Natalie Strynadka

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

View record

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

View record

The bacterial type III secretion system, or injectisome, is a syringe-shaped nanomachine essential for the virulence of many pathogenic Gram-negative bacteria. A major functional subcomplex of the injectisome, the needle complex, is a 3.5MDa complex formed by more than ten unique proteins. The needle complex forms a continuous channel spanning both the inner and outer membranes of Gram-negative pathogens, created by three highly oligomerized inner and outer membrane hollow rings and a polymerized helical needle filament. The effector proteins secreted through this channel, which vary amongst different bacterial species, are essential for subsequent pathogenicity. Thus, structural studies of this complex can provide important atomic level information for understanding complex assembly and function of the injectisome as well as potentially development of new antivirulence drugs or vaccines to combat infections in susceptible human, animal and plant hosts pathogens.The first high-resolution needle complex structures determined by cryogenic electron microscopy (cryo-EM) here shows the atomic details of the inner and outer membrane protein complex and the needle filaments. The outer membrane component of the needle complex belongs to the secretin family, a giant necessarily gated pore common and essential to other bacterial secretion systems but which had remained largely uncharacterized at the atomic level until recent work including major contributions as outlined in this thesis. Further, the structures of the dual nested rings that form the major inner membrane structural component of the needle complex shows remarkable similarity regardless of the assembly stages, inferring a highly stable foundation for the other components of the system to pack and function within.The snapshots of multiple needle complexes at different assembly stages revealed multiple new structures, the dynamics and the sequence of the assembly. The structural information also answered several long-standing additional questions, such as the mystery of the apparent symmetry mismatch between the inner and outer membrane complex of prior structures, and the fold, span and functional role of the historically named “inner rod” protein which this thesis works shows is not a rod but an adaptor to set the needle helicity and anchor it to the stable inner membrane platform.

View record

The bacterial cell wall is a complex polymeric structure with essential roles in defense, survival, and pathogenesis. Targeting the assembly of cell wall peptidoglycan with β-lactam antibiotics is becoming increasingly ineffective due the development and spread of resistance mechanisms. New therapeutic agents are now urgently needed, and one proposed target for drug development is the biosynthesis of the Gram-positive bacterial cell wall polymer known as wall teichoic acid (WTA). WTA plays critical roles in host cell adherence, immune evasion, and regulation of essential physiological processes such as cell division and peptidoglycan assembly. Importantly, the dysregulation of peptidoglycan synthesis in the absence of WTA was found to resensitize methicillin-resistant Staphylococcus aureus strains to β-lactam antibiotics. In addition, interruptions at certain points of WTA biosynthesis is lethal due to the sequestering of lipid precursors shared with peptidoglycan synthesis. With WTA assembly validated as a drug target, it is essential to characterize the responsible enzymes to guide drug discovery and development. In this thesis, high resolution X-ray crystallographic structures of TarI, TarJ, and TarL, in the assembly of the central ribitol-phosphate polymer of S. aureus WTA, are presented. Our ensemble of TarI and TarJ crystal structures illustrate the mechanism of synthesizing CDP-ribitol, the activated donor substrate for WTA polymerization by TarL. Furthermore, our crystal structure of the uncharacterized TarL N-terminal domain discloses potential roles in protein- and substrate-binding. In addition, crystal structures of LCP enzymes were solved, providing novel insights into substrate-binding and catalysis involved in the attachment of the complete WTA polymer onto peptidoglycan. Lastly, atomic structures of GlpQ, a WTA-degrading enzyme involved in cell wall maintenance, are presented. The structures provide the basis for substrate specificity and support an exolytic mechanism of WTA degradation. Together, these structural and biochemical analyses of WTA biosynthetic and degradative enzymes provide mechanistic understanding of their activities and reveal features that can guide drug discovery and development.

