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. 2024 Nov 5;12(11):e0026524.
doi: 10.1128/spectrum.00265-24. Epub 2024 Oct 9.

Antimicrobial activity of novel symmetrical antimicrobial peptides centered on a hydrophilic motif against resistant clinical isolates: in vitro and in vivo analyses

Chaoqun Zhang #  1 , Le Fu #  1 , Yuan Zhu #  1 , Qigui Chen  2 , Zetong Chen  2 , Yung-Fu Chang  3 , Yide Li  1 , Mengjing Yao  1 , Xinyi Huang  1 , Li Jin  2 , Xue Gao  2 , Yiyu Zhang  1 , Biao Jin  1 , Shuli Chou  1 , Liang Luo  1
Affiliations

Antimicrobial activity of novel symmetrical antimicrobial peptides centered on a hydrophilic motif against resistant clinical isolates: in vitro and in vivo analyses

Chaoqun Zhang et al. Microbiol Spectr. .

Abstract

Antibiotic resistance poses a significant public health threat worldwide. The rise in antibiotic resistance and the sharp decline in effective antibiotics necessitate the development of innovative antibacterial agents. Based on the central symmetric structure of glycine-serine-glycine, combined with tryptophan and arginine, we designed a range of antimicrobial peptides (AMPs) that exhibited broad-spectrum antibacterial activity. Notably, AMP W5 demonstrated a rapid and effective sterilization against methicillin-resistant Staphylococcus aureus (MRSA), displaying both a minimum inhibitory concentration and a minimum bactericidal concentration of 8 µM. Mechanistic studies revealed that AMP W5 killed bacterial cells by disrupting the cytoplasmic membrane integrity, triggering leakage of cell contents. AMP W5 also exhibited excellent biocompatibility in both in vitro and in vivo safety evaluations. AMP W5 treatment significantly reduced skin bacterial load in our murine skin infection model. In conclusion, we designed a novel centrosymmetric AMP representing a promising medical alternative to conventional antibiotics for treating MRSA infections.

Importance: Increasing antibiotic resistance and the paucity of effective antibiotics necessitate innovative antibacterial agents. Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing bacterial infections with high incidence and mortality rates, showing increasing resistance to clinical drugs. Antimicrobial peptides (AMPs) exhibit significant potential as alternatives to traditional antibiotics. This study designed a novel series of AMPs, characterized by a glycine-serine-glycine-centered symmetrical structure, and our results indicated that AMP W5 exhibited a rapid and effective bactericidal effect against MRSA. AMP W5 also demonstrated excellent biocompatibility and a bactericidal mechanism that disrupted membrane integrity, leading to leakage of cellular contents. The notable reduction in skin bacterial load observed in mouse models reinforced the clinical applicability of AMP W5. This study provides a promising solution for addressing the increasing threat of antibiotic-resistant bacteria and heralds new prospects for clinical applications.

Keywords: MRSA; antibiotic resistance; antimicrobial peptides; symmetric sequence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Characteristics of novel designed peptides: (a) scheme chemical structural formula; (b) three-dimensional structure projections.
Fig 2
Fig 2
The MIC of the peptides.
Fig 3
Fig 3
Time-kill curves against MRSA: (a) AMP W5; (b) vancomycin.
Fig 4
Fig 4
Hemolytic analysis of peptides. Positive control: Triton X-100. Hemolysis (%) = [(Absorbance of sample − Absorbance of PBS) / (Absorbance of Triton − Absorbance of PBS)] ×100. Some negative values, possibly due to experimental variability, and also observed in other studies (41, 45). Statistical significance: *P < 0.05; **P < 0.01; ****P < 0.0001.
Fig 5
Fig 5
Cytoplasmic membrane depolarization study of AMP W5 against MRSA: (a) Comparison of AMP W5 at various concentrations versus the control group; (b) Significance analysis of AMP W5 at a concentration of 1/2 × MIC compared to the control group. Control, no peptides. Statistical significance: ns, non-significant, ****P < 0.0001.
Fig 6
Fig 6
Flow cytometric analysis. MRSA cells were exposed to various concentrations of AMP W5 for 30 minutes. The increment of the logarithmic fluorescence signal represents the PI uptake generated by peptide processing. The experimental groups included the following: (a) Absence of peptide, negative control; (b) AMP W5 at a concentration of 1/2 × MIC (4 µM); (c) AMP W5 at a concentration of 1 × MIC (8 µM); and (d) AMP W5 at a concentration of 2 × MIC (16 µM).
Fig 7
Fig 7
SEM micrographs of MRSA treated with AMP W5. SEM micrographs of MRSA: (a AND b) Control, no peptides; (c AND d) AMP W5 treated at a concentration of 1 × MIC (8 µM) for 30 minutes. Scale bar = 1 µm (a, c) or 200 nm (b, d).
Fig 8
Fig 8
TEM micrographs of MRSA treated with AMP W5. TEM micrographs of MRSA: (a and b) Control, no peptides; (c and d) AMP W5 treated at a concentration of 1 × MIC (8 µM) for 30 minutes. Scale bar = 2 µm (a, c) or 200 nm (b, d).
Fig 9
Fig 9
(a) Protocol for intravenously injected peptide W5 to determine in vivo safety. (b) Weight changes of mice from day 0 to day 5. (c) Serum indicator levels of liver and kidney function. (d) HE staining of liver and kidney tissues. ns: non-significant. Scale bar = 200 µm.
Fig 10
Fig 10
(a) A skin infection model protocol to assess the in vivo effectiveness of AMP W5. (b) Observation of the wound area in mice on day 10. (c) Evaluation of the wound area in mice on days 1, 3, 5, and 7. (d) The bacterial load in vivo. (e) Evaluation of proinflammatory cytokines in mice. (f) HE staining of mice’s skin and soft tissue. Scale bar = 100 µm. (C, the untreated group of mice without MRSA-infection; M, the MRSA-infected group; W5, the peptide-treated group.) Statistical significance: ns, non-significant, *P < 0.05, **P < 0.01.
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