Michael Murphy

Burkholderia cenocepacia is an opportunistic pathogen correlated with increased diseaseseverity and mortality in cystic fibrosis (CF) patients. It is resistant to a wide range of disinfectantsand antibiotics, and no standard treatment is available to eradicate these infections. Previousanalysis of CF sputum showed elevated levels of zinc and iron ions and lower pH (2.9-6.5),compared to healthy individuals. Burkholderia species are abundant in acidic soils and some areheavy-metal resistant. B. cenocepacia grows at acidic pH (~3.5) and persists in acid compartmentsof amoebas and macrophages. We aimed to understand the impact of acidic pH and increased zincand iron concentrations on B. cenocepacia physiology and antibiotic resistance. We modified asynthetic cystic fibrosis sputum media (SCFM) to represent the acidic pH and high zinc and ironconcentrations found in CF sputum (SCFM-FeZn). We found that elevated iron and acidic pHincreased B. cenocepacia growth rate, and more strikingly decreased susceptibility toantimicrobials used clinically to treat CF infections. We studied B. cenocepacia internal pHhomeostasis and found that it maintains a neutral internal pH when exposed to mildly acidicexternal pH (5.50). We also assessed the effect of B. cenocepacia growth on the SCFM externalpH. B. cenocepacia cultured at pH 6.8, maintained an external pH of ~6.5, and when culture at pH5.5, it increased to 6.5. Using comparative transcriptomics and metabolomics analysis, weidentified 990 differentially expressed genes, and 23 differentially abundant metabolites insupernatants at acidic compared to neutral pH. Some of these genes and metabolites were involvedin aromatic amino acid metabolism. One gene (trpE) with increased expression encodes an enzymein the tryptophan biosynthesis pathway. A trpE deletion strain showed decreased growth in SCFM-FeZn and was a tryptophan auxotroph. All in all, this work demonstrates that acidic pH in thesputum environment modulates B. cenocepacia antimicrobial susceptibility and triggers molecular mechanisms associated with pathogenicity and virulence. Understanding B. cenocepaciaphysiology and antimicrobial susceptibility in the CF nutritional environment could help improvesusceptibility testing in the clinical environment, and pave the way to design new antimicrobialtherapies against B. cenocepacia infections.

View record

The helical morphology of enteropathogenic Campylobacter jejuni is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by cleavages by the LD-carboxypeptidase Pgp2 and the O-acetyl esterase Ape1 within the periplasm. In this thesis, I used X-ray crystallography, biochemical and genetic methods to investigate the interaction interfaces between these two enzymes and PG to select sites for remodeling to generate a helical cell shape. Pgp2 uses two PG binding sites for enzyme activity. The Pgp2 structure consists of LD-carboxypeptidase (LD-CPase) and NTF2 domains, each contains a pocket formed by conserved residues. The LD-CPase pocket also contains the catalytic triad. The nucleophile Cys174 is confirmed to be essential for Pgp2 activity and helical cell shape. The NTF2 pocket, ~40 Å away from the triad, is lined with charged and hydrophobic residues important for full Pgp2 activity and helical shape. Site-directed mutagenesis demonstrates that residues in both binding sites are required for generating helical cell shape. NMR spectroscopy and PG pull-down assays unequivocally demonstrate that both pockets are PG binding sites. Since Pgp2 is likely to form a dimer in C. jejuni, I expect up to four PG binding sites in the dimer. I propose Pgp2 recognizes the tertiary structure of PG involving both the LD-CPase domain and the accessory NTF2 domain to induce a helical cell shape. The Ape1 crystal structure is composed of a SGNH hydrolase domain, a CBM35 domain, and bound acetate located next to the predicted oxyanion hole. Deacetylase activity by Ape1 is assisted by residues derived from loops on the adjacent CBM35 domain. Residues Gln105, Asn121 and Arg123 of the CBM35 domain are hydrogen bonded to the loop forming the oxyanion hole. A model of the an Ape1-hexasaccharide complex suggests an orientation of PG in the active site that diverges from other members of the SGNH superfamily. I propose Ape1 activity is dependent on the length of the PG glycan strand to modulate cell wall homeostasis. Collectively, my thesis contributes to the knowledge about selective PG binding by PG hydrolases as one mechanism to control cell wall remodeling for generating helical shape.