View record

The bacterial injectisome is an essential virulence factor for many Gram-negative pathogens. Resembling a 50-100 nm long syringe, the injectisome creates a continuous channel between the bacterial and host cytosols through which the bacterium secretes effector proteins to modulate host signalling. The aim of the following work is to contribute to the structural characterization of the injectisome, focusing on proteins involved in its assembly and substrate selection. The outer membrane pore of the injectisome, termed the secretin, relies on a pilotin protein for its localization. The X-ray crystallographic structure of the Salmonella enterica SPI-1 pilotin InvH reveals an α-helical dimer, confirmed to exist in solution through biophysical experiments. The pilotin-secretin interface, characterized by X-ray crystallography and NMR spectroscopy, is mutually exclusive with the InvH dimer and results in a 1:1 complex. The inner membrane pore protein has a vital role in injectisomal secretion hierarchy. The cryo-EM structure of the IM pore EscV from enteropathogenic Escherichia coli (EPEC) demonstrates that the protein forms a nonameric ring in solution. Of its four subdomains, two contribute to ring formation while the remaining two have some rotational freedom.The cytosolic ATPase complex is essential to separating effector proteins from their cognate chaperones prior to secretion. The cryo-EM analysis of the EPEC ATPase-stalk complex, EscN-EscO, yielded an asymmetric homohexamer structure resembling F1- and V-ATPases. Kinetic studies show that oligomerization and the presence of the stalk are required for efficient ATP hydrolysis. The similarity to rotary ATPases supports the hypothesis that the injectisomal ATPase complex is a rotary motor, with the stalk acting as the rotor. How this rotation contributes to secretion is yet to be discovered.

View record

In bacteria, the carrier lipid undecaprenyl phosphate (C₅₅P) is used as a scaffold for the synthesis of bacterial cell wall polymers such as peptidoglycan. C55P is synthesized as undecaprenyl pyrophosphate (C55PP) by undecaprenyl pyrophosphate synthase (UppS) and must be dephosphorylated by an as yet unknown mechanism before it can be used in cell wall biosynthesis. Individual subunits of cell wall polymers are assembled in the cytoplasm on C₅₅P before being flipped to the periplasmic face of the membrane where they are polymerized into the existing structure, releasing C₅₅PP as a by-product. The resultant C₅₅PP must be recycled to C₅₅P before being used in another round of cell wall polymer biosynthesis. The major protein responsible for recycling in Escherichia coli is undecaprenyl pyrophosphate phosphatase (UppP). The ongoing synthesis and recycling of undecaprenyl phosphate by UppS and UppP, respectively, are required for the survival and pathogenesis of bacteria; thus, both enzymes represent attractive targets for the development of therapeutics. In this thesis, UppP was structurally and functionally characterized, and inhibition of UppS by novel inhibitors was investigated. The X-ray crystallographic structure of the polytopic integral membrane protein membrane protein UppP was solved to 2.0 Å resolution using lipid cubic phase (LCP) crystallization. The crystal structure revealed an unexpected membrane topology and three-dimensional structure that suggests a potential role for UppP as a C₅₅P(P) lipid flippase and allowed for the rationalization of previously published site-directed mutagenesis results. An ordered monoolein molecule in the active site of the enzyme allowed us to model a C₅₅PP and propose a catalytic mechanism for C₅₅PP dephosphorylation. The crystal structure of UppS from Bacillus subtilis was solved in apo- and inhibitor bound states using X-ray crystallography, allowing us to rationalize two novel inhibitors’ superior efficacy against B. subtilis UppS versus Staphylococcus aureus or E. coli orthologues. The inhibitors bind in the hydrophobic tunnel into which the nascent C55PP product of UppS grows. Additionally, a crystal structure of B. subtilis UppS in complex with clomiphene provided a clearer structural basis of its inhibition of UppS and provides a basis for the rational design of improved UppS inhibitors.

