INVESTIGATING THE ROLE OF THE PERIPLASMIC BINDING PROTEIN, FAT B, IN THE BINDING INTERACTION OF [IRON (III)-SIDEROPHORE] COMPLEXES IN BRUCELLA SPP.

Abstract

Brucella abortus, an obligate intracellular Gram-negative bacterium, is the causative agent of brucellosis in ruminants. As an intracellular pathogen, Brucella survives inside macrophages or placental trophoblast cells (for pregnant cattle). Like most living organisms, Brucella strains require the micronutrient, iron, for their survival. Brucella abortus produces two siderophores, Brucebactin (BB), a di-catechol chelator, and 2,3-dihydroxybenzoic acid (2,3-DHBA), while proliferating in placental trophoblast to meet the higher iron demand. Previous genome profiling of Brucella abortus has identified the fatBCDE genes as the putative Fe(III)-Siderophore inner-membrane ABC-type (ATP Binding Cassette) transporter. The fatB gene encodes, bi-lobal Periplasmic Binding Protein (PBP) and is expected to bind the Fe(III)-Siderophore complexes in the periplasmic space. Although Brucella abortus does not need to express the siderophore production and uptake genes during colonization of macrophages, but the expression of this system is essential for the placental infection, which has been attributed to infertility and abortion in cattle. Moreover, biochemically, this siderophore uptake system is important as brucebactin is predicted to be a di-catechol, and unlike its tri-catechol counter parts produce coordinatively unsaturated Fe(III) complex. The mechanism of Fe(III) release from such di-catechol complexes are not well understood, creating a knowledge gap in the siderophore mediated Fe(III) uptake field. Because of these, understanding the mechanism of Fe(III) uptake through the Fat system is essential and the subject of this research work. Homology modeling and bioinformatics studies performed on FatB conducted show an α-helical hinge, typical of Fe(III)-siderophore binding proteins with conserved R, H, and Y residues. Superposition of the FatB model with the crystal structures of its homologs (CeuE, YclQ) show these conserved residues to play direct roles in Fe(III)-siderophore recognition (R) and Fe(III) binding (H and Y). Equipped with this information, I successfully cloned Brucella abortus 2308 FatB gene in pET 32a+ vector and expressed the protein from E. coli BL21 to conduct spectroscopic studies (CD, DSC, Fluorescence, UV-Vis, etc.). Data from these experiments confirm that recombinant wild-type FatB can differentiate between 2,3-DHBA (one of the native siderophores) bound Fe(III) from 3,4-DHBA bound Fe(III). Further, results from my experiments also confirm that FatB folds forming α-helix and β-sheets in solution, as predicted by the homology model, and the percent compositions of these secondary structural elements are significantly altered when Fe(III)-2,3-DHBA or Fe(III)-BB are titrated into this protein. Finally, I determined the binding affinities of Fe(III)-2,3-DHBA and Fe(III)-BB with FatB using isothermal titration calorimetry, showing µM affinities. This thesis is the first comprehensive study on the siderophore mediated Fe(III) transport in Brucella abortus.