View record

The dearth of new antibiotics and the widespread emergence of multi-drug resistant (MDR) bacteria have created a global crisis in medicine and highlighted the drastic need for novel antimicrobial agents. Natural clay minerals with a long history of therapeutic and biomedical applications have recently received increasing attention due to recent studies on their potent antibacterial activity. Kisameet clay (KC), a natural clay deposit found in British Columbia, Canada, has been used by local First Nation people for medicinal purposes for generations. This research investigates the antimicrobial properties of KC, the spectrum of activity, and the mechanism(s) underlying its antibacterial action. In order to characterize KC antimicrobial activity and define its active components, a series of integrated microbiological, chemical, and mineralogical studies have been performed. This study revealed that aqueous suspensions of KC exhibit in vitro broad-spectrum antimicrobial activity against a variety of MDR bacterial pathogens, including the ESKAPE pathogens and Cystic fibrosis clinical isolates. Moreover, two major fungal pathogens, Candida albicans and Cryptococcus neoformans were also susceptible to KC. In addition, KC aqueous leachates (KC-L) show potent bactericidal activity in which low-pH plays a key role. Treatment of KC minerals and KC-L with cation-chelating agents indicates roles for divalent and trivalent cations, more specifically iron and aluminum. Further studies suggest that the bactericidal activity of KC-L is due to multiple modes of action. The low-pH buffered environment, rich in a combination of released metal ions, can synergistically challenge treated bacteria to maintain their metal homeostasis, while aluminum-related impairment of outer membrane (OM) permeability may exacerbate this situation. KC-L can, concurrently, stress multiple bacterial components, cause metal intoxication and consequential cell damage, impair OM and destabilize the cell membrane structure. Furthermore, it induces oxidative stress, generates hydrogen peroxide, and damages DNA, which collectively leads to lethal pleiotropic effects in treated bacteria. Further studies detected KC-L-related transcriptional modulation in oxidative- and envelope- stress responses, DNA damage, metal detoxification pathways, or efflux pump function. Better understanding of the principal components of KC antibacterial activity may permit the formulation of defined, active preparations of this natural clay mineral for therapeutic applications.

View record

Staphylococcus aureus is a common member of the human microbiome, but is an opportunistic pathogen that can cause a variety of infections. Critical to the growth and survival of S. aureus during infection is acquisition of iron from the host. However, host iron availability is restricted to effectively suppress microbial growth as a type of innate, nutritional immunity. Mechanisms used by S. aureus to access the host iron pool include lysing erythrocytes to liberate hemoglobin for heme uptake and through the secretion of staphyloferrins, which are iron-chelating siderophores to scavenge iron from the host. The multiplicity of iron uptake systems S. aureus possesses is likely a reflection of the varied host environments that S. aureus can colonize or infect. However, the spatiotemporal regulatory mechanisms by which S. aureus adapts to changing iron availability over the course of infection are ill-defined. SbnI is a heme-dependent regulator of staphyloferrin B (SB) biosynthesis suggested to mediate between iron-uptake modes.In this thesis, study of the structure SbnI revealed homology to a free L-serine kinase, SerK, from Thermococcus kodakarensis. Biochemical assays and characterization of a serC mutant of S. aureus showed that SbnI is an ATP dependent L-serine kinase required for production of the SB precursor O-phospho-L-serine. SbnI kinase activity enables SB biosynthesis in environments where S. aureus catabolism is primarily reliant on amino acids, as in abscesses. Characterization of heme binding by SbnI and heme transfer reactions with IsdI and IsdG, two heme degrading enzymes, were used to construct a model of heme-binding by SbnI for regulating heme-SB uptake. This model is consistent with a modest effect of heme-binding on SbnI kinase activity. Heme transfer rates were measured from ChdC, the terminal enzyme in heme biosynthesis, to SbnI, IsdG, and IsdI to delineate an intracellular network of heme sensing and trafficking proteins that are likely required for regulation and adaptation to the dynamic host environment.