View record

The bacterial cell wall plays a crucial role in cellular viability and is an important drug target. The bacterial cell wall is constructed using a complex biosynthetic pathway, which ultimately terminates in the activity of peptidoglycan synthases. These synthases act to polymerize the lipid-linked cell wall precursors and crosslink these strands into the existing sacculus. The activity of these synthases can be modulated in two key ways. First, there are proteins which modulate the type of synthase activity. In most bacteria, the peptidoglycan synthase crosslinking reaction uses D,D-transpeptidase activity. However, an alternate crosslinking mechanism involving the formation of a complex between peptidoglycan synthases and the L,D-transpeptidase YcbB can lead to bypass of D,D-transpeptidation, β-lactam resistance, typhoid toxin release, and stress linked cell wall crosslinking. Second, there are proteins which associate with peptidoglycan synthases to stimulate or inhibit the canonical synthase activity. In E. coli, there are the positive regulators LpoA and LpoB, and the negative regulator CpoB. These proteins seen in E. coli are conserved across a large number of Gram-negative organisms, though the human pathogen P. aeruginosa is seen to have an alternative to LpoB, known as LpoP. Here, we provide insight into the structure and function of YcbB and LpoP. We show that the crystallographic structure of YcbB from E. coli, S. Typhi, and C. rodentium consists of a conserved L,D-transpeptidase catalytic domain, substrate capping loop subdomain, peptidoglycan-binding domain and scaffolding domain. Meropenem and ertapenem acylation of YcbB gives insight into inhibition by carbapenems, the singular antibiotic class with significant activity against L,D- transpeptidases. Additionally, we probe the interaction network of this pathway, assay β-lactam resistance in vivo, and provide insight into the role of YcbB in acute bacterial infection. Second, we show that the crystallographic structure of LpoP consists of tandem tetratricopeptide repeats, distinct from the structure of the canonical LpoB. Using in vitro glycosyltransferase and transpeptidase assays, we compare and contrast the structure of LpoP- and LpoB-PBP1b systems. As a whole, this thesis provides insight into both forms of modulation of peptidoglycan synthase activity and lays the groundwork for further understanding of this complex biosynthetic pathway.

View record

Methicillin-resistant Staphylococcus aureus (MRSA) infections increase mortality andmorbidity worldwide, threatening public health. MRSA is resistant to many classes of antibioticsincluding the most commonly prescribed β-lactam antibiotic class, making treatment ofinfections difficult. In MRSA β-lactam resistance is primarily mediated by PBP2a, a β-lactamresistant penicillin-binding protein and to some extent, PC1, a β-lactamase. Additionally, β-lactam resistance in S. aureus has also been recently shown to be facilitated independently ofPBP2a by mutations in the gene coding for penicillin-binding protein 4 (PBP4), though themechanisms of resistance have remained mysterious.In an effort to understand the mechanism of PBP4-mediated β-lactam resistance, twoligand-free and six acyl-enzyme intermediate X-ray crystallographic structures of mutant andwild-type PBP4 were solved. Localised within the transpeptidase active site cleft, the twosubstitutions appear to have different effects depending on the drug. Kinetic analysis shows themissense mutations impaired the KM value for ceftobiprole 150-fold, decreasing the proportion ofinhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors,possibly including mutation of the pbp4 promoter. These findings suggest PBP4 mediated β-lactam resistance is mediated by at least two separate mechanisms.The expression of the genes coding PC1 and PBP2a are controlled by two integralmembrane proteins: BlaR1 and MecR1 respectively, which consist of a zinc metalloproteasedomain and an extracellular C-terminal β-lactam sensing domain which activates the proteolyticdomain when acylated by a β-lactam antibiotic. Here, avibactam, a diazabicyclooctane β-lactamase inhibitor, was found to induce expression of pbp2a (which codes for PBP2a) and blaZ(which codes for PC1) in a clinical strain of MRSA. The X-ray crystallographic structures of theivBlaR1 and MecR1 sensor domains show avibactam binds to MecR1 as has been observed for theClass-D β-lactamases. In contrast, BlaR1 has two avibactam binding poses orientated 180° toeach other. As avibactam upregulates expression of blaZ and pbp2a antibiotic resistance genes,we suggest further research is needed to explore the effect of administering β-lactam-avibactamcombinations to treat MRSA infections.Together, these findings improve our understanding of β-lactam resistance in MRSA andprovide molecular details to facilitate improved inhibitors of MRSA.

View record

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.

View record

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.

View record

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.

View record

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.

View record

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.

View record

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.

View record