The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called “post-antibiotic era” in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting bacterial cell wall peptidoglycans to promote their osmotic lysis. However, relatively little is known regarding the spectrum of lysin bactericidal activity against Gram-negative bacteria. In this study, we present the results of in vitro activity assays of three putative and newly cloned Myoviridae bacteriophage endolysins (LysAm24, LysECD7, and LysSi3). The chosen proteins represent lysins with diverse domain organization (single-domain vs. two-domain) and different predicted mechanisms of action (lysozyme vs. peptidase). The enzymes were purified, and their properties were characterized. The enzymes were tested against a panel of Gram-negative clinical bacterial isolates comprising all Gram-negative representatives of the ESKAPE group. Despite exhibiting different structural organizations, all of the assayed lysins were shown to be capable of lysing Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Salmonella typhi strains. Less than 50 μg/mL was enough to eradicate growing cells over more than five orders of magnitude. Thus, LysAm24, LysECD7, and LysSi3 represent promising therapeutic agents for drug development.
Our previous studies demonstrated that precoating of filter fibers with biologically active tea tree oil (TTO) enhances physical collection efficiency of conventional heating, ventilation, and air conditioning (HVAC) filters, and provides cost effective and rapid inactivation of captured bacterial and fungal particles on the filter surface. The main aim of this study was to investigate the antiviral activity of two natural disinfectants, i.e., TTO and eucalyptus oil (EUO), against the influenza virus captured onto the filter surface. It was found that both tested oils possess strong antiviral properties when used as fiber coating materials, capable of inactivating captured microorganisms within 5-10 min of contact on the fiber surface. The antiviral activity of TTO was also successfully challenged in aerosol form by mixing viable airborne viral particles with oil droplets in the rotational aerosol chamber. The results look very promising for further development of virus inactivating procedures and technologies for air quality applications.
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.
Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD50) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD50 of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.
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