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.
Unique 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, Rv0203) has been identified in mycobacteria. Also, a potential cytosolic heme-degrading protein, MhuD, has been identified. Finally, two potential heme transporters have been identified, MmpL11 and MmpL3. 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 and heme-degarding protein have been determined, and we have shownt that heme is transferred from the hemophore to the soluble domains of the potential heme uptake membrane proteins. Investigation into this uptake system is on-going.
Contact Dependent Growth Inhibition
Contact-dependent growth inhibition (CDI) systems are present in a wide variety of gram-negative bacteria, including many important pathogens, where they appear to function in intra-species growth competition. CDI(plus) cells physically interact with susceptible bacteria and deliver a protein toxin that inhibits target cell growth. Inhibition is mediated by CdiA, a large filamentous protein that is expressed on the surface of inhibitor cells. The C-terminal region of CdiA (CdiA-CT) is polymorphic and contains the growth inhibition activity, suggesting that CDI systems deploy a variety of different toxins. CDI loci also encode highly variable CdiI immunity proteins, which specifically bind cognate CdiA-CT toxins to prevent autoinhibition (left panel). A survey of available bacterial genomes revealed approximately 30 distinct families of CdiA-CT/CdiI toxin/immunity pairs, with typically less than 20% amino acid sequence identity between different CdiA-CT/CdiI families, strongly suggesting that the protein-protein interactions underlying each toxin/immunity complex are unique.
Moreover, we have recently discovered that some CdiA-CT domains interact with specific target cell proteins termed permissive factors, and these binding interactions are required to activate the delivered toxins (right panel). Thus, some CdiA-CT domains interact with both their cognate CdiI immunity protein and a target cell permissive factor. Our long-term goal is to leverage the knowledge gained from this work to develop novel and targeted antimicrobial therapies. In this proposal, we focus on the structural, functional and genetic analyses of CdiA-CT toxins and their interacting protein partners. We will elucidate the mechanisms by which CdiA-CT toxins are neutralized by CdiI proteins, and activated by permissive factors, thereby yielding insights into the intricate toxin-immunity network encoded by bacterial CDI systems.
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
equired for i
roliferation) 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.