23 research outputs found

    Modeling the architecture of depolymerase-containing receptor binding proteins in Klebsiella phages

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    Klebsiella pneumoniae carries a thick polysaccharide capsule. This highly variable chemical structure plays an important role in its virulence. Many Klebsiella bacteriophages recognize this capsule with a receptor binding protein (RBP) that contains a depolymerase domain. This domain degrades the capsule to initiate phage infection. RBPs are highly specific and thus largely determine the host spectrum of the phage. A majority of known Klebsiella phages have only one or two RBPs, but phages with up to 11 RBPs with depolymerase activity and a broad host spectrum have been identified. A detailed bioinformatic analysis shows that similar RBP domains repeatedly occur in K. pneumoniae phages with structural RBP domains for attachment of an RBP to the phage tail (anchor domain) or for branching of RBPs (T4gp10-like domain). Structural domains determining the RBP architecture are located at the N-terminus, while the depolymerase is located in the center of protein. Occasionally, the RBP is complemented with an autocleavable chaperone domain at the distal end serving for folding and multimerization. The enzymatic domain is subjected to an intense horizontal transfer to rapidly shift the phage host spectrum without affecting the RBP architecture. These analyses allowed to model a set of conserved RBP architectures, indicating evolutionary linkages

    Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

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    Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools

    Drug-induced phospholipidosis – causes, effects, identification

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    Adverse reactions of drugs are a significant problem in pharmacotherapy. The observed side effects may have multiple causes. They may depend on the dose and type of medicine used. They can occur during therapy or appear with a delay, after its completion. They may disappear after discontinuation of treatment or persist. In this review, we summarize the vast literature in the area of phospholipidosis, which is a major consequence of the use of cationic amphiphilic drugs (CAD). Their specific chemical structure causes the accumulation of phospholipids inside the lysosomes, which affects their proper function, however, the mechanism of phospholipidosis at the molecular level is not fully understood. Several hypotheses on the mechanism of phospholipidosis formation have been put forward - they mainly concern: the formation of complexes between phospholipids and CAD drugs, competitive inhibition of lysosomal phospholipases by CAD and increased biosynthesis of phospholipids and cholesterol under the influence of the drug. As a result of the accumulation of phospholipids in lysosomes, the so-called lamellar bodies may appear in tissues days or weeks after in vivo CAD administration, and this process is dose-dependent. Phospholipidosis is believed to be a reversible process and is assumed to be an adaptive - but not toxic - response to drugs. If the concentration of CAD or a toxic substance accumulated in lysosomes exceeds a critical value, apoptosis and autophagy may be activated. Phospholipidosis can also be caused by drugs other than CAD, oxysterols and some nanoparticles. Phospholipidosis is one of the least known complications of pharmacotherapy - methods of its detection at the initial stage as well as the full spectrum of functional disorders of the body are still being sought. Identification of phospholipidosis in cells is possible by means of electron microscopy studies confirming the presence of lamellar bodies in tissues from biopsies or by means of real-time PCR techniques examining the expression of genes correlated with the occurrence of phospholipidosis. A potential biomarker detecting this process in the blood and urine of patients is bis(monoacylglycero)phosphate (BMP). Drug-induced phospholipidosis can be detected at the preclinical stage. However, the accumulation of phospholipids and the formation of lamellar bodies found in in vitro or in animal studies does not necessarily mean organ damage in the human body. In recent years, there has been an increase in interest in the mechanism of the phospholipidosis and in the study of new drugs in terms of causing this undesirable effect. This review presents an overview of the most important studies to date related to the mechanism of phospholipidosis formation, methods of its identification and effects at the cellular level as well as on the whole organism

    Engineering the modular receptor-binding proteins of Klebsiella phages switches their capsule serotype specificity

