The First Homologous Expression System for the ß-Lytic Protease of Lysobacter capsici VKM B-2533T, a Promising Antimicrobial Agent.

A successful homologous expression system based on Lysobacter capsici VKM B-2533T and the plasmid pBBR1-MCS5 was first developed for a promising bacteriolytic enzyme of this bacterium, ß-lytic protease (Blp). In the expression strains, blp gene expression under the regulation of the GroEL(A) and T5 promoters increased by 247- and 667-fold, respectively, as compared with the wild-type strain. After the cultivation of the expression strains L. capsici PGroEL(A)-blp and L. capsici PT5-blp, the Blp yield increased by 6.7- and 8.5-fold, respectively, with respect to the wild-type strain. The cultivation of the expression strain L. capsici PT5-blp was successfully scaled up. Under fermentation conditions the yield of the enzyme increased by 1.6-fold. The developed homologous system was used to express the gene of the bacteriolytic serine protease (Serp) of L. capsici VKM B-2533T. The expression of the serp gene in L. capsici PT5-serp increased by 585-fold. The developed homologous system for the gene expression of bacteriolytic Lysobacter enzymes is potentially biotechnologically valuable, and is promising for creating highly efficient expression strains.

Pathogenesis-based preexposure prophylaxis associated with a low risk of SARS-CoV-2 infection in healthcare workers at a designated COVID-19 hospital: a pilot study.

BACKGROUND: At present, no agents are known to be effective at preventing COVID-19. Based on current knowledge of the pathogenesis of this disease, we suggest that SARS-CoV-2 infection might be attenuated by directly maintaining innate pulmonary redox, metabolic and dilation functions using well-tolerated medications that are known to serve these functions, specifically, a low-dose aerosolized combination of glutathione, inosine and potassium. METHODS: From June 1 to July 10, 2020, we conducted a pilot, prospective, open-label, single-arm, single-center study to evaluate the safety and efficacy of preexposure prophylaxis (PrEP) with aerosolized combination medication (ACM) on the incidence of SARS-CoV-2 positivity in 99 healthcare workers (HCWs) at a hospital designated for treating COVID-19 patients. We compared SARS-CoV-2 positivity in ACM users to retrospective data collected from 268 untreated HCWs at the same hospital. Eligible participants received an aerosolized combination of 21.3 mg/ml glutathione and 8.7 mg/ml inosine in 107 mM potassium solution for 14 days. The main outcome was the frequency of laboratory-confirmed SARS-CoV-2 cases, defined as individuals with positive genetic or immunological tests within 28 days of the study period. RESULTS: SARS-CoV-2 was detected in 2 ACM users (2, 95% CI: 0.3 to 7.1%), which was significantly less than the incidence in nonusers, at 24 (9, 95% CI: 5.8 to 13.0%; P = 0.02). During the PrEP period, solicited adverse events occurred in five participants; all were mild and transient reactions. CONCLUSIONS: Our findings might be used either to prevent SARS-CoV-2 infection or to support ongoing and new research into more effective treatments for COVID-19. TRIAL REGISTRATION: ISRCTN, ISRCTN34160010 . Registered 14 September 2020 - Retrospectively registered.

Modification of Arabinogalactan Isolated from Larix sibirica Ledeb. into Sulfated Derivatives with the Controlled Molecular Weights.

The process of sulfation of arabinogalactan-a natural polysaccharide from Larix sibirica Ledeb.-with sulfamic acid in 1,4-dioxane using different activators has been studied for the first time. The dynamics of the molecular weight of sulfated arabinogalactan upon variation in the temperature and time of sulfation of arabinogalactan with sulfamic acid in 1,4-dioxane has been investigated. It has been found that, as the sulfation time increases from 10 to 90 min, the molecular weights of the reaction products grow due to the introduction of sulfate groups without significant destruction of the initial polymer and sulfation products. Sulfation at 95 °C for 20 min yields the products with a higher molecular weight than in the case of sulfation at 85 °C, which is related to an increase in the sulfation rate; however, during the further process occurring under these conditions, sulfation is accompanied by the destruction and the molecular weight of the sulfated polymer decreases. The numerical optimization of arabinogalactan sulfation process has been performed. It has been shown that the optimal parameters for obtaining a product with a high sulfur content are a sulfamic acid amount of 20 mmol per 1 g of arabinogalactan, a process temperature of 85 °C, and a process time of 2.5 h.

