Campopiano, Dominic

Dorin, Julia

Capewell, Samantha Jessica

Biotechnology and Biological Sciences Research Council (BBSRC)

2014-11-17T15:01:19Z

2014-11-17T15:01:19Z

2014-06-28

Ferric ABC Transporters. Pathogenic bacteria have evolved specialised iron acquisition systems that allow them to effectively colonise a host. One of these systems is the ferric binding protein (Fbp) complex that is a member of the ATP-Binding Cassette (ABC) superfamily of small molecule transporters. The Fbp complex is made up of three-components (FbpABC) that transports ferric iron from the periplasm to the cytoplasm of many Gram negative bacteria. FbpA binds iron in the periplasm and transports it to the FbpB transporter complex that permeates the cytoplasmic membrane. Here the iron is actively transported by FbpB through the membrane that is powered by ATP hydrolysis catalysed by FbpC, the cytoplasmic ATPase. Burkholderia cenocepacia is an opportunist pathogen that colonises the lungs of cystic fibrosis patients and is particularly resistant to antibiotic treatment. In this study the iron uptake system of B. cenocepacia strain J2315 is investigated. A putative FbpA from B. cenocepacia J2315 was expressed in the periplasm of Escherichia coli cells and the recombinant FbpA B. cenocepacia protein purified. The structural and electrochemical properties of native FbpA B. cenocepacia were investigated using UV Visible spectroscopy, spectro-electrochemistry, mass spectrometry and crystallographic techniques. It appears that FbpA B. cenocepacia is a novel member of the FbpA superfamily that selectively utilises citrate as an exogenous anion in ferric iron co-ordination. This is the first instance that a recombinant ferric binding protein has been documented as preferentially utilising citrate in this manner. The putative ATPase from B. cenocepacia (FbpC B. cenocepacia) was also expressed in E. coli but it was found to be insoluble. A number of expression systems were tested but none were found to be successful in generating sufficient quantities of FbpC B. cenocepacia for structural studies. Human β-defensin 2. Despite daily contact with a range of microorganisms, mammals do not regularly succumb to pathogenic invasion. One reason is the presence of an important defence mechanism uses a reservoir of antimicrobial peptides (AMPs) that are expressed in eukaryotes as a means of innate immunity. The AMP superfamily is composed of over 900 members, displays broad structural and sequence diversity and is active against a wide range of bacteria, fungi and viruses. β-defensins are small (3-5 kDa), cationic peptides that display antimicrobial activity against a range of microbes and have also been shown to act as chemo-attractants (chemokines) within the adaptive immune system. In this study we obtained milligram amounts of pure human β-defensin 2 (HBD2) for functional studies by the development of a method for the rapid expression and purification of the recombinant peptide. A clone encoding a thioredoxin-HBD2 fusion protein was designed for the expression of soluble peptide in E. coli cells that was purified by simple affinity chromatography. The HBD2 peptide was cleaved from the fusion by an efficient protease step and further purified to yield pure HBD2. This recombinant HBD2 defensin was shown to be active against a Mycobacterium tuberculosis mutant strain.

http://hdl.handle.net/1842/9636

en

The University of Edinburgh

Arnold MFF, Haag AF, Capewell S, Boshoff HI, James EK, McDonald R, Mair I, Mitchell AM, Kerscher B, Mitchell TJ, Mergaert P, Barry CE, Scocchi M, Zanda M, Campopiano DJ, Ferguson GP. (2013) Partial complementation of Sinorhizobium meliloti bacA mutant phenotypes by the Mycobacterium tuberculosis BacA protein. J. Bacteriol, 195(2), 389-398.

ferric binding protein

iron uptake system

B. cenocepacia

recombinant HBD2 defensin

Structural and functional studies of protein targets at the host-pathogen interface.

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy