Boosting Geranyl Diphosphate Synthesis for Linalool Production in Engineered Yarrowia lipolytica.

Linalool is a pleasant-smelling monoterpenoid widely found in the essential oils of most flowers. Due to its biologically active properties, linalool has considerable commercial potential, especially in the food and perfume industries. In this study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce linalool de novo. The (S)-linalool synthase (LIS) gene from Actinidia argute was overexpressed to convert geranyl diphosphate (GPP) into linalool. Flux was diverted from farnesyl diphosphate (FPP) synthesis to GPP by introducing a mutated copy of the native ERG20F88W-N119W gene, and CrGPPS gene from Catharanthus roseus on its own and as part of a fusion with LIS. Disruption of native diacylglycerol kinase enzyme, DGK1, by oligo-mediated CRISPR-Cas9 inactivation further increased linalool production. The resulting strain accumulated 109.6 mg/L of linalool during cultivation in shake flasks with sucrose as a carbon source. CrGPPS expression in Yarrowia lipolytica increased linalool accumulation more efficiently than the ERG20F88W-N119W expression, suggesting that the increase in linalool production was predominantly influenced by the level of GPP precursor supply.

Biofilm matrix proteome of clinical strain of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit.

Extracellular matrix plays a pivotal role in biofilm biology and proposed as a potential target for therapeutics development. As matrix is responsible for some extracellular functions and influence bacterial cytotoxicity against eukaryotic cells, it must have unique protein composition. P. aeruginosa is one of the most important pathogens with emerging antibiotic resistance, but only a few studies were devoted to matrix proteomes and there are no studies describing matrix proteome for any clinical isolates except reference strains PAO1 and ATCC27853. Here we report the first biofilm matrix proteome of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit. We have identified the largest number of proteins in the matrix among all published studies devoted to P. aeruginosa biofilms. Comparison of matrix proteome with proteome from embedded cells let us to identify several enriched bioprocess groups. Bioprocess groups with the largest number of overrepresented in matrix proteins were oxidation-reduction processes, proteolysis, and transmembrane transport. The top three represented in matrix bioprocesses concerning the size of the GO annotated database were cell redox homeostasis, nucleoside metabolism, and fatty acid synthesis. Finally, we discuss the obtained data in a prism of antibiofilm therapeutics development.

On the possibility of photodynamic inactivation of tracheobronchial tree pathogenic microbiota using methylene blue (in vitro study).

BACKGROUND: The treatment of patients after mechanical ventilation of lungs suffering from a multi-species infection of the tracheobronchial tree can be complicated.. The situation is aggravated in patients with post-intubation tracheal stenosis, where infection plays a leading pathogenetic role in damage to the tracheal wall. As a result of such a pathological process, cicatricial stenosis of the trachea of purulent-inflammatory infectious genesis or infected tracheal stenosis (ITS) may occur. METHODS: In this work, we studied the possibility of photodynamic inactivation of pathogenic microbiota typical for patients with ITS using methylene blue (MB) as a photosensitizer. RESULTS: 13 clinical isolates of 8 species of bacteria from 9 patients were susceptible to photodynamic inactivation with MB. 30 µM of MB at a light irradiation dose of 25 J/cm2 and incubation with MB for 15 min allows to completely inactivate bacteria found in the tracheobronchial secretions of patients with ITS. CONCLUSIONS: MB retains its optico-physical properties in the range of 3-30 µM and provides effective inactivation of isolated Gram-positive and Gram-negative bacteria, including multi- and pan-resistant to antibiotics.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Integrated into Environmental Biofilm Chromobacterium vaccinii Survives Winter with Support of Bacterial Community.

Chromobacterium species are common in tropical and subtropical zones in environmental samples according to numerous studies. Here, we describe an environmental case of resident Chromobacterium vaccinii in biofilms associated with Carex spp. roots in Moscow region, Russia (warm-summer humid continental climate zone). We performed broad characterization of individual properties as well as surrounding context for better understanding of the premise of C. vaccinii survival during the winter season. Genome properties of isolated strains propose some insights into adaptation to habit and biofilm mode of life, including social cheaters carrying ΔluxR mutation. Isolated C. vaccinii differs from previously described strains in some biochemical properties and some basic characteristics like fatty acid composition as well as unique genome features. Despite potential to modulate membrane fluidity and presence of several genes responsible for cold shock response, isolated C. vaccinii did not survive during exposure to 4 °C, while in the complex biofilm sample, it was safely preserved for at least half a year in vitro at 4 °C. The surrounding bacterial community within the same biofilm with C. vaccinii represented a series of psychrophilic bacterial species, which may share resistance to low temperatures with other species within biofilm and provide C. vaccinii an opportunity to survive during the cold winter season.

Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice.

The bacterium Clostridium botulinum is the causative agent of botulism-a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel C. botulinum immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD50) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD50. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A.

Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa.

Induction of a robust and long-lived mucosal immune response during vaccination is critical to achieve protection against numerous pathogens. However, traditional injected vaccines are generally poor inducers of mucosal immunity. One of the effective strategies to improve vaccine efficacy is incorporation of adjuvant molecules that enhance and polarize adaptive immune reactions. Effects of Syk-coupled lectin receptor agonists as adjuvants to induce mucosal immune reactions during parenteral immunization are not fully studied. We now report that the agonists trehalose-6,6-dibehenate (TDB), curdlan, and furfurman, which stimulate Dectin-1, Dectin-2, and Mincle, respectively, activate transcription factors (NF-κB, NFAT, and AP-1) to various extents in murine RAW 264.7 macrophages, even though similar pathways are activated. The agonists also elicit differential expression of maturation markers in bone marrow-derived dendritic cells, as well as differential cytokine secretion from these cells and from splenic mononuclear cells. In vivo assays also show that agonists of Dectin-1 and Dectin-2, but not Mincle, induce heavy IgA secretion in intestinal mucosa even when delivered parenterally. Strikingly, this effect appears to be formulation-independent. Collectively, the data suggest that adjuvants based on Dectin-1 and Dectin-2 agonists may significantly improve the efficacy of parenteral vaccines by inducing robust local immune reactions in intestinal mucosa.

Preconcentration and detection of mercury with bioluminescent bioreporter E. coli ARL1.

Cell wall envelopes treated with sodium hydroxide and spray-dried were used as mercury sorbents. The sorbent having sorption capacity 17.7 ± 0.1 µmol/g determined was employed for preconcentration of mercury containing 1-10 ng/L. After preconcentration, bioavailable mercury was detected in samples of soil, stream, and tap water via induction of bioluminescence of E. coli ARL1. Iron and manganese at concentrations of tenth microgram per liter interfered bioluminescence detection of mercury. In tap water was detected semiquantitatively 0.127 ± 0.1 nmol/L by the induction of bioluminescence of E. coli ARL1 in medium with tryptone after preconcentration using a method of standard addition.