GLRCE: Research Projects on MRSA
Research Project 3 (GLRP003) : Therapies for anthrax and MRSA.
Bacterial growth is dependent on the acquisition of iron from the surrounding environment. In the Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus, this process relies heavily on the biosynthesis of siderophores. These small secondary metabolites are excellent chelating agents and we apply both genetic and biochemical approaches to discovering how these compounds are synthesized and utilized by the cell to scavenge for iron and promote pathogen survival.
Petrobactin is a "stealth siderophore" produced by B. anthracis which evades host defenses against similar iron chelators; this molecule is essential for pathogenesis and spore outgrowth in the causative microbe of anthrax. Up-regulation of biosynthetic gene clusters under host-like conditions leads to expression of involved "siderophore synthases". These proteins form complexes establishing unique non-ribosomal peptide synthetase (NRPS)-independent machinery reliant on multiple condensation reactions for production of a secondary metabolite. Previous characterization of products encoded by the Bacillus asb operon has demonstrated petrobactin only requires three simple, diffusible precursors: 3,4-dihydroxybenzoic acid, spermidine, and citrate. The petrobactin biosynthetic pathway is however more complex than first anticipated, with multiple routes toward the complete siderophore's synthesis demonstrated in vitro. Additionally, in recent collaboration with GLRCE members at the Joachimiak and Hanna labs, the origin of 3,4-DHBA, the unusual moiety conferring petrobactin's stealth ability and iron-chelating effect has been determined through mechanistic and structural elucidation of the dehydroshikimate dehydratase AsbF (structure pictured).
Lessons learned from petrobactin have been applied to study secondary metabolic reactions by iron-restricted S. aureus strains. Unique activity of individually purified biosynthetic proteins may prove applicable in future engineering of new compounds, creation of useful microbial strains, or isolation of inhibitors for iron sequestration in virulent species. Upon characterization of the pathways pathogens rely on for iron homeostasis within the host, we search for candidate inhibitors of these processes by capitalizing on the expanding chemical libraries housed onsite at the Center for Chemical Genomics within the Life Sciences Institute. Results of these studies indicate further characterization of siderophore biosynthesis and utilization pathways to be promising avenues in exploring new therapeutics capable of shutting down nutrient acquisition mechanisms associated with dangerous bacterial infection.
Research Project 6 (GLRP006) : Therapies against MRSA SuperAntigens
Staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) are exceptionally toxic to humans, with doses as low as 0.1 ug/human causing symptoms of toxic shock syndrome through superantigen activities. The major staphylococcal superantigens associated with serious human diseases include TSST-1 and enterotoxin serotypes A-E and Q. This project, through the collaborative interaction of investigators at the University of Minnesota and University of Illinois, develops soluble high-affinity T cell receptor chains that neutralize the toxicities of these superantigens, as measured in rabbit models of diseases. Rabbit models include intravenous, subcutaneous, and intra-pulmonary administration of both superantigens and high-affinity T cell receptor chains. The serum half-life of each high-affinity T cell receptor chain and ability to rescue animals from superantigen-induced serious illness will be assessed. Studies are also planned to evaluate the ability of the high-affinity T cell receptor chains to neutralize superantigens made in vivo in rabbits by methicillin-resistant S. aureus, which have recently been determined to be the most significant causes of serious infectious diseases in the United States.
Research Project 7 : Selective Lysis of B anthracis and MRSA
Our long-term goal is to develop potent agents for prevention and treatment of anthrax and methicillin-resistant S. aureus (MRSA) by engineering phage endolysins. PlyG, a lysin of 25 kDa molecular weight encoded by gamma phage. PlyG contains a T7 lysozyme-like catalytic domain capable of hydrolyzing B. anthracis cell wall peptidoglycan, resulting in bacterial cell lysis, attached to a ~75 amino acid C- terminal domain. The C-terminal domain is a dimeric carbohydrate recognition module that targets the enzyme specifically to vegetative B. anthracis cells and germinating spores. We hypothesize that full- length PlyG exists in a monomeric inactive state stabilized by specific contacts between the N- and C-terminal domains, and that binding of the C-terminal (regulatory) domain to carbohydrates unique to the B. anthracis cell wall releases the autoinhibitory interaction and promotes formation of the fully active, dimeric PlyG enzyme. Phage endolytic enzymes like PlyG have the potential to serve as novel and powerful antibiotic agents, termed 'enzybiotics'. We broadened this approach to include MRSA by assembling an enzybiotic from other phage lysins with specific S. aureus activity. ClyS is a chimeric protein containing an N-terminal catalytic domain and a C-terminal cell wall targeting domain. The N-terminal catalytic domain is an endopeptidase of 184 amino acids and the C-terminal cell wall targeting domain is 94 residues. In aim 1, we will determine the NMR structure of full-length (inactive) PlyG to reveal the molecular basis of lysin autoinhibition for this class of antibacterial enzymes, and solve structures of the ClyS lysin and its component domains. In Aim 2, the basis for specific anthrax and MRSA targeting will be elucidated using NMR to monitor interactions between cell wall components and the PlyG and ClyS binding domains. Aim 3 will exploit this structural knowledge to engineer isoforms of PlyG and ClyS with enhanced stability in vivo. Because the bacterium must alter the basic construction of the cell wall to evade an enzybiotic, the probability that PlyG- and ClyS- resistant strains will emerge is low. These studies will provide important mechanistic insights into a novel class of antibacterial compounds moving toward clinical application.
