Yana A. Khlusevich

Staphylococcus aureus is an ESKAPE pathogen, defined by its ability to evade antibacterial treatment, and together with S. epidermidis is a leading Gram-positive cause of wound infection. The emergence of methicillin- and vancomycin-resistant strains further limits conventional antibiotic treatment. The staphylococcal endolysin LysSte134_1 was produced recombinantly, its proper folding confirmed by zymography, and its lytic activity demonstrated against planktonic cultures of both S. aureus and S. epidermidis. The combination of LysSte134_1 with 10% 1,3-propanediol and Zn²⁺ increased CFU reduction in both species relative to LysSte134_1 with Zn²⁺ alone. In a murine wound model, topical application of LysSte134_1 significantly reduced the bacterial burden in wound tissue, achieving a 2–3-log reduction in MRSA and below the detection limit for S. epidermidis. Treatment was accompanied by normalization of peripheral leukocyte counts and a shift in the serum cytokine profile from a sustained pro-inflammatory state with elevated IL-6, IL-17 and IL-10 toward levels comparable to non-infected controls. These results suggest the potential utility of LysSte134_1 as a component of topical antibacterial formulations and support further investigation in models of staphylococcal wound infection.

Multidrug resistance (MDR) is a global problem for the healthcare system, complicating the therapy of bacterial infections. It is noted that patients infected with MDR strains often require prolonged hospitalization, have a high risk of mortality, and remain vulnerable to reinfection after recovery. In this study, recombinant phage-encoded depolymerase DepKP144 was produced using the Escherichia coli expression system and purified. The depolymerase DepKP144 protein was able to reduce viable bacterial counts following capsule degradation in 95% of the tested strains of type K1 and 85% of the tested strains of type K2 Klebsiella pneumoniae. The depolymerase DepKP144 was active against K. pneumoniae K1-type and K2-type planktonic cells and destroyed the biofilms formed by clinical MDR strains of K. pneumoniae. In in vivo experiments, DepKP144 at a dose of 180 μg/mouse resulted in a 50% survival of the mice infected with K2-type K. pneumoniae and in a 17% survival of the mice infected with K1-type K. pneumoniae. This depolymerase is promising for further development of prevention and therapeutic candidates against MDR K. pneumoniae.

Staphylococcus aureus, including methicillin- and vancomycin-resistant variants (MRSA, VRSA), causes infections that are increasingly difficult to treat with conventional antibiotics. One of the approaches to developing new therapeutics to treat staphylococcal infections is the use of bacteriophages specific to these bacteria or the lytic enzymes of such bacteriophages, which are capable of hydrolyzing the cell walls of these bacteria. Phage-encoded endolysins offer an alternative promising class of antimicrobial agents. In this study, LysSA120, a 250-amino-acid endolysin encoded by the S. aureus podophage vB_SauP_120, was cloned, expressed in Escherichia coli, and characterized. The domain organization and tertiary structure of LysSA120 were predicted. Recombinant LysSA120 hydrolyzed cell walls were obtained from S. aureus, S. epidermidis, S. haemolyticus, S. warneri, S. auricularis and S. saprophyticus. It was shown that treatment of S. aureus planktonic cells with endolysin LysSA120 led to reduced viability. Furthermore, LysSA120 could hydrolyze mature biofilm formed by VRSA. The lytic spectrum and antibiofilm activity of LysSA120 warrant its further evaluation as an enzybiotic against drug-resistant staphylococcal infections.

Klebsiella pneumoniae, a member of the ESKAPEE group of priority pathogens, has become one of the most challenging bacterial pathogens in modern clinical practice, largely due to its multidrug resistance and the immune-evasive effect of its capsular polysaccharide (CPS). Phage-encoded depolymerases, which selectively degrade the capsular polysaccharide, have emerged as promising antimicrobial agents capable of restoring bacterial susceptibility to both immune clearance and phage infection. The fragment corresponding to the C-terminal region of a putative depolymerase of bacteriophage KlebP_144, namely DepKP144ΔC, was cloned, expressed in E. coli, and purified using immobilized metal affinity chromatography. DepKP144ΔC displays an enzymatic activity against capsular polysaccharides of 100% K1 capsular-type strains and 85% K2 capsular-type strains, including classical and hypervirulent isolates. It was demonstrated that this protein is capable of inhibiting K. pneumoniae biofilm formation, but it is unable to disrupt mature biofilms. In vivo experiments using a murine K. pneumoniae infection model further confirmed its therapeutic potential: treatment with DepKP144ΔC improved survival rate in mice infected with K2-type K. pneumoniae, indicating significant attenuation of bacterial virulence. Therefore, these results demonstrate the potential role of the C-terminal domain of the bacteriophage KP144 tail-fiber protein in phage entry and show that its carbohydrate-recognition motifs possess enzymatic activity against the Klebsiella capsular polysaccharides.

Orthoflavivirus omskense (Omsk hemorrhagic fever virus, OHFV) is a tick-borne flavivirus that causes Omsk hemorrhagic fever (OHF), a severe zoonotic disease endemic to Western Siberia. Despite the fact that the role of NS1 proteins of various mosquito-borne flaviviruses in pathogenesis was investigated and their ability to affect human endothelial permeability was shown, the role of the NS1 protein of OHFV in pathogenesis is unstudied. In this work, the ability of OHFV NS1 to induce human endothelial permeability was investigated for the first time. It was shown that recombinant OHFV NS1 produced in eucaryotic cells directly affects both human lung microvascular endothelial cells (HLMVEC) and human umbilical vein endothelial cells (HUVEC) in vitro. RNAseq of endothelial cells treated with OHFV NS1 indicated that OHFV NS1 enhances the expression of genes associated with cellular stress responses, vascular signaling, and cell–cell junction regulation, resulting in a nonspecific increase in the endothelial permeability of various vessels. These results suggest that the NS1 protein may contribute to OHFV pathogenesis by disrupting endothelial barrier function and promoting vascular leakage, potentially playing a role in the hemorrhagic manifestations of Omsk hemorrhagic fever.

