Nano-encapsulated HHC10 host defense peptide (HDP) reduces the growth of Escherichia coli via multimodal mechanisms

Abstract

The eradication of several pathogenic drug resistant "Superbug" such as Escherichia coli became difficult especially in chronic infections using existing antibiotics due to the emergence of antibiotic resistance. Owing to their unique antibacterial properties, host defense peptides (HDP) have gained significant attention to combat colonization of bacteria. This study aims designing delivery systems for HHC10 peptide to target bacteria inside the cells might be a promising approach by protecting from degradation, controlling the release, enhancing the susceptibility of target microbes and improving bioavailability. Nano-formulated HHC10 was evaluated for its efficacy (CFU assay) and possible mechanism of action (membrane interaction and apoptosis) against E. coli. Dose-dependent inhibition of E. coli growth is observed for nano-encapsulated and bare HHC10 and encapsulated form remain non-toxic to macrophage mouse cells (RAW264.6) up to 20 μM. Mechanistic analyses using transmission electron microscopy and flow cytometry techniques revealed that bactericidal activity of HHC10-NP progresses via a multimodal mechanism of bacterial cell death by cell-membrane lysis on direct interaction with bacteria while through induction of the apoptotic death pathway inside the host cells. These results offer an insight on future strategies for the development and application of antimicrobial peptides as antibacterial alternatives. [Formula: see text] Controlled delivery of HHC10 peptide from PLGA-NP kills bacteria by two different mechanism: (i) direct killing: HHC10 disintegrate the cell membrane of bacteria by electrostatic interactions and (ii) indirect killing: induction of apoptosis in bacteria infect cells.

Defensin–lipid interactions in membrane targeting: mechanisms of action and opportunities for the development of antimicrobial and anticancer therapeutics

“…Currently, in vivo studies typically utilise a subcutaneous injection to deliver defensins [ 20 , 98 ] which is less desirable clinically when compared with oral delivery mechanisms [ 102 ]. A novel delivery mechanism is desirable for defensins to address systemic delivery concerns as well as reduce potential toxicities, allowing the defensins to only be exposed to the host cellular environment upon arrival at the target site [ 103 ]. Such a delivery system may aid in increasing the bioavailability of defensins, thus making them an incredibly attractive area of research for the defensin field [ 103 ].…”

Bacterial Aggregation Triggered by Fibril Forming Tryptophan-Rich Sequences: Effects of Peptide Side Chain and Membrane Phospholipids

“…HHC-10 (Lys-Arg-Trp-Trp-Lys-Trp-Ile-Arg-Trp-NH 2 ) and its Har-rich analog ( 4Har HHC-10; Har-Har-Trp-Trp-Har-Trp-Ile-Har-Trp-NH 2 ) are Trp-rich synthetic AMPs with selectivity and excellent activities against various bacterial superbugs in vitro and in vivo . − While conserving the activity against bacteria, the presence of Har residue in 4Har HHC-10 may overcome the associated shortcomings with the peptide proteolytic instability. , The short length of these HHC sequences and the presence of multiple charged and Trp residues is presumably responsible for their strong activity . In this regard, Sharma et al suggested membrane disruption as the direct antimicrobial mechanism of HHC-10 activity against E. coli . However, the detailed contributions of peptide concentration, oligomerization state, and morphological characteristics of self-assembled nanostructures of HHC-10, as well as the peptide sensing of the intrinsic curvature of bacterial cytoplasmic phospholipids especially in the context of induction of cell aggregation, have yet to be considered.…”

“…26 In this regard, Sharma et al suggested membrane disruption as the direct antimicrobial mechanism of HHC-10 activity against E. coli. 27 However, the detailed contributions of peptide concentration, oligomerization state, and morphological characteristics of self-assembled nanostructures of HHC-10, as well as the peptide sensing of the intrinsic curvature of bacterial cytoplasmic phospholipids especially in the context of induction of cell aggregation, have yet to be considered.…”

Current Advances in Lipid and Polymeric Antimicrobial Peptide Delivery Systems and Coatings for the Prevention and Treatment of Bacterial Infections

“…Furthermore, Gomez et al showed the encapsulation of synthetic AMPs, namely GIBIM-P5S9K (G17) and GAM019 (G19), in PLGA resulted in a 3-fold and 1-fold decrease of the minimum inhibitory concentration to inhibit 50% of bacterial growth (MIC 50 ) of MRSA for G17NPs and G19NPs, respectively; and the MIC 50 of E. coli showed a 2-fold decrease for both formulations 8 h post-treatment [93]. Additionally, Sharma et al demonstrated enhanced in vitro killing of E. coli with PLGA-encapsulated HHC10 in a concentration-dependent manner and higher cellular uptake in mouse macrophages compared with a peptide solution 12 h post-treatment [94]. The bactericidal activity of PLGA-encapsulated HHC10 occurred via multimodal interactions with bacteria, which involved cell-membrane lysis on direct interaction with bacteria, and activation of apoptotic death pathway (cathepsin-B) upon internalization in E. coli-infected cells in vitro.…”