Diagnostic markers of allergic bronchopulmonary aspergillosis in patients with severe asthma.

INTRODUCTION: Allergic bronchopulmonary aspergillosis (ABPA) is a lung disease in patients with asthma or cystic fibrosis (CF) caused by chronic allergic inflammation to Aspergillus spp. antigens. The role of different immunological mediators in the formation of chronic allergic inflammation in patients with ABPA is not sufficiently explored. OBJECTIVES: This study aimed to investigate serum levels of thymic stromal lymphopoietin (TSLP), thymus and activated chemokine (TARC) as well as IL-8 in patients with ABPA, and to evaluate their diagnostic and monitoring value in the disease. PATIENTS/METHODS: Prospective study included 21 patients with ABPA, 25 patients with severe asthma with fungal sensitisation (SAFS), 37 patients with severe asthma without fungal sensitisation (SAwFS), and 16 healthy people. In patients with ABPA, the serum levels of biomarkers were determined at baseline and after 12 weeks of itraconazole therapy. Serum levels of total IgE, Aspergillus-fumigatus-specific IgE, TSLP, TARC, IL-8 were analysed by enzyme-linked immunosorbent assay. RESULTS: In patients with ABPA we established significantly higher serum levels of TARC, IL-8, total IgE, Aspergillus-fumigatus-specific IgE and peripheral blood eosinophil counts, compared to patients with SAwFS. There were no differences in TSLP levels between the examined groups of patients. Serum TARC levels were positively correlated to serum total IgE levels, A fumigatus-specific IgE levels and peripheral blood eosinophil counts and also negatively correlated to lung function (FEV1 ). Longitudinally, serum levels TARC, total IgE and peripheral blood eosinophil counts significant decreased after treatment of ABPA. CONCLUSION: Thymus and activated chemokine is a useful test in diagnosing and monitoring response to the antifungal treatment of patients with ABPA.

Genotyping of Russian isolates of fungal pathogen Trichophyton rubrum, based on simple sequence repeat and single nucleotide polymorphism.

BACKGROUND: The Trichophyton rubrum species group consists of prevalent causative agents of human skin, nail and hair infections, including T rubrum sensu stricto and T violaceum, as well as other less well-established or debatable taxa like T soudanense, T kuryangei and T megninii. Our previous study provided limited evidence in favour of the existence of two genetic lineages in the Russian T rubrum sensu stricto population. OBJECTIVES: We aimed to study the genetic structure of the Russian population of T rubrum and to identify factors shaping this structure. METHODS: We analysed the polymorphism of 12 simple sequence repeat (SSR or microsatellite) markers and single nucleotide polymorphism in the TERG_02941 protein-coding gene in 70 T rubrum isolates and performed a phylogenomic reconstruction. RESULTS: All three types of data provided conclusive evidence that the population consists of two genetic lineages. Clustering, performed by means of microsatellite length polymorphism analysis, was strongly dependent on the number of nucleotide repeats in the 5'-area of the fructose-1,6-bisphosphate aldolase gene. Analysis of molecular variance (AMOVA) on the basis of SSR typing data indicated that 22%-48% of the variability was among groups within T rubrum. There was no clear connection of population structure with types of infection, places of geographic origin, aldolase gene expression or urease activity. CONCLUSION: Our results suggest that the Russian population of T rubrum consists of two cosmopolitan genetic lineages.

Influence of Membrane Receptor Lateral Diffusion on the Short-Term Depression of Acetylcholine-Induced Current in Helix Neurons.

We have studied how various drugs increasing the rate of nicotinic acetylcholine receptors (nAChRs) lateral diffusion affect the depression of ACh-induced current in land snail Helix lucorum neurons responsible for defensive behavior. The acetylcholine (ACh) iontophoretic application protocol imitated the behavioral habituation protocol for the intact animal. We found that the drugs decreasing cholesterol level in cell membranes as methyl-ß-cyclodextrin 1 mM and Ro 48-8071 2 µM, and polyclonal antibodies to actin-binding proteins as spectrin 5 µg/ml and merlin 2.5 µg/ml have changed the dynamic of ACh-current depression. The nAChRs lateral diffusion coefficient was obtained by fluorescence recovery after photobleaching. A curve fitting model specially created for analysis of short-term choline sensitivity depression in snail neurons helped us evaluate separately the contribution of nAChRs lateral diffusion, their endocytosis and exocytosis to observed effects during electrophysiological experiments. Taken together, we hypothesize that nAChRs lateral diffusion plays an important role in the cellular correlate of habituation in land snail Helix lucorum neurons.