View record

Staphylococcus aureus requires iron as a nutrient and uses uptake systems to extract iron from the human host. S. aureus produces the iron-chelating siderophore staphyloferrin B (SB) to scavenge for available iron under conditions of low iron stress. Upon iron-siderophore re-entry into the cell, iron is separated from the siderophore complex to initiate assimilation into metabolism. To gain insight into how SB biosynthesis is integrated into S. aureus central metabolism, the three SB precursor biosynthetic proteins, SbnA, SbnB, and SbnG, were biochemically characterized. SbnG is a citrate synthase analogous to the citrate synthase enzyme present in the TCA cycle. The crystal structure of SbnG was solved and superpositions with TCA cycle citrate synthases support a model for convergent evolution in the active site architecture and a conserved catalytic mechanism. Since L-Dap is an essential precursor for SB, the biosynthetic pathway for L-Dap was elucidated. A combination of X-ray crystallography, biochemical assays and biophysical techniques were used to delineate the reaction mechanisms for SbnA and SbnB, demonstrating that SbnA performs a β-replacement reaction using O-phospho-L-serine (OPS) and L-glutamate to produce N-(1-amino-1-carboxy-2-ethyl)-glutamic acid (ACEGA). Oxidative hydrolysis of ACEGA catalyzed by SbnB produces α-ketoglutarate and L-Dap. Detailed analysis of the substrate specificity of SbnA revealed that OPS binding and conversion to the PLP-α-aminoacrylate intermediate in SbnA induced a conformational change and formation of a second substrate binding pocket for L-glutamate. Furthermore, L-cysteine was identified as a competitive inhibitor of SbnA activity, revealing a link between iron uptake and the oxidative stress response in S. aureus. IruO was examined for its role in Fe(III)-siderophore reduction. Utilizing a combination of visible spectroscopy and enzyme kinetics, a mechanism for electron transfer was proposed. IruO was demonstrated to reduce iron bound hydroxamate-type siderophores to release Fe(II) using NADPH as the electron donor. Under anaerobic conditions, IruO formed a stable FAD semiquinone intermediate that mediates a single electron transfer from the FAD to the Fe(III)-siderophore complex. These studies have shown how SB precursors are synthesized and led to the development of models for SB biosynthesis integration into central metabolism under conditions of low iron stress.

View record

Gram-positive Staphylococcus aureus is a common member of the normal human flora, but can also cause serious infections. Survival and growth of S. aureus is dependent on the acquisition of iron from the host, wherein the majority of iron occurs as part of the heme molecule in the oxygen-carrier protein hemoglobin (Hb). S. aureus possesses a system of proteins designed to use heme and hemoglobin as an iron source: the iron-regulated surface determinant (Isd) system. IsdB is the primary Hb receptor and extracts heme from Hb at the cell surface for transfer to IsdA or IsdC, which then transfer it to the membrane transporter for internalization. IsdB contains two NEAT domains (IsdB-N1 and IsdB-N2) which were hypothesized to carry out the Hb-binding, heme binding and heme transfer functions of the protein.Heme binding by IsdB-N2 was characterized biochemically and the crystal structure of heme-reconstituted IsdB-N2 was solved. IsdB-N2 bore the canonical eight-stranded β-sandwich NEAT domain fold and used a conserved Tyr residue to coordinate heme-iron, as well as a non-conserved Met residue, resulting in a novel Tyr-Met hexacoordinate heme-iron. Biochemical differences between equivalent mutations produced in IsdBN² and IsdBN¹N² introduced the possibility of intraprotein domain interactions.The molecular mechanism for heme transfer from IsdB-N2 to IsdA-N1 was investigated using stopped-flow spectroscopy and the kinetics of heme transfer from IsdB-N2 to IsdA-N1 were modeled. The rate of heme transfer between the isolated NEAT domains was similar to that measured for the full-length proteins.Only a recombinant construct with both domains in a contiguous unit (IsdBN¹N²) could bind Hb with high affinity. Spectroscopic analysis demonstrated that both domains were also required to extract heme from Hb. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed heme transfer from Hb to IsdA at a rate 370-fold slower than heme transfer from IsdBN² to IsdAN¹, revealing that heme transfer from Hb to IsdB is the rate-limiting step in this pathway. Finally, the serum Hb-binding protein haptoglobin blocked heme uptake from Hb by IsdB, revealing new areas for exploration of function. These studies provide insight into mechanisms of host-pathogen interactions during infection.

