Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast  Pichia pastoris  system

Zhao, Hua; Tang, Jieyuan; Cao, Lei; Jia, Gang; Long, Dingbiao; Liu, Guangmang; Chen, Xiaoling; Cai, Jingyi; Shang, Haiying

doi:10.1016/j.enzmictec.2015.06.001

Cited by 18 publications

(14 citation statements)

References 48 publications

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“…This hypothesis could explain that lower concentrations of BP100 combined with lysozyme presented similar activity than BP100 alone four times concentrated. These results are in agreement with the report that antimicrobial activity of peptide parasin I was enhanced by lysozyme [ 57 ]. The authors suggest that lysozyme could allow the access of parasin I to the cytoplasmic membrane thanks to the lysis of peptidoglycan.…”

Section: Discussionsupporting

confidence: 94%

Lysozyme enhances the bactericidal effect of BP100 peptide against Erwinia amylovora, the causal agent of fire blight of rosaceous plants

Cabrefiga

Seguí

2017

BMC Microbiol

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BackgroundFire blight is an important disease affecting rosaceous plants. The causal agent is the bacteria Erwinia amylovora which is poorly controlled with the use of conventional bactericides and biopesticides. Antimicrobial peptides (AMPs) have been proposed as a new compounds suitable for plant disease control. BP100, a synthetic linear undecapeptide (KKLFKKILKYL-NH2), has been reported to be effective against E. amylovora infections. Moreover, BP100 showed bacteriolytic activity, moderate susceptibility to protease degradation and low toxicity. However, the peptide concentration required for an effective control of infections in planta is too high due to some inactivation by tissue components. This is a limitation beause of the high cost of synthesis of this compound. We expected that the combination of BP100 with lysozyme may produce a synergistic effect, enhancing its activity and reducing the effective concentration needed for fire blight control.ResultsThe combination of a synhetic multifunctional undecapeptide (BP100) with lysozyme produces a synergistic effect. We showed a significant increase of the antimicrobial activity against E. amylovora that was associated to the increase of cell membrane damage and to the reduction of cell metabolism. Combination of BP100 with lysozyme reduced the time required to achieve cell death and the minimal inhibitory concentration (MIC), and increased the activity of BP100 in the presence of leaf extracts even when the peptide was applied at low doses. The results obtained in vitro were confirmed in leaf infection bioassays.ConclusionsThe combination of BP100 with lysozyme showed synergism on the bactericidal activity against E. amylovora and provide the basis for developing better formulations of antibacterial peptides for plant protection.

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Section: Discussionsupporting

confidence: 94%

Lysozyme enhances the bactericidal effect of BP100 peptide against Erwinia amylovora, the causal agent of fire blight of rosaceous plants

Cabrefiga

Seguí

2017

BMC Microbiol

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“…This can be achieved by selecting species with relatively simple genomes, such as bacteria and fungi, and employing restriction nucleases or ultrasonic disruption to generate a large number of DNA fragments. These fragments can be subsequently incorporated into microbial expression vectors and screened using microbial expression systems, such as the yeast expression system ( 60 , 61 ) and the B. subtilis expression system ( 62 ). Second, we recommend the integration of bioinformatics tools and genome databases like Ensembl to predict regions within fragments that may possess antimicrobial properties.…”

