J Andrew Alexander
Doctor of Philosophy in Biochemistry and Molecular Biology (PhD)
Understanding and combating antibiotic resistance
No abstract available.
Bacterial diseases have an enormous impact on human health. The most widespread class of human antibacterials is the β-lactams that target the transpeptidase activity of penicillin-binding proteins, which are responsible for cross-linking the peptidoglycan cell-wall. However, bacteria have gained resistance to all major classes of β-lactams. To protect the clinical utility of the β-lactams it is essential to understand the structural basis for this resistance. This thesis aims to better understand the molecular details governing extended-spectrum β-lactamase mediated β-lactam resistance, and to gain insights into inhibition of these emerging resistance factors. Recently, a novel resistance factor known as the New Delhi Metallo-β-Lactamase-1 has been found to confer enteric pathogens such as Escherichia coli and Klebsiella pneumoniae with nearly complete resistance to all β-lactams. The 2.1Å resolution crystal structure of K. pneumoniae holo-NDM-1 revealed an expansive active site, which we propose leads to a broader β-lactam substrate specificity. Furthermore, NDM-1 localizes to the bacterial outer-membrane by sucrose density gradient centrifugation. The structural details underpinning the broad-spectrum resistance of NDM-1 was further investigated by analysis of the protein in complex with hydrolyzed β-lactams as well as bound to the inhibitor L-captopril. An analysis of the NDM-1 active site in these structures reveals key features important for the informed design of novel inhibitors of NDM-1 and other metallo-β-lactamases. The novel diazabicyclooctane (DBO) avibactam inhibits a wider range of serine β-lactamases than has been previously observed with clinical β-lactamase inhibitors. To understand the molecular basis and spectrum of inhibition by avibactam, we provide structural and mechanistic analysis of the compound in complex with important class A and D serine β-lactamases. A kinetic analysis of key active-site mutants for class A β-lactamase CTX-M-15 allows us to propose a validated mechanism for avibactam-mediated β-lactamase inhibition including a unique role for S130, which acts as a general base. We then show that avibactam derivatives retain β-lactamase inhibitory properties but also exhibit considerable antimicrobial activity against clinically relevant bacteria via targeting penicillin-binding proteins. Our results provide evidence that structure-activity relationship studies for the purposes of drug discovery must consider both β-lactamases and penicillin-binding proteins as targets.
Many Gram-negative pathogens use a type III secretion system (T3SS) to inject effector proteins into the host cytoplasm, where they manipulate host processes to the advantage of the bacterium. The T3SS is composed of a cytoplasmic export apparatus, a membrane-spanning basal body with a central channel formed by the inner rod, an extra-cellular needle filament and a translocon complex that inserts in the host membrane. In this thesis, proteins involved in T3SS assembly, as well as a T3SS effector protein were structurally and functionally characterized. The structure of EtgA, a T3SS-associated peptidoglycan (PG)-cleaving enzyme from enteropathogenic Escherichia coli (EPEC) was solved. The EtgA active site has features in common with lytic transglycosylases (LTs) and hen egg-white lysozyme (HEWL). EtgA contains an aspartate that aligns with lysozyme Asp52 (a residue critical for catalysis), a conservation not observed in LT families to which the conserved T3SS enzymes were presumed to belong. Mutation of the EtgA catalytic glutamate conserved across LTs and HEWL, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in T3SS assembly. EtgA forms a complex with the T3SS inner rod component, which enhances PG-lytic activity of EtgA in vitro, providing localization and regulation of the lytic activity to prevent cell lysis. After assembly of the basal body and needle, the gatekeeper protein ensures the translocon assembles at the needle tip prior to secretion of effector proteins. The gatekeeper from EPEC (SepL) was crystallized and it was shown that it has three X-bundle domains, which likely mediate protein-protein interactions to control translocon and effector secretion. Comparison of SepL to structurally characterized orthologs revealed several conserved residues, which may be required to regulate secretion of translocators or effectors. Finally, SopB, a Salmonella effector protein, in complex with host Cdc42, an Rho GTPase that regulates critical events in eukaryotic cytoskeleton organization and membrane trafficking was structurally characterized. Structural and biochemical analysis of the SopB/ Cdc42 complex shows that SopB structurally and functionally mimics a host guanine nucleotide dissociation inhibitor (GDI) by contacting key residues in the regulatory switch regions of Cdc42 and slowing Cdc42 nucleotide exchange.