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    The high specificity of bacteriophages is driven by their receptor-binding proteins (RBPs). Many Klebsiella bacteriophages target the capsular exopolysaccharide as the receptor and encode RBPs with depolymerase activity. The modular structure of these RBPs with an N-terminal structural module to attach the RBP to the phage tail, and a C-terminal specificity module for exopolysaccharide degradation, supports horizontal transfer as a major evolutionary driver for Klebsiella phage RBPs. We mimicked this natural evolutionary process by the construction of modular RBP chimeras, exchanging N-terminal structural modules and C-terminal specificity modules. All chimeras strictly follow the capsular serotype specificity of the C-terminal module. Transplanting chimeras with a K11 N-terminal structural RBP module in a Klebsiella phage K11 scaffold results in a capsular serotype switch and corresponding host range modification of the synthetic phages, demonstrating that horizontal transfer of C-terminal specificity modules offers Klebsiella phages an evolutionary highway for rapid adaptation to new capsular serotypes. IMPORTANCE The antimicrobial resistance crisis has rekindled interest in bacteriophage therapy. Phages have been studied over a century as therapeutics to treat bacterial infections, but one of the biggest challenges for the use of phages in therapeutic interventions remains their high specificity. In particular, many Klebsiella phages have a narrow spectrum constrained by the high diversity of exopolysaccharide capsules that shield access to the cells. In this work, we have elaborated how Klebsiella phages deal with this high diversity by exchanging building blocks of their receptor-binding proteins

    Dynamic cerebral autoregulation is compromised in ischaemic stroke of undetermined aetiology only in the non-affected hemisphere

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    Background and purpose To assess dynamic cerebral autoregulation (CA) in patients with acute ischaemic stroke of undetermined aetiology, within 72h of stroke onset. Materials and methods In 6 patients with ischaemic stroke of undetermined aetiology (aged 66±9 years, National Institutes of Health Stroke Scale [NIHSS] score on admission: 4.0, range: 4–11), selected based on screening of 118 consecutive ischaemic stroke patients and in 14 volunteers (aged 62±10 years), we continuously monitored RR intervals (RRI), mean arterial pressure (MAP) by means of photoplethysmography, mean cerebral blood flow velocity (CBFV) using transcranial Doppler ultrasonography, end-tidal CO2 (ETCO2) and respiration during 2-min deep breathing paced at 6min−1 (0.1Hz). To assess CA, we evaluated the impact of breathing-induced MAP oscillations on fluctuations of CBFV in the hemispheres with stroke, the non-involved hemispheres and randomly selected hemispheres of controls by applying cross-spectral analysis and calculating coherence, transfer function gain (CBFV–MAP gain) and phase shift angle between the two oscillating signals. Results Phase shift angle between MAP and CBFV oscillations showed values >0 and was significantly reduced in the hemispheres without stroke as compared to controls (0.39±0.95 vs. −1.59±0.33rad, p=0.015), whereas in the hemispheres with stroke, phase shift angle did not differ significantly from that observed in the control hemispheres. Clinical status of stroke patients significantly improved at discharge from the hospital (NIHSS: 2.0, range: 1–8, p=0.028). Conclusions During the first days of ischaemic stroke of undetermined aetiology, dynamic cerebral autoregulation is compromised in the non-affected hemisphere, but not in the hemisphere with ischaemic lesion

    Dynamic cerebral autoregulation is compromised in ischaemic stroke of undetermined aetiology only in the non-affected hemisphere

