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Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials
Published on: April 10, 2016
Identification and antibacterial activity of a novel phage-type lysozyme from the freshwater mussel Hyriopsis
Hui Xu1, Wenjuan Dai2, Zhengyu Xiong1
1Science & Technology Normal University of Jiangxi, Nanchang, 330013, China.
Abstract:
A cDNA encoding a phage-type lysozyme, designated as HcPLYZ, was successfully cloned from Hyriopsis cumingii. The full-length cDNA sequence of HcPLYZ was determined to be 896 base pairs in length. Analysis revealed the absence of a signal peptide at its N-terminus, and identified two highly conserved phage-type lysozyme activity sites, Glu20 and Asp29, within the deduced amino acid sequence of HcPLYZ. The results of the cloning and sequencing symbiotic bacteria in tissues were consistent with those obtained using tissue cDNA as the template, suggesting that HcPLYZ may originate a symbiotic bacterium. The expression levels of HcPLYZ mRNA exhibited significant variations across different tissues. Successful expression was induced using IPTG, and the native recombinant protein was subsequently purified through affinity chromatography employing Ni2+, and the optimal pH and temperature of which were determined to be 5.5 and 50 °C, respectively. Following exposure to Aeromonas hydrophila, there was a significant increase in the levels of HcPLYZ mRNA in the hemocytes, hepatopancreas, and gills. HcPLYZ was demonstrated the inhibition activity of 55% and 83% against Micrococcus lysodeikticus under pH 5.5 and 50 °C conditions, respectively. These results suggested that HcPLYZ possessed antibacterial activity against both A. hydrophila and M. lysodeikticus.
Insights
A novel phage-type lysozyme, HcPLYZ, was identified in Hyriopsis cumingii, showing significant antibacterial activity against Aeromonas hydrophila and Micrococcus lysodeikticus. This finding highlights its potential role in the mollusk
Area of Science:
- Molecular Biology
- Biochemistry
- Immunology
Background:
- Hyriopsis cumingii is a freshwater mussel with potential immune functions.
- Lysozymes are crucial enzymes in innate immunity, involved in bacterial cell wall degradation.
- Understanding novel lysozymes can reveal new mechanisms of host defense.
Purpose of the Study:
- To clone and characterize a phage-type lysozyme (HcPLYZ) from Hyriopsis cumingii.
- To investigate the expression patterns and antibacterial activity of HcPLYZ.
- To explore the potential role of HcPLYZ in the immune response of H. cumingii.
Main Methods:
- cDNA cloning and sequencing of HcPLYZ.
- Recombinant protein expression and purification (IPTG induction, Ni2+ affinity chromatography).
- Determination of optimal pH and temperature for enzyme activity.
- Quantitative analysis of HcPLYZ mRNA expression in different tissues and after bacterial challenge.
- In vitro antibacterial assays against Micrococcus lysodeikticus and Aeromonas hydrophila.
Main Results:
- A full-length cDNA of 896 bp encoding HcPLYZ was obtained, lacking a signal peptide and containing conserved lysozyme active sites.
- HcPLYZ mRNA expression varied across tissues and significantly increased in hemocytes, hepatopancreas, and gills upon Aeromonas hydrophila exposure.
- Recombinant HcPLYZ exhibited optimal activity at pH 5.5 and 50°C, demonstrating 55% and 83% inhibition against Micrococcus lysodeikticus under these conditions.
- HcPLYZ showed antibacterial activity against both A. hydrophila and M. lysodeikticus.
Conclusions:
- HcPLYZ is a phage-type lysozyme potentially originating from symbiotic bacteria within H. cumingii.
- HcPLYZ plays a role in the innate immune defense of H. cumingii against bacterial infections.
- The characterized HcPLYZ has potential applications in aquaculture and disease control.
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