Structural insights into thrombolytic activity of destabilase from medicinal leech

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.</p

A fluorescent microspheres-based microfluidic test system for the detection of immunoglobulin G to SARS-CoV-2

Background: The pandemic of the new coronavirus infection, COVID-19, is currently ongoing in the world. Over the years, the pathogen, SARS-CoV-2, has undergone a series of mutational genome changes, which has led to the spread of various genetic variants of the virus. Meanwhile, the methods used to diagnose SARS-CoV-2, to establish the disease stage and to assess the immunity, are nonspecific to SARS-CoV-2 variants and time-consumable. Thus, the development of new methods for diagnosing COVID-19, as well as their implementation in practice, is currently an important direction. In particular, application of systems based on chemically modified fluorescent microspheres (with a multiplex assay for target protein molecules) opens great opportunities. Aim: development of a microfluidic diagnostic test system based on fluorescent microspheres for the specific detection of immunoglobulins G (IgG) to SARS-CoV-2. Methods: A collection of human serum samples was characterized using enzyme-linked immunosorbent assay (ELISA) and commercially available reagent kits. IgG to SARS-CoV-2 in the human serum were detected by the developed immunofluorescent method using microspheres containing the chemically immobilized RBD fragment of the SARS-CoV-2 (Kappa variant) viral S-protein. Results: The level of IgG in the blood serum of recovered volunteers was 9-300 times higher than that in apparently healthy volunteers, according to ELISA (p0.001). Conjugates of fluorescent microspheres with the RBD-fragment of the S-protein, capable of specifically binding IgG from the blood serum, have been obtained. The immune complexes formation was confirmed by the fluorescence microscopy data; the fluorescence intensity of secondary antibodies in the immune complexes formed on the surface of microspheres was proportional to the content of IgG (r 0.963). The test system had a good predictive value (AUC 70.3%). Conclusion: A test system has been developed, based on fluorescent microspheres containing the immobilized RBD fragment of the SARS-CoV-2 S-protein, for the immunofluorescent detection of IgG in the human blood serum. When testing the system on samples with different levels of IgG to SARS-CoV-2, its prognostic value was shown. The obtained results allow us to present the test system as a method to assess the level of immunoglobulins to SARS-CoV-2 in the human blood serum for the implementation in clinical practice. The test system can also be integrated into various microfluidic systems to create chips and devices for the point-of-care diagnostics

Targeting of Silver Cations, Silver-Cystine Complexes, Ag Nanoclusters, and Nanoparticles towards SARS-CoV-2 RNA and Recombinant Virion Proteins

Background: Nanosilver possesses antiviral, antibacterial, anti-inflammatory, anti-angiogenesis, antiplatelet, and anticancer properties. The development of disinfectants, inactivated vaccines, and combined etiotropic and immunomodulation therapy against respiratory viral infections, including COVID-19, remains urgent. Aim: Our goal was to determine the SARS-CoV-2 molecular targets (genomic RNA and the structural virion proteins S and N) for silver-containing nanomaterials. Methods: SARS-CoV-2 gene cloning, purification of S2 and N recombinant proteins, viral RNA isolation from patients’ blood samples, reverse transcription with quantitative real-time PCR ((RT)2-PCR), ELISA, and multiplex immunofluorescent analysis with magnetic beads (xMAP) for detection of 17 inflammation markers. Results: Fluorescent Ag nanoclusters (NCs) less than 2 nm with a few recovered silver atoms, citrate coated Ag nanoparticles (NPs) with diameters of 20–120 nm, and nanoconjugates of 50–150 nm consisting of Ag NPs with different protein envelopes were constructed from AgNO3 and analyzed by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), ultraviolet-visible light absorption, and fluorescent spectroscopy. SARS-CoV-2 RNA isolated from COVID-19 patients’ blood samples was completely cleaved with the artificial RNase complex compound Li+[Ag+2Cys2−(OH−)2(NH3)2] (Ag-2S), whereas other Ag-containing materials provided partial RNA degradation only. Treatment of the SARS-CoV-2 S2 and N recombinant antigens with AgNO3 and Ag NPs inhibited their binding with specific polyclonal antibodies, as shown by ELISA. Fluorescent Ag NCs with albumin or immunoglobulins, Ag-2S complex, and nanoconjugates of Ag NPs with protein shells had no effect on the interaction between coronavirus recombinant antigens and antibodies. Reduced production of a majority of the 17 inflammation biomarkers after treatment of three human cell lines with nanosilver was demonstrated by xMAP. Conclusion: The antiviral properties of the silver nanomaterials against SARS-CoV-2 coronavirus differed. The small-molecular-weight artificial RNase Ag-2S provided exhaustive RNA destruction but could not bind with the SARS-CoV-2 recombinant antigens. On the contrary, Ag+ ions and Ag NPs interacted with the SARS-CoV-2 recombinant antigens N and S but were less efficient at performing viral RNA cleavage. One should note that SARS-CoV-2 RNA was more stable than MS2 phage RNA. The isolated RNA of both the MS2 phage and SARS-CoV-2 were more degradable than the MS2 phage and coronavirus particles in patients’ blood, due to the protection with structural proteins. To reduce the risk of the virus resistance, a combined treatment with Ag-2S and Ag NPs could be used. To prevent cytokine storm during the early stages of respiratory infections with RNA-containing viruses, nanoconjugates of Ag NPs with surface proteins could be recommended

Isolation and Characterization of the First <i>Zobellviridae</i> Family Bacteriophage Infecting <i>Klebsiella pneumoniae</i>

In order to address the upcoming crisis in the treatment of Klebsiella pneumoniae infections, caused by an increasing proportion of resistant isolates, new approaches to antimicrobial therapy must be developed. One approach would be to use (bacterio)phages and/or phage derivatives for therapy. In this study, we present a description of the first K. pneumoniae phage from the Zobellviridae family. The vB_KpnP_Klyazma podovirus, which forms translucent halos around the plaques, was isolated from river water. The phage genome is composed of 82 open reading frames, which are divided into two clusters located on opposite strands. Phylogenetic analysis revealed that the phage belongs to the Zobellviridae family, although its identity with the closest member of this family was not higher than 5%. The bacteriophage demonstrated lytic activity against all (n = 11) K. pneumoniae strains with the KL20 capsule type, but only the host strain was lysed effectively. The receptor-binding protein of the phage was identified as a polysaccharide depolymerase with a pectate lyase domain. The recombinant depolymerase protein showed concentration-dependent activity against all strains with the KL20 capsule type. The ability of a recombinant depolymerase to cleave bacterial capsular polysaccharides regardless of a phage’s ability to successfully infect a particular strain holds promise for the possibility of using depolymerases in antimicrobial therapy, even though they only make bacteria sensitive to environmental factors, rather than killing them directly