Mycobacterium tuberculosis uses the ESX-1 type VII secretion system to export proteins to its cell surface, which permeabilize the host macrophage phagosomal membrane, allowing the bacterium to escape and spread to new cells. The structure of the type VII membrane complex and how it mediates this function is unknown, but it is hypothesized that some of the secreted proteins form an extracellular appendage that facilitates membrane lysis or direct secretion of virulence factors into the host cytoplasm. This thesis investigates the structural relationship between one of these secreted proteins, EspB, and a protease that processes it, MycP1. The x-ray crystallographic structures of both proteins are determined and described. EspB is shown to form a multimer with heptameric stoichiometry, and an EM reconstruction of this multimer is generated and used to create a model of the oligomer using symmetric Rosetta docking. The final model is supported by mass spectrometry-based detection of chemically cross-linked peptides from adjacent subunits. We use mass spectrometry to determine how EspB is proteolytically processed during secretion and discuss the effect of this processing event on the EspB ultrastructure. Finally, the structure of one of the membrane apparatus proteins, EccB1 is determined, revealing structural homology to a phage lysin. The combination of x-ray crystallography, EM, modeling, and mass-spectrometry provides an exciting first glimpse at the structure and function of the type VII secretion system - a critical factor in the TB pathogenesis cycle.
Mucins are proteins that contain dense clusters of α-O-GalNAc-linked carbohydrate chainsand are the major component of the mucosal barrier that lines the mammaliangastrointestinal tract from mouth to gut. A critical biological function of mucins is to protectthe underlying epithelial cells from infection. Enterohemorrhagic Escherichia. coli O157:H7(EHEC), a bacterial pathogen that causes severe food and water borne disease, is capable ofbreaching this barrier and adhering to intestinal epithelial cells during infection. StcE(secreted protease of C1-esterase inhibitor) is a ~100 kDa zinc metalloprotease virulencefactor secreted by EHEC and plays a pivotal role in remodelling the mucosal lining duringEHEC pathogenesis. StcE also dampens the host immune response by targeting the mucinlikeregion of C1-INH, a key complement regulator of innate immunity. To obtain furthermechanistic insight into StcE function, I have determined the crystal structure of the fulllengthprotease to 2.5Å resolution. This structure shows that StcE adopts a dynamic, multidomainarchitecture featuring an unusually large substrate binding cleft. Electrostatic surfaceanalysis reveals a prominent polarized charge distribution highly suggestive of anelectrostatic role in substrate targeting. The observation of key conserved motifs in the activesite allows us to propose the structural basis for the specific recognition of α-O-glycancontaining substrates, which have been confirmed by glycan array screening to be Oglycosylationof the mucin-type. Complementary biochemical analysis employing domainvariants of StcE further extends our understanding of the substrate binding stoichiometry anddistinct substrate specificity of this important virulence-associated metalloprotease.