View record

A novel ferritin was identified in marine pennate diatoms, unicellular photosynthetic organisms that play a major role in global primary production and carbon sequestration in the deep ocean. The expression of the iron storage and detoxifying protein ferritin is thought to facilitate the blooming of pennate diatoms after iron fertilization in the open ocean.X-ray structures of Pseudo-nitzschia multiseries ferritin (PmFTN) from crystals soaked for various durations in ferrous iron and zinc sulfate revealed three distinct metal binding sites; sites A and B comprise the catalytic ferroxidase centre, and site C forms a pathway leading toward the central cavity where iron storage occurs. In contrast, crystal structures derived from anaerobically grown and ferrous iron soaked crystals revealed only one ferrous ion occupying site A. Together with kinetic analysis, these studies suggest a model of stepwise iron binding to the ferroxidase centre of PmFTN followed by a very fast iron oxidation phase and partial mobilization of iron from the ferroxidase centre. Using a combination of rapid reaction kinetics and high resolution crystallography, the function of site C was investigated with site C and site B/C variants. Glu130, a site B/C ligand, functions in stabilizing Fe(III) bound at the ferroxidase centre and as a consequence reducing the rate of mineralization. Furthermore, Glu44, a site C ligand, is shown to be important for limiting the rate of post-oxidation reorganisation of iron coordination. Iron was observed within the B-channels, first identified in prokaryotic ferritins and BFRs, of the E44Q variant of PmFTN and provides the first evidence that these channels are possible routes for Fe(II) entry into the cavity. The anaerobic crystal structure of the bacterioferritin from E. coli (EcBFR) revealed two Fe(II) at the ferroxidase centre sites A and B. In comparison with PmFTN, differences in ferrous iron binding and reaction rates are further evidence that in EcBFR a distinct mechanism is in operation.Clearly, PmFTN shows some characteristics of bacterial ferritins. Moreover, retention of iron at the ferroxidase centre at the expense of mineralization points to a role for this diatom ferritin in facilitating short term rather than long term iron storage.

View record

Campylobacter jejuni and Escherichia coli strain F11 are two Gram-negative pathogens with a versatile armament of iron uptake systems to cope with the fluctuating host nutrient environment. Our current understanding of Gram-negative iron uptake systems focuses heavily on a prototypical scheme involving a TonB-dependent outer membrane receptor and an ABC transporter, with little knowledge on systems that do not fall neatly into this paradigm. The primary focus of this thesis is the characterization of three such atypical iron uptake proteins from C. jejuni (ChaN and P19) and pathogenic E. coli (FetP).C. jejuni ChaN is a 30 kDa, iron-regulated lipoprotein hypothesized to be involved in iron uptake. The crystal structure of ChaN reveals that it can bind two cofacial heme groups in a pocket formed by a ChaN dimer. Each heme iron is coordinated by a single tyrosine from one monomer and the propionate groups are hydrogen bonded by a histidine and a lysine from the other monomer. Analytical ultracentrifugation studies demonstrate heme-dependent dimerization in solution. Cell fractionation of C. jejuni shows that ChaN is localized to the outer membrane. Based on these findings, the predicted in vivo role of ChaN in iron uptake is discussed.C. jejuni cFtr1-P19 and E. coli FetMP are homologous iron-regulated systems also proposed to be iron transporters. Through growth studies in both organisms, we show that P19 and FetMP are required for optimal growth under iron-limited conditions. Furthermore, metal binding analysis demonstrates that recombinant P19 and FetP bind both copper and iron. Dimerization of P19 is shown to be metal dependent in vitro and is detected in vivo by cross-linking. Through x-ray crystallography, we have determined the structures of P19 and FetP with various metals bound, thus revealing the locations of the highly conserved copper and iron binding sites. Additionally, the crystal structure of FetP reveals two copper positions in each binding site that is likely functionally important. Through mutagenesis, residues contributing to the alternative copper positions were identified. Together, these studies provide insight into the mechanism of iron transport by the two systems and allow for the development of functional models.

