chamados glicoproteínas ou glicolipídios e interagem de modo específico com proteínas denominadas lectinas. A lectina isolada das sementes de Erythrina cristagalli L.(Fabaceae) interage seletivamente com D-galactose e N-acetil-lactosamina. Muitas lectinas reconhecem padrões de carboidratos expressos por microrganismos invasores. Vários microrganismos têm desenvolvido estratégias para explorar o reconhecimento em seu benefício e invadir células de defesa do sistema imunológico. Tem sido proposto que esse o Vírus da Imunodeficiência Humana (VIH), considerado agente etiológico da Síndrome da Imunodeficiência Adquirida (SIDA), utiliza uma interação com a lectina DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) para ser transportado aos sítios linfóides. Essa lectina reconhece D-manose e Lfucose por um processo multivalente. A DC-SIGN é um importante alvo para a ação de anti-adesinas, substâncias capazes de bloquear a interação entre oligossacarídeos e lectinas, capazes de mimetizar esses componentes de membranas. Entre essas substâncias se encontram os glicodendrímeros. Neste trabalho são apresentadas as sínteses de derivados monoméricos, diméricos e tetraméricos da D-galactose, planejados como possíveis antiadesinas. São apresentadas também as sínteses de derivados monoméricos e tetraméricos de D-manose e as tentativas de obtenção de tetrâmeros derivados de D-alactose, D-manose e D-glicose por métodos alternativos. Dos derivados sintetizados, dois monômeros (N-(2-hidroxietil)-4-(b-Dgalactopiranosiloxi) benzenopropanamida e N-(2- hidroxieti l)-4-(b-Dgalactopiranosiloxi)- 3-metoxibenzenopropanamida) e dois dímeros (N,N-bis-[4-b- D-galactopiranosiloxi)benzenopropanoil]-1,4-butanodiamina e N,N-bis-[3-(4-b-Dgalactopiranosiloxi- 3-metoxifenil)propanoil]-1,4-butanodiamina) foram avaliados quanto a sua atividade inibitória da hemaglutinação mediada pela lectina isolada de Erythrina cristagalli L. (Fabaceae). O tetrâmero tetra-[3-[[[1-(4-metoxifenil)-6-desoxi-a-Dmanopiranosid- 6-il]amino]carbonil]propanoato] de pentaeritritila foi avaliado quanto à sua interação com a lectina DC-SIGN, por análise de ressonância de superfície plasmônica.The Cell surface is composed of proteins, lipides and carbohydrates. The carbohydrates of cell surface are conjugated with proteins and lipids, forming glycoproteins or glycolopides. These macromolecular glycoconjugates interact with proteins named lectins in a specific way. Seeds of ERYTHRINA CRISTAGALLI L. (Fabaceae) have a lectin which interacts specifically with D- galactose and N- acetil-lactosamine. Many lectins are involved in the recognition of the carbohydrates present in the cell surface of invasive microorganisms. On the other hand, many microorganisms have evolved strategies to explore these recognition processes and use it to invade cells of the immune system. Some studies have proposed that the Human Immunodeficiency Virus (HIV), assumed as the etiologic agent of the acquired Immunodeficiency Syndrome (AIDS), interacts with the DC-SIGN (dendrict cell-specific ICAM-3-grabbing non-integrin) lectin to be transported to the lymphoid sites. This lectin recognize D- mannose and L-fucose by multivalence. DC-SIGN is an important target to the anti-adhesins, a class of substances with block the interaction between oligossacharides and lectins, since they can mimic these membrane constituents. Glycodendrimers are anti-adhesins molecules. In this work are described the syntheses of monomeric, dimeric and tetrameric D- galactose derivates designed as anti-adhesins. It´s also described the synthesis of monomeric and tetrameric D-manose and D-glucose by alternative methods. From the derivates obtained by synthesis, two monomers (n-(2-hydroxyethyl)-4-(b-d galactopyranosyloxy) benzenepropanamide and N-(2- hydroxieth)- 4-(b-d galactopyranosyloxy) -3-ethoxybenzenepropanamide) and two dimmers (N,N-bis-{4-B-D- galactopyranosyloxy)benzenepropanoyl]-1,4- butanediamine and N,N-bis-[3-(4-B-D-galactopyranosyloxi-3-methoxyphenyl)propanoyl}-1-4-butanediamine) were evaluated as inhibitors of the hemmaglutination process mediated by the lectin of Erythrina cristagalli. The interaction between the tetramer pentaerythrityl tetra [3-[[[1-(4-methylphenyl)-6-deox-alfa-D-MANNOPYRANOSID-6-YL]amino]carbonyl]propanoate] and the lectin D-SIGN were evaluated by surface Plasmon resonance
Synthesis of dimeric aryl β-D-galactopyranosides for the evaluation of their interaction with the Erythrina cristagalli lectin
5 páginas, 2 figuras, 1 tabla.The synthesis of two new D-galactose-based dimers having a 1,4-butanediamine spacer is reported aiming at the evaluation of their interaction with the Erythrina cristagalli lectin. The title compounds were prepared in four and five steps from 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside bromide, in 20 % and 15 % overall yield, respectively, using the Doebner modification of the Koenavenagel reaction as the key sep. The lectin-carbohydrate interaction could be evaluated for only one dimer, due to solubility problems. A twofold enhancement of affinity was observed, compared to the corresponding monovalent ligand.Ao CNPq, CAPES e FAPEMIG a concessão de auxílio financeiro, bolsa de doutorado (R. C. Figueiredo) e bolsa de produtividade (M. A. F. Prado e R. J. Alves).Peer reviewe
Synthesis of dimeric aryl β-D-galactopyranosides for the evaluation of their interaction with the Erythrina cristagalli lectin
5 páginas, 2 figuras, 1 tabla.The synthesis of two new D-galactose-based dimers having a 1,4-butanediamine spacer is reported aiming at the evaluation of their interaction with the Erythrina cristagalli lectin. The title compounds were prepared in four and five steps from 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside bromide, in 20 % and 15 % overall yield, respectively, using the Doebner modification of the Koenavenagel reaction as the key sep. The lectin-carbohydrate interaction could be evaluated for only one dimer, due to solubility problems. A twofold enhancement of affinity was observed, compared to the corresponding monovalent ligand.Ao CNPq, CAPES e FAPEMIG a concessão de auxílio financeiro, bolsa de doutorado (R. C. Figueiredo) e bolsa de produtividade (M. A. F. Prado e R. J. Alves).Peer reviewe
Rapid and efficient synthesis of α(1 – 2) mannobiosides.
α(1,2) mannobiosides with different substituents at the reducing end have been synthesized by a common strategy using benzoyls as the permanent protecting groups and an acetyl as the orthogonal protecting group at position C2 of the glycosyl acceptor. The new synthetic strategy has been performed remarkably reducing the number of purification steps, the time of synthesis (less than 72 hours) and improving the overall yield at least three times with respect to the best procedure described in the literature at the moment. Additionally, this protecting group strategy is compatible with the presence of azido groups and the use of Cu catalyzed azide alkyne cycloaddition (CuAAC) also called “click chemistry” for conjugating the α(1–2)mannobiosides to different scaffolds for the preparation of mannosyl multivalent systems