Outer membrane vesicles of Lysobacter sp. XL1: biogenesis, functions, and applied prospects.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed.

Lysobacter gummosus 10.1.1, a Producer of Antimicrobial Agents.

This work investigated the antimicrobial potential of Lysobacter gummosus 10.1.1. The culture fluid of the strain was found to contain antimicrobial agents active against Staphylococcus aureus, Micrococcus luteus, and Bacillus cereus. L. gummosus was first shown to be capable of forming outer membrane vesicles, which have a bacteriolytic effect against not only Gram-positive bacteria but also against the Gram-negative pathogen Pseudomonas aeruginosa. Transcriptomic analysis revealed the genes of almost all known bacteriolytic enzymes of Lysobacter, as well as the genes of enzymes with putative bacteriolytic activity. Also identified were genes involved in the biosynthesis of a number of secondary metabolites for which antimicrobial activities are known. This research is indicative of the relevance of isolating and studying L. gummosus antimicrobial agents.

Hardening of Bimetallic Wires from Secondary Materials Used in the Construction of Power Lines.

Copper-sheathed steel wires combine the conductivity of copper and the traction resistance of steel, which makes a bimetallic wire an ideal material for the construction of power lines. Currently, there is a small number of studies devoted to the change in the microstructure of steel-copper wire during its strain. Since steel and copper have different mechanical properties, these metals at the interface can be deformed in different ways. Therefore, the present research is devoted to the study of ECAP-drawing process impacts on the properties of bimetallic steel-copper wire. During the conducted studies, the possibility and efficiency of using the combined strain technology for the formation of ultrafine grained structure and increased strength properties of steel-copper wire have been proved.

Whole-Genome Sequencing of Lysobacter capsici VKM B-2533T and Lysobacter gummosus 10.1.1, Promising Producers of Lytic Agents.

Lysobacter capsici VKM B-2533T and Lysobacter gummosus 10.1.1 are promising strains for use in biomedicine as sources of new antimicrobial agents. Here, we report the whole-genome sequences of both strains (total lengths, 6,239,188 bp and 6,056,609 bp, respectively), obtained using the Illumina and Nanopore platforms.

Deletion of alpB Gene Influences Outer Membrane Vesicles Biogenesis of Lysobacter sp. XL1.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria constitute important factors in defining interactions with the extracellular milieu. Lysobacter sp. XL1 produces OMVs capable of lysing microbial cells due to the presence in their cargo of bacteriolytic protease L5 (AlpB). Although protein L5 has been functionally and biochemically characterized (including aspects of its packing into OMVs), its role in vesicle biogenesis through genetic deletion of alpB had not been studied previously. Here, we have successfully deleted alpB by allelic replacement and show that the alpB deletion mutant produces a significantly lower amount of OMVs that lack bacteriolytic activity and display altered ultrastructural characteristics in relation to the OMVs produced by the wild-type strain. These results confirm that, as previously proposed, protein L5 participates in OMV production through a mechanism that is not yet fully understood.

Draft Genome Sequence of the Type Strain Lysobacter capsici VKM B-2533.

Lysobacter capsici VKM B-2533T is a promising strain for isolation of new lytic agents. Here, we report a draft genome sequence of this strain, consisting of 131 scaffolds with a total length of 6,196,943 bp. The results obtained will aid in the discovery and study of biologically active compounds important for biomedicine.

ß-Lytic Protease of Lysobacter capsici VKM B-2533T.

