Antibiotic resistance has become a global health problem in both clinical and community settings. Our lab is interested in the structure-based design of inhibitors that either block existing antibiotic-resistance mechanisms or provide novel antibiotic therapies by targeting macromolecular assemblies essential to bacterial viability and/or pathogenesis. To illuminate the molecular basis of antibiotic resistance and viability/pathogenicity afforded by the proteins involved, we use a multidisciplinary structural biology approach that relies on x-ray crystallography, NMR, and electron microscopy, complemented by phenotypic analysis in vitro and in vivo. Many of the bacterial proteins and protein complexes we study are membrane associated, adding to the challenge of achieving their structural, biochemical, and in vivo characterization.
We are focusing on the bacterial mechanisms that facilitate broad-spectrum resistance to the classic family of beta-lactam antibiotics, such as the penicillins and cephalosporins. These mechanisms have three general modes of action: hydrolysis of the antibiotic, efflux of the antibiotic by pumps that efficiently and specifically extrude the drug from the cell before they reach their designated target, and acquisition of altered and drug-insensitive targets. Our group has contributed to the understanding and treatment of each of these three mechanisms in various clinical pathogens. For example, we have determined atomic resolution structures of the newly emerged plasmid-mediated metallo-beta-lactamase NDM-1 in the presence of a wide variety of the penicillin and cephalosporin substrates it rapidly degrades. This information has led to insights into the molecular basis for the observed broad-spectrum specificity of NDM-1 and allowed for the design of new classes of inhibitors to thwart its drug-resistance effects.
Another example centers on the key determinant of broad-spectrum beta-lactam resistance in the notorius clinical superbug methicillin-resistant Staphylococcus aureus (MRSA), the membrane-spanning, penicillin-binding protein 2a (PBP2a), a transpeptidase component of the multi-enzyme bacterial cell wall–synthesizing assembly. Transpeptidases (the target of beta-lactam antibiotics) are required for production of peptide cross-links that give the cell wall its necessary strength and rigidity. Because of its low affinity for beta-lactams, PBP2a provides cross-linking transpeptidase activity at beta-lactam concentrations that inhibit the other cell wall transpeptidases; such peptidases are normally produced by S. aureus and other pathogenic bacteria. We have determined, to high resolution, the crystal structures of native MRSA PBP2a as well as those of acyl-enzyme complexes with various beta-lactam antibiotic substrates. Analysis of PBP2a's active site reveals the structural basis of its broad-spectrum resistance to the clinically used beta-lactam antibiotics and identifies features important for high-affinity binding. This information is being used in structure-based inhibitor design strategies aimed at combating MRSA resistance, as highlighted by recent analysis of PBP2a with new generations of cephalosporin compounds such as Ceftobiprole.
We have also determined the structure of the first bifunctional glycosyltransferase/transpeptidase enzyme component of the cell wall assembly, PBP2 from S. aureus, which polymerizes/cross-links the lipidated glyco-peptide–containing building blocks of the cell wall. We have analyzed the structure of this membrane-anchored enzyme in the presence of the substrate analogue inhibitor moenomycin, a highly potent natural product antibiotic commonly used to treat livestock. Our structures provided new insight into the molecular basis of processive polymerization/cross-linking by these enzymes as well as the essential features of glycosyltransferase-targeted inhibition by moenomycin. We are using information from these structural findings to create modified forms of moenomycins and to pursue leads for new antibiotic treatment of human infections.
In work on other novel targets, our laboratory has made significant progress in the structural elucidation of proteins and subcomplexes that compose bacterial protein secretion systems, including the type III secretion apparatus (T3SS), which is common and essential to the pathogenicity of many Gram-negative pathogens, such as enteropathogenic Escherichia coli, Salmonella, Shigella, Bordetella, Chlamydia, and Pseudomonas.
The needle-like T3SS allows specific and direct injection of bacterial virulence proteins into the cytoplasm of human host cells, where they mediate a wide range of pathogenic effects by manipulating host cytoskeletal proteins, signaling machinery, and other core cellular processes. The injected virulence proteins differ from bacterial pathogen to pathogen, but the T3SS apparatus is highly conserved and thus presents an excellent target for the design of antimicrobials and vaccines that disable only pathogenic bacteria, leaving "good bacteria" to continue to colonize in the patient. The T3SS is composed of approximately two dozen proteins that create an oligomerized set of membrane-spanning rings and connecting hollow filaments reaching from the bacterial cytoplasm to the host cytoplasm.
We developed a combination of customized x-ray crystallography, NMR, electron microscopy, mass spectroscopy, Rosetta-based molecular modeling, and cellular microbiology approaches to study this massive multimembrane-spanning assembly. This effort has contributed major insights to the current high-resolution models of the T3SS needle complex and mode of action, including the cytoplasmic ATPase and inner- and outer-membrane rings, which provide a foundation for all subsequent T3SS assembly and the extended, hollow translocation filaments/pore-forming complex, which together allow delivery of T3SS bacterial virulence effectors directly into the host cytoplasm. This work provides the foundation for understanding the molecular details of this process, which is unique to pathogenic variants, as well as for the design of novel antimicrobials and vaccine epitopes to combat the clinically notorious bacteria that encode the T3SS.
Our studies are also supported by grants from the Canadian Institutes of Health Research, the Canadian Foundation for Innovation, and the Michael Smith Foundation for Health Research.
As of September 26, 2012