Orthoflavivirus encephalitidis (tick-borne encephalitis virus, TBEV) is of high concern due to its ability to cause severe neurological manifestations. Despite the fact that the role of NS1 proteins from various mosquito-borne flaviviruses in pathogenesis and their ability to affect human endothelial permeability have been investigated, TBEV NS1 has thus far been insufficiently studied. In this study, human endothelial permeability was assessed using TEER and transwell permeability assays. Signaling pathways were determined by RNAseq. The ability of the NS1 protein of TBEV to affect human endothelial permeability was investigated for the first time. It was shown that recombinant TBEV NS1 produced in eucaryotic cells directly affected human lung microvascular endothelial cells (HLMVECs) in vitro but not human umbilical vein endothelial cells (HUVECs). It was indicated that TBEV NS1 induced endothelial hyperpermeability of HLMVECs through activating TNF-α and other inflammatory signaling pathways.

Orthoflavivirus omskense (Omsk hemorrhagic fever virus, OHFV) is a tick-borne flavivirus that causes Omsk hemorrhagic fever (OHF), a severe zoonotic disease endemic to Western Siberia. Despite the fact that the role of NS1 proteins of various mosquito-borne flaviviruses in pathogenesis was investigated and their ability to affect human endothelial permeability was shown, the role of NS1 protein of OHFV in pathogenesis is unstudied. In this work, the ability of OHFV NS1 to induce human endothelial permeability was investigated for the first time. It was shown that recombinant OHFV NS1 produced in eucaryotic cells directly affects both human lung microvascular endothelial cells (HLMVEC) and human umbilical vein endothelial cells (HUVEC) in vitro. RNAseq of endothelial cells treated with OHFV NS1 indicated that OHFV NS1 enhances the expression of genes associated with cellular stress responses, vascular signaling, and cell–cell junction regulation, resulting in a nonspecific increase in the endothelial permeability of various vessels.

Background/Objectives: Orthoflavivirus encephalitidis (tick-borne encephalitis virus, TBEV) is of high concern due to its ability to cause severe neurological manifestations. Despite the fact that the role of NS1 proteins of various mosquito-borne flaviviruses in pathogenesis and their ability to affect human endothelial permeability was investi-gated, TBEV NS1 is insufficiently studied. Methods: Human endothelial permeability was assessed using PEER and transwell permeability assays. Signaling pathways were determined by RNAseq. Results: The ability of NS1 protein of TBEV to affect human endothelial permeability was investigated for the first time. It was shown that recombinant TBEV NS1 produced in eucaryotic cell directly affected on human lung microvascular endothelial cells (HLMVEC) in vitro but not on human umbilical vein endothelial cells (HUVEC). Conclusions: it was indicated that TBEV NS1 induced endothelial hyperpermeability of HLMVEC through activation TNF-α and other inflammatory signaling pathways.

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis virus (TBEV) complex of the Flaviviridae family. Currently, there are no data on the cross-reactivity of antibodies to the NS1 proteins of OHFV and TBEV. Such data are of major interest for monitoring viral encephalitis of unknown etiology due to the increasing geographical distribution of OHFV. In this study, a recombinant OHFV NS1 protein was produced using the Escherichia coli expression system and purified. The recombinant OHFV NS1 protein was recognized by specific mice immune ascetic fluids to the native OHFV NS1 protein. A Western blot analysis and ELISA of the recombinant NS1 proteins of OHFV and TBEV were used to study the cross-reactivity of antibodies from immune ascites fluid obtained from OHFV-infected mice and mAbs against TBEV NS1. Anti-TBEV NS1 mouse monoclonal antibodies (mAbs) have been shown to not be cross-reactive to the OHFV NS1 protein. Sera from patients with confirmed tick-borne encephalitis (TBE) were examined by ELISA using recombinant OHFV NS1 and TBEV NS1 proteins as antigens. It was shown for the first time that cross-reactive antibodies to the OHFV NS1 protein were not detected in the sera of TBE patients, whereas the sera contained antibodies to the TBEV NS1 protein.

Omsk hemorrhagic fever virus (OHFV) is a member of the tick-borne encephalitis virus (TBEV) complex of the Flaviviridae family. Currently, there is no data on cross-reactivity of antibodies to the NS1 proteins of OHFV and TBEV. Such data are of major interest for monitoring viral encephalitis of unknown etiology due to the increasing geographical distribution of OHFV. In this study, recombinant OHFV NS1 protein was produced using Escherichia сoli expression system and purified. The recombinant OHFV NS1 protein was recognized by specific mice immune ascetic fluids to the native OHFV NS1 protein. Western blot analysis and ELISA of recombinant NS1 proteins of OHFV and TBEV were used to study the cross-reactivity of antibodies from immune ascites fluid obtained from OHFV-infected mice and mAbs against TBEV NS1. Anti-TBEV NS1 mouse monoclonal antibodies (mAbs) have been shown not to be cross-reactive to OHFV NS1 protein. Sera from patients with confirmed tick-borne encephalitis (TBE) were examined by ELISA using recombinant OHFV NS1 and TBEV NS1 proteins as antigens. It was shown for the first time that cross-reactive antibodies to OHFV NS1 protein were not detected in the sera of TBE patients whereas the sera contained antibodies to TBEV NS1 protein.