The lipidation and glycosylation enabling bioactivity enhancement and structural change of antibacterial peptide G3.

Bacterial resistance has led to increased interest in the use of antibacterial peptides (AMPs), but their clinical application is limited by poor stability and solubility, as well as complex cytotoxicity. Chemical modification is a common strategy to modulate AMPs. In this study, a de novo designed AMP (G3) was modified by adding an alkyl acid at the N-terminal and a monosaccharide at the C-terminal. Bio-activity assays demonstrated that conjugation with n-caprylic acid increased the peptide's antibacterial activity and permeabilized the membrane. Attachment of glucose or galactose at the C-terminal improved its biofilm inhibitory capacity and marginally reduced cytotoxicity. The hybrid peptide, containing both n-caprylic acid and galactose, exhibited excellent antibacterial and antibiofilm activity, as well as permeabilized the outer membrane.

Antimicrobial peptides fight against Pseudomonas aeruginosa at a sub-inhibitory concentration via anti-QS pathway.

The broad-spectrum antimicrobial ability of de novo designed amphiphilic antimicrobial peptides (AMPs) G(IIKK)3I-NH2 (G3) and C8-G(IIKK)2I-NH2 (C8G2) have been demonstrated. Nonetheless, their potential as anti-quorum-sensing (anti-QS) agents, particularly against the opportunistic pathogen Pseudomonas aeruginosa at subinhibitory concentrations, has received limited attention. In this study, we proved that treating P. aeruginosa PAO1 with both AMPs at subinhibitory concentrations led to significant inhibition of QS-regulated virulence factors, including pyocyanin, elastase, proteases, and bacterial motility. Additionally, the AMPs exhibited remarkable capabilities in suppressing biofilm formation and their elimination rate of mature biofilm exceeded 95%. Moreover, both AMPs substantially downregulated the expression of QS-related genes. CD analysis revealed that both AMPs induced structural alterations in the important QS-related protein LasR in vitro. Molecular docking results indicated that both peptides bind to the hydrophobic groove of the LasR dimer. Notably, upon mutating key binding sites (D5, E11, and F87) to Ala, the binding efficiency of LasR to both peptides significantly decreased. We revealed the potential of antibacterial peptides G3 and C8G2 at their sub-MIC concentrations as QS inhibitors against P. aeruginosa and elucidated their action mechanism. These findings contribute to our understanding of the therapeutic potential of these peptides in combating P. aeruginosa infections by targeting the QS system.

Effects of hydroxyl group in cyclo(Pro-Tyr)-like cyclic dipeptides on their anti-QS activity and self-assembly.

We investigated the influence of hydroxyl groups on the anti-quorum-sensing (anti-QS) and anti-biofilm activity of structurally similar cyclic dipeptides, namely cyclo(L-Pro-L-Tyr), cyclo(L-Hyp-L-Tyr), and cyclo(L-Pro-L-Phe), against Pseudomonas aeruginosa PAO1. Cyclo(L-Pro-L-Phe), lacking hydroxyl groups, displayed higher virulence factor inhibition and cytotoxicity, but showed less inhibitory ability in biofilm formation. Cyclo(L-Pro-L-Tyr) and cyclo(L-Hyp-L-Tyr) suppressed genes in both the las and rhl systems, whereas cyclo(L-Pro-L-Phe) mainly downregulated rhlI and pqsR expression. These cyclic dipeptides interacted with the QS-related protein LasR, with similar binding efficiency to the autoinducer 3OC12-HSL, except for cyclo(L-Pro-L-Phe) which had lower affinity. In addition, the introduction of hydroxyl groups significantly improved the self-assembly ability of these peptides. Both cyclo(L-Pro-L-Tyr) and cyclo(L-Hyp-L-Tyr) formed assembly particles at the highest tested concentration. The findings revealed the structure-function relationship of this kind of cyclic dipeptides and provided basis for our follow-up research in the design and modification of anti-QS compounds.

An additive-free multifunctional ß-glucan-peptide hydrogel participates in the whole process of bacterial-infected wound healing.

Bacterial infections and excessive inflammation can impede the healing of wounds. Hydrogels have emerged as a promising approach for dressing bacterial-infected injuries. However, some antibacterial hydrogels are complex, costly, and even require assistance with other instruments such as light, making them unsuitable for routine outdoor injuries. Here, we developed an in-situ generating hydrogel via hybridizing oxidized ß-D-glucan with antimicrobial peptide C8G2 through the Schiff base reaction. This hydrogel is easily accessible and actively contributes to the whole healing process of bacterial-infected wounds, demonstrating remarkable antibacterial activity and biological compatibility. The pH-sensitive reversible imine bond enables the hydrogel to self-heal and sustainably release the antibacterial peptide, thereby improving its bioavailability and reducing toxicity. Meanwhile, the immunoregulating ß-D-glucan inhibits the release of inflammatory factors while promoting the release of anti-inflammatory factors. In methicillin-resistant Staphylococcus aureus (MRSA)-infected full-thickness skin wound models, the hybrid hydrogel showed superior antibacterial and anti-inflammatory activity, enhanced the M2 macrophage polarization, expedited wound closure, and regenerated epidermis tissue. These features make this hydrogel an appealing wound dressing for treating multi-drug-resistant bacteria-infected wounds.

Glycosylation increases the anti-QS as well as anti-biofilm and anti-adhesion ability of the cyclo (L-Trp-L-Ser) against Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections pose a huge threat to cystic fibrosis patients, as well as those suffering from immunodeficiency. Antimicrobial resistance, especially multi-drug resistance, due to its ability to aggregate the compact biofilm, makes it more inefficient to treat this pathogen with traditional antibiotics. Biofilm and quorum sensing (QS) have become the alternative targets for treating P. aeruginosa infections. Previously, a cyclic dipeptide cyclo(L-Trp-L-Ser) has been identified as a QS inhibitor of P. aeruginosa. On the other hand, some monosaccharides have been proved lectin-targeting behavior and to mediate biofilm formation and adhesion of P. aeruginosa. We constructed novel cyclic dipeptide-carbohydrate conjugates as a low molecular weight dual-functional QS inhibitor, which can not only enhance its anti-QS activity but also enable good anti-biofilm and anti-adhesion ability. The IC50 of galactosylated c(WS) on biofilm formation and glass adhesion was 1/6 and 1/4 of that of the unmodified cyclic dipeptide, respectively. And the ability to eliminate the preformed biofilm was increased 10-fold. Furthermore, the carbohydrate conjugates can increase the germicidal efficiency of clinical antibiotic azithromycin when used synergistically. Our results provide a novel scaffold for developing anti-virulence adjuvants when taken with clinical antibiotics.