Characterization of the sulfate capture/assimilation pathway in Mycobacterium tuberculosis: strategies for the development of inhibitors and potential vaccinal and diagnosis targets

Author(s)

Andreia Navarro Cerone

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-07-10

Examining board members

Andrea Balan; Ana Carolina de Mattos Zeri; Adriana Santos Soprano; Carla Cristina Polo; Juliana Helena Costa Smetana

• Advisor: Andrea Balan

Abstract

Tuberculosis is a disease caused by Mycobacterium tuberculosis and it is considered a serious worldwide problem due to its high incidence in the population, peculiar characteristics of the pathogen and treatment of difficult success. It is estimated that currently 2 billion people are infected, a figure that covers about 30% of the global population. The poli-therapy used against Koch's bacillus is increasingly ineffective due to the proliferation of multi-resistant strains. The present project aims to identify new targets for the development of drugs that are more effective in blocking this pathogen. Considering that ABC transport systems (ATP-Binding Cassette) type importers plays a fundamental role in the uptake and transport of several substances essential to the survival of organisms, which are poorly characterized in Mycobacterium tuberculosis, the elucidation of structures and molecular models of action focusing in a possible inhibition of the viability of the microorganism, become an alternative as a way to obtain new targets. In this scenario, the targets chosen were the ABC system involved in sulfate uptake (SubICysWTA1) and its assimilation pathway consisting of 8 proteins (CysHDNK1K2MA2NirA). Sulfate is involved in sulfur metabolism, fundamental to bacterium and whose importance for infection and pathogenesis has been functionally demonstrated. In developing the design, two proteins became major targets: SubI and CysA2. SubI was expressed and purified and had its three-dimensional structure resolved in the presence of the anion as well as the biophysics of the sulfate interaction. Additionally, using thermalshift, fluorescence, thermophoresis and nuclear magnetic resonance techniques, small molecules capable of interacting with the protein were identified for their inhibition. Docking experiments were used to characterize the location of the interactions. CysA2 previously described as sulfurtransferase, acts secondary in the oxide-reducing pathway of sulfate in the cellular cytoplasm. After the kinetic characterization of the protein, its ability to use previously unknown thiosulphate and 2-mercaptopyruvate substrates was demonstrated. Additionally, the protein was evaluated for immunogenic and vaccine potential. CysA2 has been shown to be a highly immunogenic protein and an excellent vaccinal and diagnostic target for the treatment of the disease (AU)

• FAPESP's process: 14/20921-6 - Identification and development of new molecules against Mycobacterium tuberculosis based inhibition of protein metabolism of sulfur -Based methodology using fragments
• Grantee: Andreia Navarro Cerone
• Support Opportunities: Scholarships in Brazil - Doctorate