STUDIES OF ANTIOXIDANT ENZYME PEROXIREDOXINS FROM CANDIDATUS LIBERIBACTER ASIATICUS AND ITS HOST CITRUS SINENSIS

Abstract

Citrus greening or huanglongbing (HLB), one of the most devastating diseases of citrus worldwide, is caused by a phloem-limited unculturable Gram-negative fastidious α-proteobacteria. It is classified on the basis of its geographical origin and 16S rDNA sequence into three species, Candidatus Liberibacter asiaticus (Las), Candidatus Liberibacter africanus (Laf), and Candidatus Liberibacter americanus (Lam). Among three species, Candidatus Liberibacter asiaticus is the most virulent strain and is transmitted by Asian citrus psyllid (Diaphorina citri Kuwayama). An early symptom of HLB in citrus is blotchy mottling with green islands on leaves. As the disease progresses, infected shoots are undersized and the branches slowly die. The fruit from infected trees may be small, lopsided, dark aborted seeds, with deprived coloration, and tends to drop prematurely. Till date, no effective control strategies have been developed for the management of HLB disease and to stop it from spreading to disease-free new citrus-production areas. The current disease control strategies include minimizing the psyllid population chemically and biologically and preventing trees from becoming infected by removing the infected tree. However, targeting some of the crucial proteins which are essential for the survival of the bacterium holds a promising strategy to combat the bacterium. Proteins are the most versatile biological macromolecules found in living systems, performing a wide range of activities in almost every biological process. They serve as antioxidant defense systems, catalysts, mechanical support, immunological defense, nerve transmission, storage of other molecules like oxygen, and regulation of growth and differentiation processes. Antioxidant defense systems are known to reduce oxidative stress in living organisms. The oxidative stresses can damage and alters the functions of the membranes and biomolecules like proteins, nucleic acids, carbohydrates, lipids, etc. Peroxiredoxins are important antioxidant enzymes that play a significant role in ROS scavenging and reduction of the peroxides level in bacteria. Prxs are highly conserved, thiol-based antioxidant enzyme that belongs to the peroxidase family and is found in almost all organisms from archaebacteria to mammals. Depending on the number of cysteine residues present on the active site, enzymes are categorized as 1-Cys and 2-Cys Prxs. The catalytic residue is called peroxidatic cysteine. 1-Cys Prxs lacks resolving cysteine. In 2-Cys Prxs, the typical and atypical ones are differentiated on the basis of intermolecular or intramolecular disulfide-bond formation between peroxidatic cysteine (CPSH) and resolving cysteine (CRSH) respectively. The active site in peroxiredoxins family proteins is mostly conserved with a PXXXTXXC catalytic motif and conserved Arg residue. To maintain the cellular peroxides level in the cytosol, peroxiredoxins decline more than 90% peroxide and showed catalytic rates of ~107M-1 s-1. Peroxiredoxins substrates include different kinds of peroxide such as hydrogen peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, etc. The antioxidant defense enzyme peroxiredoxin is critical for the survival and pathogenesis of Candidatus Liberibacter asiaticus (CLa). In CLa, the bacterioferritin comigratory protein (CLaBCP) and 2-Cys peroxiredoxin from Candidatus Liberibacter asiaticus (Prx) genes are expressed. Previously, in our lab, basic characterization of CLaBCP have done. The CLaBCP results analysis of biochemical studies, secondary structure analysis, and the ROS scavenger potential revealed that they have significant antioxidant potential. The different studies suggested that peroxiredoxin is a defense protein in pathogens and is used as a drug target. The CLa host Citrus sinensis also have a defense-related peroxiredoxin protein in their cellular and sub-cellular organelles. In the present work, we performed the biochemical and biophysical characterization of CLaBCP and CsPrx. Subsequently, the comparative binding studies of inhibitor conoidin and celastrol were done against CLaBCP and its host peroxiredoxin from Citrus sinensis (CsPrx) to develop potential inhibitor molecules against CLaBCP. The 3-dimensional model was used for further virtual screening, molecular docking, and molecular dynamic simulation to identify the small molecule inhibitors against CLaBCP and Prx. The work has been divided into six chapters.