Overview

The Goulding lab is building a systems-based perspective of aspects of Mycobacterium tuberculosis, informed by proteomic and crystallographic groundwork. Until recently, the focus of structural biology has been on single, isolated proteins. By focusing on molecular assemblies, we add an additional dimension to our molecular view of living systems. The utility of this approach is in yielding potential drug targets at the level of protein interactions, an approach inaccessible from an atomistic view of isolated proteins.

Projects

Novel Mycobacterial Heme Uptake System

Iron is an essential metal for life. Mycobacteria must import iron from its host. Molecules involved in iron chelating pathways are well characterized, such as those involving exochelins and siderophores. Recently, it has been shown that during the early stages of S. aureus infection the major source of nutrient iron is heme rather than transferrin iron. A potential mycobacterial secreted hemophore (heme scavenging protein) has been identified in mycobacteria. Also, a potential cytosolic heme-degrading protein has been identified. Hence, a novel mcobacterial heme uptake system may exisit. My laboratory will be dissecting this pathway both structrually and biochemically. Thus far, the X-ray crystal structure of the potential hemophore has been deteremined, and a potential heme uptake membrane protein has been identified by mass spectrometry. Investigation into this uptake system is on-going.

Disulfide Bond Isomerase System

Disulfide bond-forming (Dsb) proteins have been shown to be involved in virulence in many pathogenic bacteria. They are oxidoreductase proteins that have a variety of functions including chaperone activity, electron transfer and disulfide bond isomerase activity. It has been predicted that of the 161 potential secreted proteins of M. tuberculosis approximately 60 % of these contain at least one disulfide bon. Hence, Dsb proteins which assist in folding secreted proteins into their correct conformation and assist in disulfide bond formation are of great importance for the survival of M. tuberculosis. Utilizing bioinformatics, two secreted Dsb proteins have been identified in M. tuberculosis, one of which is my target protein to investigate the secreted disulfide bond isomerase system of mycobacteria. The secreted proteins which these two Dsb proteins interact with is presently under investigation.

Homologous systems in Yersinia pestis

A major factor for the potency of bubonic plague-causing Yersinia pestis is it's ability, shared with Mycobacterium tuberculosis, to proliferate in human macrophages. The pgm (pigmentation) gene locus on the Y. pestis genome is required for this ability, encoding multiple genes over 34kb (PubMed ref.). Two genes from the pgm locus, ripA and ripB (required for intracellular proliferation) function in some way to lower levels of macrophage generated NO, a host anti-pathogen strategy, and have homologs in pathogenicity islands in Salmonella. Intriguingly, the third member of the three-gene rip operon, ripC / y2383, is a homolog of Mycobacterium tuberculosis CitE that we recently solved the structure of.