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    Background and purpose: To assess dynamic cerebral autoregulation (CA) in patients with acute ischaemic stroke of undetermined aetiology, within 72 h of stroke onset. Materials and methods: In 6 patients with ischaemic stroke of undetermined aetiology (aged 66 9 years, National Institutes of Health Stroke Scale [NIHSS] score on admission: 4.0, range: 4–11), selected based on screening of 118 consecutive ischaemic stroke patients and in 14 volunteers (aged 62 10 years), we continuously monitored RR intervals (RRI), mean arterial pressure (MAP) by means of photoplethysmography, mean cerebral blood flow velocity (CBFV) using transcranial Doppler ultrasonography, end-tidal CO2 (ETCO2) and respiration during 2-min deep breathing paced at 6 min1 (0.1 Hz). To assess CA, we evaluated the impact of breathing-induced MAP oscillations on fluctuations of CBFV in the hemispheres with stroke, the non-involved hemispheres and randomly selected hemispheres of controls by applying cross-spectral analysis and calculating coherence, transfer function gain (CBFV–MAP gain) and phase shift angle between the two oscillating signals. Results: Phase shift angle between MAP and CBFV oscillations showed values >0 and was significantly reduced in the hemispheres without stroke as compared to controls (0.39 0.95 vs. 1.59 0.33 rad, p = 0.015), whereas in the hemispheres with stroke, phase shift angle did not differ significantly from that observed in the control hemispheres. Clinical status of stroke patients significantly improved at discharge from the hospital (NIHSS: 2.0, range: 1–8, p = 0.028). Conclusions: During the first days of ischaemic stroke of undetermined aetiology, dynamic cerebral autoregulation is compromised in the non-affected hemisphere, but not in the hemisphere with ischaemic lesio

    Characterization and in vitro engineering of exopolysaccharide (EPS) depolymerases originated from Klebsiella phages

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    A blueprint of tail fiber modularity and its relationship with host specificity for STEC serovars

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    Shiga toxin-producing E. coli (STEC) is a foodborne pathogen causing around 2.8 million annual infections worldwide. Antimicrobial treatment is debated as treatment for STEC infections as the activated SOS response may lead to increased toxin production. Phages, as the natural predator of bacteria, therefore offer great potential in STEC treatment. The phage-host relationship is very specific and complex, where tail fibers or tailspikes of the phages are the first phage proteins initiating the infection process. These proteins bind to various outer membrane structures including O-antigen, a serovar specific sugar-based component on the outer lipopolysaccharide layer. Here we introduce a bio-informatics pipeline to find and investigate this phage-host relationship, namely the tail fiber and O-antigen interaction, using public databases and online tools. Both temperate and lytic phages were screened for the presence of tail fiber O-antigen specific genes. The O-antigen specificity of the tail fibers was confirmed for multiple phage groups, such as genera Uetake-, Lederberg- and kutterviruses. Tail fibers specific for O-antigen types O26, O103, O104, O111, O145, O146 and O157 were identified. Additionally, the current hurdles of this pipeline are disclosed. This method of screening for new O-antigen-specific tail fibers is highly interesting to develop serotype-targeting microbials, especially in current times where antimicrobial resistance is a serious threat to global health and development

    Modelling the architecture of depolymerase-containing receptor binding proteins in Klebsiella phages

    No full text
    Klebsiella pneumoniae carries a thick and diverse polysaccharide capsule that plays an important role in its virulence. Many Klebsiella bacteriophages recognize this substrate with a receptor binding protein (RBP) that contains a depolymerase domain. This domain degrades the capsule to initiate phage infection. RBPs are highly specific and thus largely determine the host spectrum of the phage. A majority of known Klebsiella phages have only one or two RBPs, but phages with up to eleven RBPs with depolymerase activity and a broad host spectrum have been identified. A detailed bioinformatic analysis shows that Klebsiella phages extensively recycle structural RBP domains for attachment of an RBP to the phage tail (anchor domain) or for branching of RBPs (T4gp10-like domain). Structural domains determining the RBP architecture are located at the N-terminus, while the depolymerase is located in the center of protein. Occasionally, the RBP is complemented with a chaperone domain at the distal end serving for folding and multimerization. The enzymatic domain is subjected to an intense horizontal transfer to rapidly shift the phage host spectrum without affecting the RBP architecture. These analyses allowed us to model a set of conserved RBP architectures that are not limited to taxonomic borders, indicating evolutionary linkages
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