Background
The problem of increasing resistance against conventional antibiotics has drawn people’s attention. Therefore, the development of novel antibacterial agents with effective and safe therapeutic effects is imminent. Antimicrobial peptides (AMPs) are considered a promising class of antibacterial agents due to their broad antibacterial spectrum.
Results
In this study, on the basis of our previously studied peptide PMAP-37(F34-R), a novel antimicrobial peptide Chol-37(F34-R) was developed by N-terminal cholesterol modification to increase hydrophobicity. We observed that the N-terminal cholesterol-modified Chol-37(F34-R) showed higher antimicrobial activity than PMAP-37(F34-R) in vitro. Chol-37(F34-R) also exhibited effective anti-biofilm activity and may kill bacteria by improving the permeability of their membranes. Chol-37(F34-R) exerted high stability in different pH, salt, serum, and boiling water environments. Chol-37(F34-R) also showed no hemolytic activity and substantially low toxicity. Furthermore, Chol-37(F34-R) exhibited good potency of bacteria eradication and promoted wound healing and abscess reduction in infected mice. Meanwhile, in S. aureus ATCC25923-infected peritonitis model, Chol-37(F34-R) exhibited an impressive therapeutic effect by reducing the decrease in systemic bacterial burden and alleviating organ damage.
Conclusions
Our findings suggested that the N-terminal cholesterol modification of PMAP-37(F34-R) could improve antibacterial activity. Chol-37(F34-R) displayed excellent bactericidal efficacy and impressive therapeutic effect in vivo. Thus, Chol-37(F34-R) may be a candidate for antimicrobial agents against microbial infection in the clinic.
Bacterial resistance induced by the use of antibiotics has provided a chance for the development of antimicrobial peptides (AMPs), and modification of AMPs to enhance the antibacterial activity or stability has become a research focus. PMAP‐37 is an AMP isolated from porcine myeloid marrow, and studies on its modification have not yet been reported. In this study, three PMAP‐37 analogs named PMAP‐37(F9‐R), PMAP‐37(F34‐R), and PMAP‐37(F9/34‐R) were designed by residue substitution to enhance the positive charge. The antimicrobial activity of PMAP‐37 and its analogs in vitro and in vivo were detected. The results showed that compared with PMAP‐37, PMAP‐37(F9‐R) and PMAP‐37(F9/34‐R) exhibited antibacterial activity against S. flexneri CICC21534. Although PMAP‐37(F34‐R) had no antibacterial activity against S. flexneri CICC21534, its minimal inhibitory concentrations (MICs) were significantly lower than those of PMAP‐37 against most bacterial strains. Besides, all PMAP‐37 analogs were pH stable, retaining stable antibacterial activity after treatment with solution from pH 2 to pH 8/9. In addition, the PMAP‐37 analogs displayed increased thermal stability, and PMAP‐37(F34‐R) retained >60% antibacterial activity after boiling for 2 hours. Furthermore, the PMAP‐37 analogs exhibited impressive therapeutic efficacy in bacterial infections by reducing bacterial burden and inflammatory damage in the lung and liver, resulting in a reduction in mortality. Notably, the therapeutic effect of PMAP‐37(F34‐R) was comparable to that of ceftiofur sodium, and even superior to antibiotics in L. monocytogenes CICC21533 infection model. In conclusion, the PMAP‐37(F34‐R) may be a candidate for the treatment of bacterial infections in the clinic.
As the important molecular machinery for membrane protein sorting in eukaryotic cells, the endosomal sorting and transport complexes (ESCRT-0/I/II/III and VPS4) usually participate in various replication stages of enveloped viruses, such as endocytosis and budding. The main subunit of ESCRT-I, Tsg101, has been previously revealed to play a role in the entry and replication of classical swine fever virus (CSFV). However, the effect of the whole ESCRT machinery during CSFV infection has not yet been well defined. Here, we systematically determine the effects of subunits of ESCRT on entry, replication, and budding of CSFV by genetic analysis. We show that EAP20 (VPS25) (ESCRT-II), CHMP4B and CHMP7 (ESCRT-III) regulate CSFV entry and assist vesicles in transporting CSFV from Clathrin, early endosomes, late endosomes to lysosomes. Importantly, we first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II) and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Further analyses reveal these subunits interact with CSFV nonstructural proteins (NS) and locate in the endoplasmic reticulum, but not Golgi, suggesting the role of ESCRT in regulating VRC assembly. In addition, we demonstrate that VPS4A is close to lipid droplets (LDs), indicating the importance of lipid metabolism in the formation of VRC and nucleic acid production. Altogether, we draw a new picture of cellular ESCRT machinery in CSFV entry and VRC formation, which could provide alternative strategies for preventing and controlling the diseases caused by CSFV or other Pestivirus.
In summary, BPP-IV did not have an effect on specific immune responses, therefore it could not reduce pathological damage in mice lungs; BPP-III provided resistance to the antigen through the cell-mediated immune response; BPP-V enhanced both humoral and cellular immune responses, therefore provided protections against the H9N2 Avian Influenza Virus (AIV). Altogether, BPP-III and BPP-V could be adjuvant candidates for the H9N2 avian influenza vaccine.
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