The use of recombinant endolysins is a promising approach for antimicrobial therapy capable of counteracting the spread of antibiotic-resistant strains. To obtain the necessary biotechnological product, diverse peptide tags are often fused to the endolysin sequence to simplify enzyme purification, improve its ability to permeabilize the bacterial outer membrane, etc. We compared the effects of two different types of protein modifications on endolysin LysECD7 bactericidal activity in vitro and demonstrated that it is significantly modulated by specific permeabilizing antimicrobial peptides, as well as by widely used histidine tags. Thus, the tags selected for the study of endolysins and during the development of biotechnological preparations should be used with the appropriate precautions to minimize false conclusions about endolysin properties. Further, modifications of LysECD7 allowed us to obtain a lytic enzyme that was largely devoid of the disadvantages of the native protein and was active over the spectra of conditions, with high in vitro bactericidal activity not only against Gram-negative, but also against Gram-positive, bacteria. This opens up the possibility of developing effective antimicrobials based on N-terminus sheep myeloid peptide of 29 amino acids (SMAP)-modified LysECD7 that can be highly active not only during topical treatment but also for systemic applications in the bloodstream and tissues.

Bovine coronaviruses (BCoVs), which cause gastrointestinal and respiratory diseases in cattle, and are genetically related to the human coronavirus HCoV-OC43, which is responsible for up to 10% of common colds, attract increased attention. We applied the method of photodynamic inactivation with cationic photosensitizers (PSs) to reduce the titers of BCoV and studied the morphological structure of viral particles under various modes of photodynamic exposure. The samples of virus containing liquid with an initial virus titer of 5 Log₁₀ TCID50/mL were incubated with methylene blue (MB) or octakis(cholinyl)zinc phthalocyanine (Zn-PcChol₈₊) at concentrations of 1–5 μM for 10 min in the dark at room temperature. After incubation, samples were irradiated with LED (emission with maximum at 663 nm for MB or at 686 nm for Zn-PcChol₈₊) with light doses of 1.5 or 4 J/cm². Next, the irradiation titrated virus containing liquid was studied using negative staining transmission electron microscopy. MB and Zn-PcChol₈₊ at concentrations of 1–5 μM, in combination with red light from LED sources in the low doses of 1.5–4.0 J/cm², led to a decrease in BCoV titers by at least four orders of magnitude from the initial titer 5 Log₁₀ TCID50/mL. Morphological changes in photodamaged BCoVs with increasing PS concentrations were loss of spikes, change in shape, decreased size of virus particles, destruction of the envelope, and complete disintegration of viruses. BCoV has been found to be sensitive to MB, which is the well-known approved drug, even in the absence of light.

Along with their excellent safety profiles, subunit vaccines are typically characterized by much weaker immunogenicity and protection efficacy compared to whole-pathogen vaccines. Here, we present an approach aimed at bridging this disadvantage that is based on synergistic collaboration between pattern-recognition receptors (PRRs) belonging to different families. We prepared a model subunit vaccine formulation using an influenza hemagglutinin antigen incorporated into poly-(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles adjuvanted with monophosphoryl lipid A (TLR4 agonist) and muramyl dipeptide (NOD2 agonist). The efficacy studies were conducted in comparison to control vaccine formulations containing individual PRR agonists. We show that the complex adjuvant based on TLR4 and NOD2 agonists potentiates proinflammatory cell responses (measured by activity of transcription factors and cytokine production both in vitro and in vivo) and enhances the phagocytosis of vaccine particles up to comparable levels of influenza virus uptake. Finally, mice immunized with vaccine nanoparticles containing both PRR agonists exhibited enhanced humoral (IgG, hemagglutination-inhibition antibody titers) and cellular (percentage of proliferating CD4+ T-cells, production of IFNɣ) immunity, leading to increased resistance to lethal influenza challenge. These results support the idea that complex adjuvants stimulating different PRRs may present a better alternative to individual PAMP-based adjuvants and could further narrow the gap between the efficacy of subunit versus whole-pathogen vaccines.

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.

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows.

Background. Coagulase-negative staphylococci (CNS), primarily Staphylococcus epidermidis, predominate in the normal microflora of the eye. However, due to irrational antibiotic therapy, resistant strains are widely distributed among CNS. Objective. To study the sensitivity of the antibiotic resistant CNS isolates to the antiseptic picloxydine dihydrochloride ("Vitabact"). Methods. Species, sensitivity to antibiotics and antiseptic picloxydine dihydrochloride were determined for 39 isolates of bacteria obtained from the conjunctival swabs. The cells morphology under the influence of antiseptic was studied by electron microscopy. Results. 33 isolates of S. epidermidis (17 sensitive or resistant to drugs of no more than 2 classes of antibiotics and 16 MDR), 2 S. haemolyticus (1 resistant to 2 classes of antibiotics and 1 MDR), 3 S. hominis (1 sensitive and 2 MDR), 1 S. caprae (MDR) were characterized. In vitro tests, the antiseptic picloxydine dihydrochloride showed high efficiency in suppressing the growth of staphylococci regardless of their sensitivity to antibiotics, as well as bactericidal activity at concentration of 31.2 g/ml. In this concentration, the antiseptic had a pronounced destructive effect on the surface structures of bacterial cells. Conclusion. Antiseptic picloxydine dihydrochloride is effective against antibiotic resistant coagulase-negative staphylococci.