Enterococcus faecalis: Cluster epa

Author(s)

Lívia Oliveira Dantas Clementino

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2023-05-19

Examining board members

Alessandro Silva Nascimento; Kate Cristina Blanco; Beatriz Gomes Guimarães; Marco Antonio Seiki Kadowaki; Mario de Oliveira Neto

• Advisor: Alessandro Silva Nascimento

Abstract

Bacterial resistance to antibiotics is a public health problem, with a tendency to worsen over time. The consequence of this is reflected in the amount of deaths caused by such microorganisms, estimated at 7.7 million in 2019, a number with tendency to increase. In this context, the study of four enzymes present in the epa gene cluster began. Epa proteins synthesize and export constituent carbohydrates of the cell wall of Enterococcus, also involved in the composition of biofilms. The enzymes EpaI, EpaOX, EpaB and EpaE of Enterococcus faecalis DSM20478 were cloned, expressed and purified. Despite different strategies applied to improve the expression and purification of these proteins, EpaE was the only one to present reasonable yield and stability to be submitted to a screening involving multiple crystallization conditions. A dataset was collected on the Manacá line of the Brazilian synchrotron ring, Sirius. The space group of the crystal is P21, presenting twinning pseudomerohedral fraction. The refined EpaE structure was determined at resolution of 2.85 Å. It has a tetrameric conformation in the asymmetric unit, with a Rossman-type fold. Analyzes carried out by the PDBePISA server revealed that the interfaces formed by the chains A and B, and C and D are more stable than those formed by chains B and C, and A and D, suggesting that it is a dimer of dimers. The catalytic site of EpaE was identified from the structural alignment with another RmlA of higher resolution. During the refinement of EpaE, was noticed the presence of an electron density corresponding to the thymidine molecule, present in the probable allosteric site of EpaE. The established protocol for measuring RmlAs activity was applied, it detects phosphate in solution using the malachite green reagent. However, it was observed that the malachite green reagent is not a good colorimetric solution because its absorbance curves intensity arent proportional to the phosphate concentration in the medium. To solve this problem, the molybdenum blue phosphate detection technique was tested to measure the specific activity of EpaE. Finally, the AlphaFold2 program predicted the structures of the other enzymes present in the cluster epa, enabling the elucidation of some of these enzymes functions in the synthesis and transport of the enterococcal polysaccharide antigen of E. faecalis. In this thesis, we determined the crystal structure of the EpaE enzyme, the first crystal structure of a member of the epa cluster, and also made advances in the identification of a potential inhibitor of this enzyme. (AU)

• FAPESP's process: 17/24901-8 - Structural studies for drug discovery against human infectious diseases
• Grantee: Lívia Oliveira Dantas Clementino
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)