Title：Biomimetic Peptide Nanonets: Exploiting Bacterial Entrapment and Macrophage Rerousing for Combatting Infections
Journal: ACS NANO
IF: 15.8
Original link：https://doi.10.1021/acsnano.4c03669
Reporter：Menghuan Sun-25-master
In order to improve the effectiveness of peptide based antimicrobial therapy, this study proposes a molecular design strategy that combines bacterial capture peptides with immune regulatory peptides. This dual approach aims to selectively enhance the immune system, stimulate chemotaxis and phagocytosis towards bacteria. The purpose of this study is to establish an upgraded multidimensional integrated antibacterial system based on human defense peptide 6. The system has widely cross-linked nanofibers that provide dual functions of capturing and moderately killing bacteria, as well as activating immune phagocytes, stimulating phagocytic chemotaxis, and enhancing bacterial phagocytosis.
The alarming rise in global antimicrobial resistance highlights the urgent need for effective antibiotics. Antibiotic resistance (AMR) causes more deaths than HIV/AIDS or malaria every year, and the total economic cost is expected to reach 100 trillion dollars by 2050. Discovering effective antibacterial agents may be a potential way to address the challenge of bacterial resistance. Fortunately, in the innate immune system, human defense factor 6 (HD6) in the lumen undergoes self-assembly into a nanonet to capture pathogens. This process significantly reduces the invasion of pathogens into intestinal mucosal cells, distinguishing HD6 from general human antimicrobial agents with broad-spectrum antibacterial activity. In the innate immune system, when HD 6 captures bacterial pathogens in the lumen, various host defense factors, including certain antimicrobial agents and recruited phagocytic cells, collectively inhibit the bacterial pathogens. Multiple defense factors work together to construct the intestinal defense system.
Previous studies have mainly focused on the capture function of HD 6 mimetic peptides targeting only bacteria, while ignoring their interactions within the host immune system. In order to improve the effectiveness of peptide based antimicrobial therapy, this study proposes a molecular design strategy that combines bacterial capture peptides with immune regulatory peptides. This dual approach aims to selectively enhance the immune system, stimulate chemotaxis and phagocytosis towards bacteria. The purpose of this study is to establish an upgraded multidimensional integrated antibacterial system based on HD 6 peptide. The system has widely cross-linked nanofibers that provide dual functions of capturing and moderately killing bacteria, as well as activating immune phagocytes, stimulating phagocytic chemotaxis, and enhancing bacterial phagocytosis.
1.
The antibacterial system includes two key functional domains:
(i) Using the principle of peptide self-assembly, a nanofiber network is constructed by alternating hydrophilic hydrophobic residues. Subsequently, polar uncharged residues were strategically positioned between charged residues for charge separation, promoting robust fiber formation under physiological conditions.
(ii) Introduce the six residue sequence from the 18 loop region (RKVRGPP) of human lactoferrin (hLF) into the C-terminal region of a peptide based antimicrobial system. This modification is used to improve immune cell migration and phagocytosis, while reducing unwanted inflammation, ultimately enhancing the host's defense against bacterial infections.
(iii) From the structural insights of traditional antimicrobial peptides (AMPs), it can be concluded that these two regions are linked to GSGS to create a holistic system that may produce unexpected antimicrobial properties. RFQF4 exhibits excellent bacterial capture ability and moderate antibacterial activity. Transcriptomic analysis showed that RFQF4 promotes energy dissipation, biosynthesis of membrane related components, and restricts flagellar movement. Detailed in vivo evaluations indicate that the nanonet forming peptide exhibits significant antimicrobial efficacy, while showing no signs of systemic toxicity. Here, capturing and killing bacteria while inducing immune regulation undoubtedly improves the efficiency of bacterial infection treatment.
2.
Most peptides exhibit a comprehensive bacterial agglutination efficiency of over 80% against Escherichia coli. It is worth noting that RFQF4 exhibits the highest comprehensive bacterial agglutination efficiency, exceeding 90%, and shows a time - and concentration dependent relationship. RFQF4 not only intercepts and agglutinates bacteria, but also exhibits potential bactericidal properties by reducing the survival rate of captured bacteria.
3.
