Structure of T6SS components and assessment of potential Gram-ve drug targets

The structure and potential function of two components of the type VI secretion system in Serratia marcescens have been investigated by X-ray crystallography. Chapters 1 and 2 describe the background of the type VI secretion system and experimental methods respectively. In chapter 3, the solution of the structure of S. marcescens Lip is presented. This is an essential outer membrane lipoprotein that is conserved among bacterial species. It is part of a membrane-spanning sub-assembly that is thought to anchor the remaining components to the cell envelope. The crystal structure of SmLip was determined using single-wavelength anomalous dispersion methods targeting iodides that were present in the crystallisation solution. The structure was refined to 1.92 Å and displays a fold similar to that of transthyretin; a protein that binds the hormone thyroxine. Despite the sequence identity between the two proteins being low, SmLip can be considered a new member of the transthyretin-like protein family. A role is suggested for loop 1 being involved in protein-protein interactions with other components of the type VI secretion system and therefore a potential drug target. <br/>In chapter 4, the structure of S. marcescens Rap1b is described. This protein is thought to be part of a set of periplasmic resistance proteins that protect S. marcescens from succumbing to the effects of its own secreted toxins. The crystal structure of Rap1b was determined using single-wavelength anomalous dispersion methods targeting both iodides from a halide soak and endogenous sulfurs. The structure was refined to 1.88 Å and displays a helical rich fold not previously characterised. This structure is described and is suggested to represent this family of resistance proteins. In addition to determining the structure of components of the type VI secretion system, the assessment of two drug targets have been investigated by biochemical techniques. The need for new antimicrobials and the importance of target selection in the drug discovery process is described in chapter 5. PBP3 from both Pseudomonas aeruginosa and Burkholderia pseudomallei is the focus of chapter 6. This protein is essential in bacteria as it catalyses the cross-linking of peptidoglycan. Peptidoglycan provides bacteria with structural rigidity and strength to withstand pressures that would otherwise rupture the cell. Sequence identity between the two homologues is approximately 40 %, with the catalytic residues conserved. The production of soluble protein and the results of screening for novel chemical fragments using several techniques are presented. The characterisation of P. aeruginosa IMPDH, an essential protein that is present in nearly all living organisms is described in chapter 7. This protein controls the pool of guanine nucleotides within cells. Fragments were identified using bio-layer interterformetry, a label free optical analytical technique that monitors biomolecular interactions in real time. Fragments were assessed for inhibitory effects using enzyme kinetics. The crystal structure of PaIMPDH was determined by molecular replacement and refined to 2.25 Å. This structure displays the catalytic domain with the active site loop fully ordered and in a conformation rarely observed. <br/>

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2012

Author dc:creator

• Rao, Vincenzo A.

Advisor dc:contributor.advisor

• Hunter, William

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/ec37df62-b65a-4f10-ba48-e8edde10e454

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/ec37df62-b65a-4f10-ba48-e8edde10e454