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35Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is 36 prevalent worldwide, but in vitro model for virus replication is lacking. Interaction between the 37 coronaviral spike (S) protein and its receptor is the major determinant of virus tissue and host 38 specificity, but virus entry is a complex process requiring a concerted action of multiple cellular 39 elements. Here, we show that KLK13 is required for the infection of the human respiratory 40 epithelium and is sufficient to mediate the entry of HCoV-HKU1 to non-permissive RD cells. 41 We also demonstrated HCoV-HKU1 S protein cleavage by KLK13 in the S1/S2 region, proving 42 that KLK13 is the priming enzyme for this virus. Summarizing, we show for the first time that 43 protease distribution and specificity predetermines the tissue and cell specificity of the virus 44 and may also regulate interspecies transmission. It is also of importance that presented data may 45 be relevant for the emerging coronaviruses, including SARS-CoV-2 and may help to understand 46 the differences in their zoonotic potential. : bioRxiv preprint 48Coronaviruses are the largest group within the order Nidovirales. Mainly, they cause 49 respiratory and enteric diseases in humans and animals, but some can cause more serious 50 conditions such as hepatitis, peritonitis, or neurological disease. Seven coronaviruses infect 51 humans, four of which (human coronavirus [HCoV]-229E, HCoV-NL63, HCoV-OC43, and 52 HCoV-HKU1) cause relatively mild upper and lower respiratory tract disease and two (SARS-53 CoV and MERS-CoV) are associated with severe, life-threatening respiratory infections and 54 multiorgan failure (1-6). Furthermore, in December 2019 a novel coronavirus SARS-CoV-2 55 emerged in Hubei province, China, causing pneumonia. To date, almost 90,000 cases were 56 identified and 3,000 patients died worldwide. 57 Coronaviral infection is initiated by interaction between the trimeric spike (S) protein 58 and its receptor, which is expressed on the surface of the susceptible cell. A number of adhesion 59 and entry receptors have been described for coronaviruses. For example, HCoV-229E (similar 60 to many other alphacoronaviruses) utilizes aminopeptidase N (APN) as the primary entry port 61 (7). Surprisingly, its cousin HCoV-NL63 shares receptor specificity with the evolutionarily 62 distant SARS-CoV and SARS-CoV-2: all hijack angiotensin-converting enzyme 2 (ACE2) (8-63 11). HCoV-NL63 was also shown to use heparan sulfate as a primary attachment site (12-14). 64 A very different receptor is recognized by MERS-CoV, which binds to dipeptidyl-peptidase 4 65 (DPP4) (9, 15, 16). Another betacoronavirus, HCoV-OC43, binds to N-acetyl-9-O-66 acetylneuraminic acid (17, 18). HCoV-HKU1 remains the great unknown because its cellular 67 receptor has not been identified and all efforts to culture the virus in vitro have failed.68 HCoV-HKU1 was identified in Hong Kong in 2004. The virus was present in a sample 69 obtained from an elderly patient with severe pneumonia (...

The work describes purification and biochemical characterization of two inducible antimicrobial peptides from the hemolymph of Galleria mellonella. The peptides were isolated by a sequence of reversed-phase chromatography steps from the hemolymph of larvae immunized with viable bacteria. The first peptide is a member of the cecropin family while the second one is rich in proline residues and has a unique sequence.

Galleria mellonella cationic protein 8 (GmCP8) is a hemolymph protein previously identified as an opsonin and an inhibitor of fungal proteases. In this work, we showed its bactericidal activity toward Pseudomonas entomophila, Pseudomonas aeruginosa, Bacillus thuringiensis, Staphylococcus aureus, and Escherichia coli and against yeast-like fungi Candida albicans. The activity against E. coli was correlated with bacterial membrane permeabilization. In turn, in the case of P. entomophila, B. thuringiensis, and C. albicans, the atomic force microscopy analysis of the microbial surface showed changes in the topography of cells and changes in their nanomechanical properties. GmCP8 also showed the inhibitory activity toward the serine protease trypsin and the metalloproteinase thermolysin. The expression of the gene encoding the GmCP8 protein did not increase either in the gut or in the fat body of G. mellonella after oral infection with P. entomophila. Similarly, the amount of GmCP8 in the hemolymph of G. mellonella did not change in immune-challenged insects. However, when GmCP8 was injected into the G. mellonella hemocel, a change in the survival curve was observed in the infected larvae. Our results shed new light on the function of GmCP8 protein in insect immunity, indicating its role in humoral defence mechanisms.

The increasing antibiotic resistance of pathogenic bacteria calls for the development of alternative antimicrobial strategies. Possible approaches include the development of novel, broad-spectrum antibiotics as well as specific targeting of individual bacterial virulence factors. It is impossible to decide currently which strategy will prove more successful in the future since they both promise different advantages, but also introduce diverse problems. Considering both approaches, our laboratory's research focuses on the evaluation of hemocidins, broad-spectrum antibacterial peptides derived from hemoglobin and myoglobin, and staphostatins, specific inhibitors of staphopains -- Staphylococcus aureus secreted proteases that are virulence factors regarded as possible targets for therapy. The article summarizes recent advances in both fields of study and presents perspectives for further development and possible applications.