Section: Discussionmentioning

confidence: 99%

BsR1, a broad-spectrum antibacterial peptide with potential for plant protection

Song,

Zhao,

Zhu

et al. 2023

Microbiol Spectr

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The urgent need for new antibacterial drugs arises from the emergence of bacterial multidrug resistance due to antibiotic overuse. Antimicrobial peptides, crucial components of innate immunity in animals and plants, exhibit effectiveness against multidrug-resistant bacteria while minimizing the development of drug resistance. In this study, the antibacterial peptide BsR1, comprising 21 amino acids, was finalized by predicting the active site of an antifungal gene and sequence optimization. BsR1 displayed broad-spectrum antibacterial activities against diverse Gram-positive and Gram-negative bacteria, with a low minimum inhibitory concentration of 4.25–17 μM. Stability experiments demonstrated that BsR1 has high resistance to thermal, ultraviolet, and acid-base conditions, while revealing increased sensitivity to divalent ions Ca 2+ and Mg 2+ . The mode of action of BsR1 involved cell membrane damage, leading to bacterial cell structure disruption and subsequent death. Secondary structure prediction indicated a linear helical conformation with a positive charge of +7.5, facilitating its interaction with the target cell membrane. BsR1 exhibited excellent biological safety, as it did not induce necrosis in tobacco leaves, and the low observed hemolytic effect on mammalian cells with a value of 3.26%. Additionally, BsR1 demonstrated the ability to enhance disease resistance in rice and effectively curbed the spread of rice bacterial blight. This research presents BsR1 as a novel approach and potential medication in the development of antibacterial drugs, offering a valuable tool in combating pathogenic microorganisms, particularly in plants. IMPORTANCE This study addresses the critical need for new antibacterial drugs in the face of bacterial multidrug resistance resulting from antibiotic overuse. It highlights the significance of antimicrobial peptides as essential components of innate immunity in animals and plants, which have been proven effective against multidrug-resistant bacteria and are difficult to develop resistance against. This study successfully synthesizes a broad-spectrum antibacterial peptide, BsR1, with strong inhibitory activities against various Gram-positive and Gram-negative bacteria. BsR1 demonstrates favorable stability and a mode of action that damages bacterial cell membranes, leading to cell death. It also exhibits biological safety and shows potential in enhancing disease resistance in rice. This research offers a novel approach and potential medication for antibacterial drug development, presenting a valuable tool in combating pathogenic microorganisms, particularly in plants.

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“…He et al (2015) fused cathelicidin from Bungarus fasciatus to chitin binding domain and expressed in a Bacillus subtilis secretory expression system; the fusion peptide was purified by affinity chromatography and intein self-cleavage; about 0.5 mg peptide was obtained from 1 L culture medium; the purified peptide showed antimicrobial activity similar to the synthetic cathelicidin. Zhao H et al (2015) expressed the AMP parasin I (PI) fused with human lysozyme (hLY) in P. pastoris; PI released after the enterokinase hydrolysis exhibited antimicrobial activities against B. subtilis, Staphylococcus aureus and E. coli. In our paper, phytase-lactoferricin fusion gene was expressed in rice endosperm specifically, which had the double benefits: while the fusion protein broke down the phytate in rice seeds resulting in high Pi contents, its intestinal digestion should set free LfcinB that could play a role in bacteriostatic, immune-modulatory and growth promotion, and release the phytase to improve P availability from phytate in feed further on.…”

Section: Discussionmentioning

confidence: 99%

Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity

Wang

Deng

LuShui

et al. 2017

Journal of Integrative Agriculture

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The developing trends of livestock production are efficiency, safety and sustainability, which face two major challenges: low availability of phytate phosphorus and abuse of antibiotics. As a solution phytases and antimicrobial peptides are applied as feed additives. However, phytases and antimicrobial peptides are susceptible to proteases, costly by fermentation and potential toxic to production hosts. We transformed an optimized phytase-lactoferricin fusion gene PhyLf driven by an endosperm-specific promoter Gt13aP and Bar (bialaphos resistance) gene as a selection maker into rice. The Bar and PhyLf genes were integrated into the rice genome, stably inherited and expressed. Their phosphinothricin acetyl transferase (PAT) protein content of transgenic plants with glufosinate resistance varied between 50.45-93.39 μg g-1. Fusion protein expressed especially in the seeds of transgenic rice had a summit phytase activity at 32.30 U g-1 , which increased by 61.71-fold compared to the control/check group (CK) and 7.54-fold compared to un-optimized transgenic plant. The highest inorganic phosphorus (Pi) content of the transgenic seeds reached 13.15 mg g-1 , increased by 12.77-fold compared to that of CK. Preliminary antibacterial experiments showed that the enterokinase hydrolysate product of fusion protein could inhibit the growth of Escherichia coli DH5α. These results indicated that the protein PhyLf has the potential to increase availability of feed phytate phosphorus, improve consumer's immunity and reduce the use of antibiotics.

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Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system

Cited by 18 publications

References 48 publications

Lysozyme enhances the bactericidal effect of BP100 peptide against Erwinia amylovora, the causal agent of fire blight of rosaceous plants

Lysozyme enhances the bactericidal effect of BP100 peptide against Erwinia amylovora, the causal agent of fire blight of rosaceous plants

BsR1, a broad-spectrum antibacterial peptide with potential for plant protection

Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity

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