[논문]Anti-Endotoxin 9-Meric Peptide with Therapeutic Potential for the Treatment of Endotoxemia

Krishnan, Manigandan; Choi, Joonhyeok; Choi, Sungjae; Kim, Yangmee

doi:10.4014/jmb.2011.11011

Anti-Endotoxin 9-Meric Peptide with Therapeutic Potential for the Treatment of Endotoxemia 원문보기

Journal of microbiology and biotechnology, v.31 no.1, 2021년, pp.25 - 32

Krishnan, Manigandan (Department of Bioscience and Biotechnology, Konkuk University) , Choi, Joonhyeok (Department of Bioscience and Biotechnology, Konkuk University) , Choi, Sungjae (Department of Bioscience and Biotechnology, Konkuk University) , Kim, Yangmee (Department of Bioscience and Biotechnology, Konkuk University)

Abstract ▼ AI-Helper

Inflammatory reactions activated by lipopolysaccharide (LPS) of gram-negative bacteria can lead to severe septic shock. With the recent emergence of multidrug-resistant gram-negative bacteria and a lack of efficient ways to treat resulting infections, there is a need to develop novel anti-endotoxin agents. Antimicrobial peptides have been noticed as potential therapeutic molecules for bacterial infection and as candidates for new antibiotic drugs. We previously designed the 9-meric antimicrobial peptide Pro9-3 and it showed high antimicrobial activity against gram-negative bacteria. Here, to further examine its potency as an anti-endotoxin agent, we examined the anti-endotoxin activities of Pro9-3 and elucidated its mechanism of action. We performed a dye-leakage experiment and BODIPY-TR cadaverine and limulus amebocyte lysate assays for Pro9-3 as well as its lysine-substituted analogue and their enantiomers. The results confirmed that Pro9-3 targets the bacterial membrane and the arginine residues play key roles in its antimicrobial activity. Pro9-3 showed excellent LPS-neutralizing activity and LPS-binding properties, which were superior to those of other peptides. Saturation transfer difference-nuclear magnetic resonance experiments to explore the interaction between LPS and Pro9-3 revealed that Trp3 and Tlr7 in Pro9-3 are critical for attracting Pro9-3 to the LPS in the gram-negative bacterial membrane. Moreover, the anti-septic effect of Pro9-3 in vivo was investigated using an LPS-induced endotoxemia mouse model, demonstrating its dual activities: antibacterial activity against gram-negative bacteria and immunosuppressive effect preventing LPS-induced endotoxemia. Collectively, these results confirmed the therapeutic potential of Pro9-3 against infection of gram-negative bacteria.

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p. injection of Pro9-3 (1 ㎎/㎏) for 1h followed by LPS stimulation for 16 h. The blood and lung tissues were collected and processed for analysis as described previously [25, 26]. Briefly, serum endotoxin levels were quantified according to the kit protocol using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific).

제안 방법

exerted strong antibacterial activity against gram-negative bacteria [19]. Here, we examined the antibacterial activities and cytotoxicity of Pro9-3, its Lys-substituted analog, and their enantiomers. The results confirmed that cationicity due to the Arg residues in Pro9-3 provides an effective electrostatic interaction with the membrane to cause critical damage to the bacteria, resulting in high antibacterial activity.
LPS was prepared as described previously [22]. STD-NMR experiments were then performed to assess the peptide-LPS interaction using a Bruker Avance 900 spectrometer (Bruker Corporation, USA) at Korea Basic Science Institute (Korea). The STD-NMR experiments were performed with saturation of LPS resonances at -2.
STD-NMR experiments were then performed to assess the peptide-LPS interaction using a Bruker Avance 900 spectrometer (Bruker Corporation, USA) at Korea Basic Science Institute (Korea). The STD-NMR experiments were performed with saturation of LPS resonances at -2.0 ppm selectively and 40 ppm for reference spectra, 45-ms Gaussian-shaped pulses with a 100-ms delay between each pulse, and a total saturation time of 2s. Subtraction of the on-resonance from the off-resonance spectra provided the difference spectrum.
The antimicrobial activities of Pro9-3, Pro9-3D, Pro9-3-K and Pro9-3D-K against gram-negative bacteria and MDR gram-negative bacteria were tested to determine their potency (Table 2). Pro9-3 and Pro9-3D showed much higher antibacterial activity than Pro9-3K and Pro9-3D-K, suggesting that Lys substitution in Pro9-3 for Arg lowered the antibacterial activity.
We then analyzed whether Pro9-3 can remove LPS endotoxin in vivo by using an LAL assay to measure the amount of endotoxin in the serum (Fig. 4D). Pro9-3 reduced the level of endotoxin by 43.

대상 데이터

(Korea), respectively. The MDR gram-negative strains Escherichia coli CCARM 1229, Escherichia coli CCARM 1238, Acinetobacter baumannii CCARM 12010, and Acinetobacter baumannii CCARM 12220 were obtained from the Culture Collection of Antibiotic-Resistant Microbes at Seoul Women’s University (South Korea).

데이터처리

Nonlinear regression curve fit was employed for RBC lysis, calcein dye leakage and BC-displacement analysis. Statistical significance (p < 0.05) was determined using one-way ANOVA with Tukey's test.
0 for windows (GraphPad Software, USA). The experiments were performed in triplicate and the values are expressed as mean ± SEM. Nonlinear regression curve fit was employed for RBC lysis, calcein dye leakage and BC-displacement analysis.

성능/효과

94). Furthermore, the in vitro cytotoxicity (Fig. 1B) of peptides against RAW264.7 macrophage cells revealed that Pro9-3 and Pro9-3K resulted in a 100% survival rate, whereas the control peptide melittin showed only 13.7% survival of the macrophages when provided at a concentration of 100 μM. By contrast, Pro9- 3D and Pro9-3D-K exhibited significant toxic effects with 40% and 71% survival, respectively, even at 50 μM.
Here, we examined the antibacterial activities and cytotoxicity of Pro9-3, its Lys-substituted analog, and their enantiomers. The results confirmed that cationicity due to the Arg residues in Pro9-3 provides an effective electrostatic interaction with the membrane to cause critical damage to the bacteria, resulting in high antibacterial activity. Since Pro9-3 can neutralize LPS and is a potent anti-endotoxic peptide, we then examined its potential as a therapeutic molecule to treat sepsis in vivo using an endotoxemia mouse model.

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