Structural and Functional Analysis of Urease Accessory Protein E from Vancomycin-Resistance Staphylococcus aureus
Humaira Siddiqui1, Atia-Tul-Wahab1, Aftab Ahmed2
1Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan.
Background:
An increasing prevalence of biofilm forming strains by vancomycinresistance Staphylococcus aureus (VRSA) is one of the most important causes of antimicrobial resistance. VRSA possesses various regulatory factors to form and sustain biofilm in biotic or abiotic conditions. Among them, ureolytic activity is an important factor in the stabilization of biofilms by neutralizing the acidic environment. Various urease accessory proteins are required to activate the urease enzyme inside the biofilm.
Objective:
To optimize the cloning, expression and purification of urease accessory protein E from VRSA for determination of the secondary structure, and functional characterization by using Berthelot's method.
Methods:
BAB58453.1 gene (which encodes possible urease accessory protein E), having 38% similarity to Bacillus pasteurii UreE protein, was cloned, expressed, and purified by single-step affinity chromatography for performing secondary structural studies using circular dichroism spectroscopy, and functional analysis using Berthelot's and crystal violet assay.
Results:
Structure elucidation using NMR and circular dichroism spectroscopy techniques revealed that UreE protein has a partially foldedα-helical structure. Using Berthelot's method, it was identified that the purified UreE protein has enhanced urease enzyme activity, in comparison to the control. From the results of Berthelot's and crystal violet assays, it was deduced that the selected gene (UreE protein) plays a key role in enhancing urease enzyme activity and contributes to biofilm stability.
Conclusion:
Structural studies on VRSA urease accessory proteins could aid in the identification of new drug targets or the development of effective antibiofilm strategies (in combination with other drug targets) against infections caused by biofilm-producing strains.
Insights
Vancomycin-resistant Staphylococcus aureus (VRSA) urease accessory protein E was cloned, expressed, and purified. This protein enhances urease activity and contributes to biofilm stability, offering potential new drug targets.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Rising vancomycin-resistant Staphylococcus aureus (VRSA) biofilm strains are a major antimicrobial resistance concern.
- Ureolytic activity is crucial for VRSA biofilm stabilization by neutralizing acidic environments.
- Urease accessory proteins are essential for activating urease enzymes within biofilms.
Purpose of the Study:
- Optimize cloning, expression, and purification of VRSA urease accessory protein E.
- Determine the secondary structure of VRSA UreE.
- Functionally characterize VRSA UreE using Berthelot's method.
Main Methods:
- Cloned and expressed the BAB58453.1 gene encoding UreE.
- Purified UreE using single-step affinity chromatography.
- Analyzed secondary structure via circular dichroism and NMR; assessed function using Berthelot's and crystal violet assays.
Main Results:
- UreE protein exhibits a partially folded alpha-helical structure.
- Purified UreE significantly enhanced urease enzyme activity compared to controls.
- UreE plays a key role in boosting urease activity and biofilm stability.
Conclusions:
- Structural insights into VRSA urease accessory proteins can identify novel drug targets.
- These findings may lead to effective antibiofilm strategies against VRSA infections.
- Targeting urease accessory proteins offers a promising avenue for combating biofilm-related antimicrobial resistance.
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