Bacteriolytic enzymes are promising antimicrobial agents for developing new-generation drugs. Recently, we have isolated a ß-lytic protease (BlpLc) from the culture liquid of Lysobacter capsici VKM B-2533T. This BlpLc possesses a valuable property, not described for ß-lytic proteases (Blps) earlier, of hydrolyzing living cells of Staphylococcus aureus 55 MRSA clinical isolate. This work phylogenetically characterized the BlpLc and investigated its properties. Analysis revealed a variability of pre-/pro-parts of Blp precursors. The mature BlpLc is the closest to the earlier annotated but not isolated Blp from Lysobacter sp. Root690. The biochemical characterization found conditions for the BlpLc general bacteriolytic activity relative to autoclaved S. aureus 209P cells to differ from that of earlier isolated Blp. Unexpected was the effect of serine (phenylmethylsulfonyl fluoride (PMSF)) and cysteine (p-chloromercuribenzoate (p-CMB)) protease inhibitors on BlpLc bacteriolytic and proteolytic activities. The specificity of BlpLc proteolytic action relative to hemoglobin, elastin, gelatin, collagen, azofibrin, myoglobin, ovalbumin, and ovamucoid was found. New types of peptide bonds-Gly-X, Ser-X, Lys-X, Ala-X, Val-X, Glu-X, and Phe-X-hydrolyzed by the enzyme in protein substrates were first revealed using MALDI-TOF. Turbidimetrically, the BlpLc was found to lyze living cells of S. aureus 209P, Micrococcus luteus B1819, and M. roseus B1236, which is important for expanding the enzyme's applied properties.

Antimicrobial and mechanism of antagonistic activity of Bacillus sp. A2 against pathogenic fungus and bacteria: The implication on honey's regulatory mechanism on host's microbiota.

Honey is thought to act against microbes and regulates microbiota balance, and this is mainly attributed to the enzymatic production of hydrogen peroxide, high osmolarity, and nonperoxidase factors, for example, lysozyme and botanical sources of nectar, while the effect of honey's probiotic is recently considered. The study of honey as source of beneficial microbes is understudied. The purpose of this study was to screen for the beneficial microorganisms in honey with antagonistic property against important pathogens and the mechanism of antimicrobial activity and thus play a beneficial role as probiotics. The results showed that one out of the fourteen bacterial isolates had antimicrobial activity and was identified as Bacillus Sp. A2 by 16S rRNA sequence and morphology. Antimicrobial activity of the isolate against C. albicans, E. coli, and S. aureus was confirmed by Agar well diffusion and liquid coculture assays, and the propagation of those microbes was significantly inhibited after treatment with the isolate Bacillus sp. A2 (p < .05) in comparison with untreated negative control and positive control (fluconazole, chloramphenicol, L. plantarum). The morphological changes including the distorted shape with indentations and leakages (SEM), damaged cell membrane, and cell wall with the disintegration and attachment of the Bacillus sp. A2 (TEM) in treated C. albicans were observed. Meanwhile, reactive oxygen species accumulation and decreased mitochondrial membrane potential were detected in treated C. albicans. These results revealed that the isolate Bacillus sp. A2 from honey has significant antimicrobial activity (p < .05) against C. albicans in comparison with untreated negative control and positive control L. plantarum, which depends on the accumulation of reactive oxygen species, mitochondrial damage, and the cell apoptosis. We concluded that the Bacillus sp. A2 possess the antimicrobial property, which may contribute to regulation of host's microbiota as a beneficial microbe or probiotic.

Probiotics as antifungal agents: Experimental confirmation and future prospects.

Fungal burden throughout the world is very high and it keeps escalating due to increasing numbers of immunocompromised individuals. In contrast, the drugs used in management of fungal infections are so few some with high toxicity. Furthermore, highly resistant fungal pathogens are emerging for example Candida auris, Candida glabrata, Candida gullemondii and Aspergillus species among others. Thus now, more than ever, there is a need for combined efforts and an all round search for possible solutions to curb these problems. Therefore, the role of probiotics in management of fungal infections is indispensable. In fact, the antimicrobial activity of probiotics has been screened with promising results against microbial pathogens. Although, recent reports indicated that probiotics may also contribute to protect against fungal infections, the research done in checking antifungal activity of probiotics has used varied technology. This calls for harmonization of the methods used to screen and confirm the antimicrobial activity of probiotics and other candidate microorganisms. We therefore sought to address issues of disparity in probiotic research and their outcomes. Thus this paper is in order as it comprehensively reviews' publications, provides a summary of the methods and future prospects of probiotics as antifungal agents.

Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles.