The structure of RFQF4 was analyzed, and at a concentration of 10 μ M, RFQF4 showed a significant increase in ANS fluorescence intensity, indicating a transition from monomer to assembled structure. The transmission electron microscope was used to observe the extensive fibrous structure of RFQF4 aggregates composed of short and thin nanofibers at twice the concentration of CAC. Scanning electron microscopy images also consistently showed the homogeneous nanofiber structure of peptides, and it was observed that the nanofiber density of RFQF4 was relatively high. The circular dichroism spectroscopy confirmed the spontaneous self-assembly of RFQF4 , revealing an unexpected tendency towards alpha helical structure. Analyze the chemical bonding state of RFQF4 through XPS and FTIR. The XPS results confirm that RFQF4 forms a multiple network of covalent crosslinking and hydrogen bonding interactions. FTIR also confirmed the existence of hydrogen bonds. These findings indicate that the nanofiber structure of RFQF4 is constructed through hydrogen bonding and π - π interactions. The Zeta potential results showed that the peptide carries a large amount of positive surface charge, which helps it to utilize the large amount of negative charge on the bacterial surface to bind and trap bacteria.
4.
Use RNA sequencing technology to analyze the potential mechanism information of RFQF4. It is worth noting that RFQF4 treatment increased the expression of 656 genes in the volcano plot and decreased the expression of 602 genes. KEGG analysis shows that 358 DEGS are enriched in 20 KEGG pathways related to key biological processes such as metabolism, genetic information processing, cellular processes, and environmental information processing. Importantly, the two-component signaling system (TCS) helps regulate various physiological processes in bacteria and is closely related to metabolism and virulence. Except for a few downstream genes regulated by metabolism, the expression of most pathogenic related genes regulated by TCS is downregulated. The effective adhesion and invasion of intestinal epithelial cells require flagella or flagellar movement, which is regulated by approximately 50 genes in Escherichia coli. After RFQF4 treatment, the expression of key genes involved in regulating flagellar dependent cell movement was significantly reduced. This effect on cell movement explains well the mechanism by which RFQF4 captures bacteria and inhibits bacterial invasion.
5.
Inject RFQF4 nanofibers into mice to evaluate their in vivo biosafety. Biochemical assessment of liver toxicity includes measuring serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST), and evaluating renal toxicity using serum creatinine (CRAE) and blood urea nitrogen (BUN). No increase in these five parameters was observed to ensure internal balance and normal physiological state. Due to the significant barrier effect of liver and kidney injury on drug delivery in the body, histopathological examination was performed on the liver and kidneys. Compared with the control group, there were no pathological changes observed in the liver and kidneys treated with RFQF4 nanofibers of different concentrations, and there was no significant difference in organ indices (liver, spleen, lungs, and kidneys) between the injection group and the control group. These results indicate that RFQF4 nanofibers have significant biocompatibility and potential application prospects. Next, the therapeutic efficacy of RFQF4 was further explored in a mouse model of peritonitis sepsis. The staining results showed significant organ lesions in the sepsis group. In contrast, the RFQF4 treatment group showed observable reductions or even non-existent symptoms. The RFQF4 treatment group had relatively normal organ structures. Overall, the reduction in bacterial load and alleviation of organ lesions in systemic bacterial infections indicate that RFQF4 has great potential in the treatment of sepsis and bacterial infections.
Taking inspiration from the bacterial capture mechanism of human defense factor 6, we have developed a biomimetic peptide nanonet composed of multiple functional fragments for the eradication of bacteria. These biomimetic peptide nanonets aim to address the challenge of antibiotic resistance through a dual approach strategy. Firstly, the resulting nanofiber network captures bacteria, and then kills them by loosening the membrane structure, dissipating proton dynamics, and causing various metabolic disturbances. Secondly, these trapped bacterial clusters interact with ECM receptors through the PI3K-AKT signaling pathway, reactivating macrophages through enhanced chemotaxis and phagocytosis to clear bacteria. In vivo results have shown that treatment with biomimetic peptide nanonets can alleviate systemic bacterial infections without causing significant systemic toxicity. As expected, the proposed strategy can address stubborn infections by capturing bacteria and awakening antibacterial immune responses. This method can serve as a guide for the design of biologically inspired materials for future clinical applications.