Sialic acid plays vital roles in various biological processes including cellular recognition and cell adhesion. The biosynthesis and post-synthetic processing of sialic acid is particularly important to host-pathogen interactions because many virulent bacteria decorate their cell surfaces with sialic acid-containing molecules in order to evade the host’s immune response. Neisseria meningitidis, a highly invasive human pathogen that causes bacterial meningitis, produces a capsular polysaccharide comprised of polysialic acids that protect the bacteria from the host’s immune system by mimicking the sialic acid-containing cell surface structures. The biosynthesis of sialic acid is catalyzed by sialic acid synthase NeuB. We report the structural and biochemical analysis of the first potent inhibitor of sialic acid synthase from N. meningitidis. The inhibitor was synthesized as a mixture of stereoisomers, which mimics the tetrahedral intermediate of the NeuB reaction. Based on the crystallographic and kinetic analysis of the inhibitor binding, an improved mechanism is proposed. Capsular polysaccharides of certain strains of N. meningitidis can be further acetylated by sialic acid-specific O-acetyltransferases, a modification that correlates with the virulence in bacterial infection. In the second part of the thesis, we report the first kinetic and structural analysis of bacterial sialic acid O-acetyltransferase OatWY from N. meningitidis. Crystals of OatWY were obtained in complex with either CoA, acetyl-CoA, or nonhydrolyzable donor analogue S-(2-oxopropyl)-CoA. Structural analysis in combination with kinetic and mutagenesis studies elucidates the mechanistic features and substrate specificity of this enzyme. Campylobacter jejuni, a leading causative agent of bacterial diarrhea and gastroenteritis, expresses sialylated lipooligosaccharide, which mimics the carbohydrate structure of human gangliosides. Sialyltransferase Cst-II is the enzyme responsible for the lipooligosaccharide sialylation in C. jejuni as a means of evading the host's immune system. The last part of the thesis describes the first ternary complex of Cst-II with the donor analogue CMP and the terminal trisaccharide (Neu5Ac-α-2,3-Gal-β-1,3-GalNAc) of its natural acceptor. Site-directed mutagenesis of acceptor binding residues was performed and mutants were characterized by enzyme kinetics. Our results reveal the structural basis for the binding of a physiologically relevant natural acceptor and provide additional insight into the mechanism and acceptor specificity of this enzyme.
Beta-lactam antibiotics have achieved phenomenal success in the treatment ofinfections by inhibiting the transpeptidase enzymes that cross-link the bacterial cell wall.Beta-lactamase-producing pathogenic bacteria and multi-drug-resistant “superbugs” such asmethicillin-resistant Staphylococcus aureus (MRSA) have emerged, however. Overcomingresistance factors is thus a research priority.BLIP (Beta-Lactamase Inhibitory Protein) from Streptomyces clavuligerus binds a varietyof beta-lactamase enzymes with widely ranging specificity. Its interaction with Escherichia colibeta-lactamase TEM-1 is a well-established model system for protein-protein interactionstudies. Presented in Chapter 2 are crystal structures of two BLIP relatives: BLIP-I (a highaffinityinhibitor, alone and in complex with TEM-1) and BLP (which appears not to inhibitbeta-lactamases). Substantial variation appears possible in the sub-nanomolar binding ofTEM-1 by two homologous proteinaceous inhibitors and such favorable interactions can benegated by a few, strongly unfavorable interactions.OXA-10 is a Pseudomonas aeruginosa beta-lactamase that is resistant to inhibitors inclinical use. Cyclobutanone beta-lactam mimics could be used instead. Chapter 3 reports thecrystal structure of OXA-10 covalently modified at its catalytic serine nucleophile with acyclobutanone inhibitor to form a hemiketal. Favorable and unfavorable contacts made atthe active site are examined with a view to improved inhibitor design.PBP2a is the resistant transpeptidase that allows MRSA to maintain the bacterial cellwall in the presence of beta-lactam antibiotics. Ceftobiprole is the most clinically-advancedamong a new generation of beta-lactams designed to treat MRSA by targeting PBP2a itself.Chapter 4 uses the crystal structure of a truncated, soluble form of PBP2a solved in complexiiiwith ceftobiprole to explain its inhibitory power and evaluate current anti-MRSA drug designhypotheses. Its efficacy appears to arise from improved binding affinity that overcomes thedisfavored energetics of acylation.Ceftobiprole clinical trials reported no bacterial resistance, yet fully ceftobiproleresistantMRSA (MIC 128 !g/ml) were generated by passage through subinhibitoryconcentrations of ceftobiprole, discussed in Chapter 5. Resistance emerges in most cases viamutations to the gene encoding PBP2a. Computational modeling predicts that ceftobiproleresistance may be mediated in PBP2a by alteration of binding affinity, acylation efficiency, orby influencing interactions with other proteins.