Synergistic effects of antimicrobial peptide dendrimer-chitosan polymer conjugates against Pseudomonas aeruginosa.

We report herein a new chemical platform for coupling chitosan derivatives to antimicrobial peptide dendrimers (AMPDs) with different degrees of ramification and molecular weights via thiol-maleimide reactions. Previous studies showed that simple incorporation of AMPDs to polymeric hydrogels resulted in a loss of antibacterial activity and augmented cytotoxicity to mammalian cells. We have shown that coupling AMPDs to chitosan derivatives enabled the two compounds to act synergistically. We showed that the antimicrobial activity was preserved when incorporating AMPD conjugates into various biopolymer formulations, including nanoparticles, gels, and foams. Investigating their mechanism of action using electron and time-lapse microscopy, we showed that the AMPD-chitosan conjugates were internalized after damaging outer and inner Gram-negative bacterial membranes. We also showed the absence of AMPD conjugates toxicity to mammalian cells. This chemical technological platform could be used for the development of new membrane disruptive therapeutics to eradicate pathogens present in acute and chronic wounds.

Chemical modifications to increase the therapeutic potential of antimicrobial peptides.

The continued use of antibiotics has been accompanied by the rapid emergence and spread of antibiotic-resistant strains of bacteria. Antimicrobial peptides (AMPs), also known as host defense peptides, show multiple features as an ideal antimicrobial agent, including potent, rapid, and broad-spectrum antimicrobial activity, low promotion of antimicrobial resistance, potent anti-biofilm activity, and lethality against metabolically inactive microorganisms. However, several crucial drawbacks constrain the use of AMPs as clinical drugs, e.g., liability in vivo, toxicity when used systemically, and high production costs. Based on recent findings and our own experiences, here we summarize some chemical modifications and key design strategies to increase the therapeutic potential of AMPs, including 1) enhancing antimicrobial activities, 2) improving in vivo effectiveness, and 3) reduction in toxicity, which may facilitate the design and optimization of AMPs for the development of drug candidates. We also discuss the present challenges in the optimization of AMPs and future concerns about the resistance and cross-resistance to AMPs in the development of AMPs as therapeutic drugs.

Tribolium castaneum defensin 1 kills Moraxella catarrhalisin an in vitro infection model but does not harm commensal bacteria.

Moraxella catarrhalis is a bacterial pathogen that causes respiratory tract infections in humans. The increasing prevalence of antibiotic-resistant M. catarrhalis strains has created a demand for alternative treatment options. We therefore tested 23 insect antimicrobial peptides (AMPs) for their activity against M. catarrhalis in a human in vitro infection model with primary macrophages, and against commensal bacteria. Effects on bacterial growth were determined by colony counting and growth curve analysis. The inflammatory macrophage response was characterized by qPCR and multiplex ELISA. Eleven of the AMPs were active against M. catarrhalis. Defensin 1 from the red flour beetle Tribolium castaneum significantly inhibited bacterial growth and reduced the number of colony forming units. This AMP also showed antibacterial activity in the in vitro infection model, reducing cytokine expression and release by macrophages. Defensin 1 had no effect on the commensal bacteria Escherichia coli and Enterococcus faecalis. However, sarcotoxin 1 C from the green bottle fly Lucilia sericata was active against M. catarrhalis and E. coli, but not against E. faecalis. The ability of T. castaneum defensin 1 to inhibit M. catarrhalis but not selected commensal bacteria, and the absence of cytotoxic or inflammatory effects against human blood-derived macrophages, suggests this AMP may be suitable for development as a new therapeutic lead against antibiotic-resistant M. catarrhalis.

Mechanisms of antimicrobial and antiendotoxin activities of a triazine-based amphipathic polymer.

TZP4 is a triazine-based amphipathic polymer designed to mimic the amphipathic structure found in antimicrobial peptides. TZP4 showed potent antimicrobial activity comparable to melittin against antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. TZP4 showed high resistance to proteolytic degradation and low tendency to develop drug resistance. The results from membrane depolarization, SYTOX Green uptake, flow cytometry, and gel retardation revealed that the mechanism of antimicrobial action of TZP4 involved an intracellular target rather than the bacterial cell membrane. Furthermore, TZP4 suppressed the messenger RNA levels of inducible nitric oxide synthase and tumor necrosis factor-α (TNF-α) and inhibited the release of nitric oxide and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BODIPY-TR-cadaverine displacement and dissociation of fluorescein isothiocyanate (FITC)-labeled LPS assays revealed that TZP4 strongly bound to LPS and disaggregated the LPS oligomers. Flow cytometric analysis demonstrated that TZP4 inhibits the binding of FITC-conjugated LPS to RAW264.7 cells. These observations indicate that TZP4 may exert its antiendotoxic activity by directly binding with LPS and inhibiting the interaction between LPS and CD14+ cells. Collectively, TZP4 is a promising drug candidate for the treatment of endotoxic shock and sepsis caused by Gram-negative bacterial infections.