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Significance of Glutamate Racemase for the Viability and Cell Wall Integrity of Streptococcus iniae
M Muhammad1,2, J Bai1, A J Alhassan3
1College of Life Science, Hebei Normal University, Shijiazhuang, 050024, China.
Abstract:
Streptococcus iniae is a pathogenic and zoonotic bacterium responsible for human diseases and mortality of many fish species. Recently, this bacterium has demonstrated an increasing trend for antibiotics resistance, which has warranted a search for new approaches to tackle its infection. Glutamate racemase (MurI) is a ubiquitous enzyme of the peptidoglycan synthesis pathway that plays an important role in the cell wall integrity maintenance; however, the significance of this enzyme differs in different species. In this study, we knocked out the MurI gene in S. iniae in order to elucidate the role of glutamate racemase in maintaining cell wall integrity in this bacterial species. We also cloned, expressed, and purified MurI and determined its biochemical characteristics. Biochemical analysis revealed that the MurI gene in S. iniae encodes a functional enzyme with a molecular weight of 30 kDa, temperature optimum at 35°C, and pH optimum at 8.5. Metal ions, such as Cu2+, Mn2+, Co2+ and Zn2+, inhibited the enzyme activity. MurI was found to be essential for the viability and cell wall integrity of S. iniae. The optimal growth of the MurI-deficient S. iniae mutant can be achieved only by adding a high concentration of D-glutamate to the medium. Membrane permeability assay of the mutant showed an increasing extent of the cell wall damage with time upon D-glutamate starvation. Moreover, the mutant lost its virulence when incubated in fish blood. Our results demonstrated that the MurI knockout leads to the generation of S. iniae auxotroph with damaged cell walls.
Insights
Streptococcus iniae, a fish pathogen, requires glutamate racemase (MurI) for cell wall integrity. Knocking out MurI creates an essential auxotroph, offering a novel target against antibiotic-resistant strains.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Enzymology
Background:
- Streptococcus iniae is a zoonotic pathogen causing significant fish mortality and emerging antibiotic resistance.
- The enzyme glutamate racemase (MurI) is crucial for peptidoglycan synthesis and cell wall integrity in bacteria, but its specific role in S. iniae is unknown.
Purpose of the Study:
- To investigate the essentiality of glutamate racemase (MurI) for S. iniae viability and cell wall integrity.
- To characterize the biochemical properties of S. iniae MurI.
- To explore MurI as a potential therapeutic target against S. iniae infections.
Main Methods:
- Gene knockout of MurI in S. iniae.
- Cloning, expression, and purification of S. iniae MurI.
- Biochemical characterization of purified MurI, including optimal temperature, pH, and metal ion effects.
- Growth assays of MurI-deficient mutants with and without D-glutamate supplementation.
- Membrane permeability assays and virulence assessment in fish blood.
Main Results:
- S. iniae MurI encodes a functional enzyme (30 kDa) with optimal activity at 35°C and pH 8.5; inhibited by specific metal ions.
- MurI is essential for S. iniae viability and cell wall integrity.
- MurI-deficient mutants are auxotrophic for D-glutamate and exhibit increased cell wall damage upon starvation.
- The MurI knockout mutant displayed reduced virulence in a fish blood model.
Conclusions:
- The MurI enzyme is critical for maintaining cell wall integrity and viability in S. iniae.
- Targeting MurI can generate auxotrophic mutants with compromised cell walls, offering a promising strategy against antibiotic-resistant S. iniae.
