Development of novel antimicrobials with engineered endolysin LysECD7-SMAP to combat Gram-negative bacterial infections.

BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.

Motility provides specific adhesion patterns and improves Listeria monocytogenes invasion into human HEp-2 cells.

Listeria monocytogenes is motile at 22°C and non-motile at 37°C. In contrast, expression of L. monocytogenes virulence factors is low at 22°C and up-regulated at 37°C. Here, we studied a character of L. monocytogenes near surface swimming (NSS) motility and its effects on adhesion patterns and invasion into epithelial cells. L. monocytogenes and its saprophytic counterpart L. innocua both grown at 22°C showed similar NSS characteristics including individual velocities, trajectory lengths, residence times, and an asymmetric distribution of velocity directions. Similar NSS patterns correlated with similar adhesion patterns. Motile bacteria, including both pathogenic and saprophytic species, showed a preference for adhering to the periphery of epithelial HEp-2 cells. In contrast, non-motile bacteria were evenly distributed across the cell surface, including areas over the nucleus. However, the uneven distribution of motile bacteria did not enhance the invasion into HEp-2 cells unless virulence factor production was up-regulated by the transient shift of the culture to 37°C. Motile L. monocytogenes grown overnight at 22°C and then shifted to 37°C for 2 h expressed invasion factors at the same level and invaded human cells up to five times more efficiently comparatively with non-motile bacteria grown overnight at 37°C. Taken together, obtained results demonstrated that (i) NSS motility and correspondent peripheral location over the cell surface did not depend on L. monocytogenes virulence traits; (ii) motility improved L. monocytogenes invasion into human HEp-2 cells within a few hours after the transition from the ambient temperature to the human body temperature.

Infectivity and Morphology of Bovine Coronavirus Inactivated In Vitro by Cationic Photosensitizers.

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.

Discovering the Potentials of Four Phage Endolysins to Combat Gram-Negative Infections.

Endolysin-based therapeutics are promising antibacterial agents and can successfully supplement the existing antibacterial drugs array. It is specifically important in the case of Gram-negative pathogens, e.g., ESKAPE group bacteria, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, and are highly inclined to gain multiple antibiotic resistance. Despite numerous works devoted to the screening of new lytic enzymes and investigations of their biochemical properties, there are significant breaches in some aspects of their operating characteristics, including safety issues of endolysin use. Here, we provide a comprehensive study of the antimicrobial efficacy aspects of four Gram-negative bacteria-targeting endolysins LysAm24, LysAp22, LysECD7, and LysSi3, their in vitro and in vivo activity, and their biological safety. These endolysins possess a wide spectrum of action, are active against planktonic bacteria and bacterial biofilms, and are effective in wound and burn skin infection animal models. In terms of safety, these enzymes do not contribute to the development of short-term resistance, are not cytotoxic, and do not significantly affect the normal intestinal microflora in vivo. Our results provide a confident base for the development of effective and safe candidate dosage forms for the treatment of local and systemic infections caused by Gram-negative bacterial species.

Development of novel darunavir amorphous solid dispersions with mesoporous carriers.

The aim of this work was to compare mesoporous carriers based on silica and magnesium aluminosilicate in the amorphous solid dispersion production. Darunavir has been selected as an active pharmaceutical ingredient that is classified as a Class 2 BCS substance and exists in two commercially available forms: crystalline ethanolate and amorphous. In the course of the study, the conditions for the preparation of amorphous samples with the selected carriers were evaluated within the framework of the most common methods for obtaining solid dispersions - hot-melt extrusion, solvent wetting, and spray drying. It was determined that the obtained dispersion properties almost completely repeat the properties of the corresponding carriers. The resulting dispersions were examined in a dissolution test and the best ones was used to formulate tablets, which were studied in an in vitro dissolution test with the original Prezista. The proposed tablet formulation showed improved dissolution compared to the original one. It was also found that silica supports have a greater positive contribution to darunavir dissolution - both ethanolate or amorphous forms.

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.

Adjuvantation of an Influenza Hemagglutinin Antigen with TLR4 and NOD2 Agonists Encapsulated in Poly(D,L-Lactide-Co-Glycolide) Nanoparticles Enhances Immunogenicity and Protection against Lethal Influenza Virus Infection in Mice.

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 IFNÉ£) 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.

Modulation of Endolysin LysECD7 Bactericidal Activity by Different Peptide Tag Fusion.

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.