View record

Iron uptake systems are paramount to the survival of many organisms. Pathogenic bacteria are faced with the especially daunting task of acquiring essential iron within their host environment. Staphylococcus aureus is a Gram-positive bacterial pathogen and one of the most common causes of bacterial infections in hospitals. In addition, multi-drug resistant S. aureus isolates are emerging and now constitute the majority of isolated strains from clinical settings. The prevalence of S. aureus is attributed, in part, to its ability to specifically use most host iron sources for growth. S. aureus uses high affinity uptake systems for many different forms of iron in the human body with the source preference varying through the time course of infection and the tissues infected. To gain insight into iron binding and import by S. aureus, surface receptors from the iron surface determinant (Isd) heme uptake system and the staphyloferrin A siderophore uptake systems (unfortunately named heme transfer system (Hts)) were studied. The systems use distinct methods for ligand import. In the Isd system, heme is received and relayed through cell wall anchored proteins (including IsdA) to the substrate binding protein (IsdE) for import through the permease. Crystal structures of IsdA and IsdE in complex with heme, in concert with in vitro heme transfer kinetics contributed to the development of a heme transfer model for NEAT domains. In contrast to heme uptake, staphyloferrin A is bound directly at the substrate binding protein (HtsA). HtsA and IsdE are homologous membrane anchored binding proteins and both receive and deliver the iron-complex to the permease. Crystal structures and ligand affinity measurements of IsdE and HtsA reveal distinct mechanisms for ligand reception and specificity. Furthermore, crystal structures of open and closed conformations of HtsA highlight unique structural changes proposed to enable discrimination by the permease of ligand-bound and -free receptor. These studies provide insight into iron import in S. aureus, which have contributed to the development of models for heme and siderophore transport from the cell surface to the permease.

View record

Pathogenic bacteria acquire essential iron using specialized iron acquisitionsystems, such as the FbpABC transport system. The periplasmic FbpA protein deliversiron to the ABC transporter. FbpA proteins have two domains with the iron binding sitelocated at the domain interface. A flexible inter-domain hinge region facilitates substrate dependent conformations. In general, the closed conformations are observed for holo FbpA proteins whereas the apo proteins exhibit increased hinge motion relative to the closed conformation. Closed conformations are likely important for initiating iron translocation across the inner membrane permease. Important bacterial pathogens such as Campylobacter jejuni and Bordetella pertussis contain previously uncharacterized FbpA proteins. Using phylogenetic analyses, six FbpA classes were defined which vary in conservation of iron site ligands and utilization of a synergistic anion. Class I includes theanion-dependent neisserial FbpA (nFbpA). This thesis characterizes the Class III FbpAfrom Campylobacterjejuni (cFbpA) and the Class II FbpA from Bordetella pertussis(bFbpA). Visible spectroscopy showed high affinity iron binding of cFbpA. X-raycrystallography showed anion-independent iron coordination by cFbpA using a histidineand four tyrosine residues. Confonnational analyses in solution by small angle x-rayscattering (SAXS) showed that cFbpA undergoes limited hinge motion in solution upon substrate binding. Furthermore, an iron uptake role is supported as a cJbpA deletionstrain, constructed from C. jejuni 81-176, exhibited impaired growth under iron-limitedconditions. Characterization of bFbpA by visible spectroscopy showed high affinity iron binding with carbonate, citrate and oxalate. Distinct holo conformations compared with the apo conformation were observed for bFbpA depending on the synergistic anion. The closed conformation holo bFbpA crystal structure shows iron coordination by carbonate and three tyrosine residues. SAXS analyses also showed that oxalate and citrate treated holo bFbpA exhibit distinct conformations from apo bFbpA in solution. Furthermore,bFbpA undergoes large hinge motion in solution similar to nFbpA. Models for irontransport are proposed in which these bob complexes of bFbpA and cFbpA arecandidates for initiating productive interactions with the permease.

View record