The Gram-negative bacterium Lysobacter sp. XL1 secretes various proteins, including bacteriolytic enzymes (L1-L5), into the culture medium. These proteins are able to degrade Gram-positive bacteria. The mechanism of secretion of extracellular proteins by Lysobacter sp. XL1 has not been studied hitherto. Electron microscopic investigations revealed the phenomenon of the formation of extracellular vesicles by Lysobacter sp. XL1. These vesicles contained components of the Lysobacter sp. XL1 outer membrane, and demonstrated bacteriolytic activity against Gram-positive and Gram-negative bacteria: Staphylococcus aureus 209-P and Erwinia marcescens EC1, respectively. Western blotting analysis with antibodies to homologous bacteriolytic endopeptidases L1 and L5 showed that endopeptidase L5 was secreted into the culture medium by means of vesicles, unlike its homolog, endopeptidase L1. When inside the vesicles, endopeptidase L5 actively lysed the Gram-negative bacterium Erwinia marcescens; outside the vesicles, it lost this ability. The secretion of bacteriolytic endopeptidase L5 through the outer membrane vesicles is of great biological significance: because of this ability, Lysobacter sp. XL1 can compete in nature with both Gram-positive and Gram-negative bacteria.

[Ultrastructural Patterns of Interactions between Murine Lung Macrophages and Yeast Cells of Cryptococcus neoformans Strains with Different Virulence].

This article presents the ultrastructural patterns of interactions between the murine lung macrophages and cells of low- (RKPGY-881, -1165, -1178) and high-virulence (RKPGY-1090, -1095, -1106) strains of Cryptococcus neoformans at the seventh post-experimental day. It was found that if macrophages ingest living yeast cells, the latter can: 1) become completely free from polysaccharide capsules, after that their contents undergo lysis, and cell wall debris are extruded from the macrophage (first scenario); 2) become partly free from their capsules, destroy the phagosomal plasma membrane and induce destructive processes inside the macrophage causing their death (second scenario); or 3) not lose their capsules and localize inside macrophage in latent state (third scenario). Macrophages can also ingest senescent and dead C. neoformans cells surrounded by capsules that are lost at the ingesting and phagosome stages (fourth scenario). The study revealed the dependence of cell-mediated immunity on the stage of development of ingested C. neoformans yeast cells. Here we describe a new mechanism of capsular polysaccharide elimination of C. neoformans yeast cells by murine macrophages.

Histone deacetylase inhibitor abexinostat affects chromatin organization and gene transcription in normal B cells and in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a rare lymphoma caused by the t(11:14) juxtaposing the cyclin D1 (CCND1) locus on chromosome 11 and the immunoglobulin heavy chain (IgH) locus on chromosome 14. Several new treatments are proposed for MCL, including histone deacetylase inhibitors (HDACi). We have studied gene expression and chromatin organization in the translocated 11q13 locus in MCL cells as compared to lymphoblastoid cell lines as well as the effect of HDACi abexinostat on chromatin organization and gene expression in the 11q13 locus. We have identified a cluster of genes overexpressed in the translocation region on chromosome 11 in MCL cells. Abexinostat provokes a genome-wide disaggregation of heterochromatin. The genes upregulated after the t(11;14) translocation react to the HDACi treatment by increasing their expression, but their gene promoters do not show significant alterations in H3K9Ac and H3K9me2 levels in abexinostat-treated cells.

Biogenesis of Lysobacter sp. XL1 vesicles.

The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained.

Gene Expression of Lytic Endopeptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonads.

Development of an efficient expression system for (especially secreted) bacterial lytic enzymes is a complicated task due to the specificity of their action. The substrate for such enzymes is peptidoglycan, the main structural component of bacterial cell walls. For this reason, expression of recombinant lytic proteins is often accompanied with lysis of the producing bacterium. This paper presents data on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonas fluorescens Q2-87, which provides for the successful secretion of these proteins into the culture liquid. In this system, the endopeptidase gene under control of the T7lac promoter was integrated into the bacterial chromosome, as well as the Escherichia coli lactose operon repressor protein gene. The T7 pol gene under lac promoter control, which encodes the phage T7 RNA polymerase, is maintained in Pseudomonas cells on the plasmids. Media and cultivation conditions for the recombinant strains were selected to enable the production of AlpA and AlpB by a simple purification protocol. Production of recombinant lytic enzymes should contribute to the development of new-generation antimicrobial drugs whose application will not be accompanied by selection of resistant microorganisms.