Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 79-95). Also available in print.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

The intracellular human pathogen Salmonella enterica serovar Typhi (S. typhi) causes the systemic disease known as typhoid fever. This disease afflicts approximately 17,000,000 people every year, of which over 600,000 cases are fatal. / Sequencing of the S. typhi genome has allowed a better understanding of the pathogenesis caused by this bacterium. In silico research on the genome sequence identified three open reading frames, termed prpZ gene cluster, present in the Ty2 and multi-drug resistant CT18 strains of S. typhi but absent in all other sequenced serovars of S. enterica. Further analysis of this gene cluster revealed that the three genes are transcribed as an operon that encodes two eukaryotic-like Ser/Thr kinases (PrkX and PrkY) and a protein phosphatase 2C (PP2C) (PrpZ). / A previous study has shown that the recombinant His-PrpZ protein has all the hallmarks of a PP2C. Typically, PP2Cs hydrolyze phosphoserine and phosphothreonine residues. In addition, His-PrpZ was found to hydrolyze phosphotyrosine residues, making it a dual specificity phosphatase. A subsequent investigation implicates the prpZ gene cluster in S. typhi virulence as the survival of a prpZ operon deletion mutant is compromised after 48 hours of macrophage infection when compared to wild type bacteria. / It is clear from these results that the prpZ operon plays a role in the pathogenesis of S. typhi. To determine the role of these three genes in virulence, an in vitro characterization of PrkY was carried out as well as an examination of the possible physiological roles of PrpZ. / We have demonstrated that PrkY is an active protein kinase capable of phosphorylating artificial substrates in the presence of Mg2+ and/or Mn2+. Optimal phosphorylation of substrates is achieved in the presence of 5mM Mg2+ at pH 8.0. In addition, we have identified a putative interaction between PrkY and PrpZ, leading to an inhibition of the kinase activity of PrkY. While exploring the possible physiological functions of PrpZ, we have found that this protein is secreted by Ty2 S. typhi in both LB and in the low pH, low phosphate and low Mg 2+ LPM medium. / These findings suggest that PrkY and PrpZ may have antagonistic effects in a S. typhi specific virulence pathway involved in the modulation of host cell signaling by secreted bacterial virulence factors.

Reduced activity of the N-methyl-D-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine site on the NR1 subunit that may be a promising therapeutic target for psychiatric illness. Recently, D-serine has been discovered to be a high-affinity endogenous activator of the NMDAR glycine site. Levels of D-serine in the brain are controlled by its synthesis enzyme serine racemase (Srr) and its catabolic enzyme D-amino acid oxidase (DAO). This work investigates the NMDAR glycine site, D-serine, and D-serine-regulatory enzymes Srr and DAO in the pathophysiology and treatment of symptomatology relevant to schizophrenia and other psychiatric disorders. Pharmacological and genetic mouse models were used to alter glycine site function and D-serine availability. Behavioral responses in these models were assessed. Administration of exogenous D-serine and the glycine transporter 1 (GlyT-1) inhibitor ALX-5407 improved performance of C57BL/6J mice in behavioral tests examining prepulse inhibition (PPI) or latent inhibition (LI). These compounds also reversed impairments induced by the NMDAR antagonist MK-801, and produced similar beneficial effects to the classical atypical antipsychotic clozapine. Mice carrying a point mutation that leads to diminished NMDAR glycine site function demonstrated abnormally persistent LI and deficits in social approach and spatial recognition that were reversible by D-serine or clozapine administration. Similarly, mutant mice that lacked Srr function and had a severe reduction in D-serine displayed impairments in sociability, PPI, spatial recognition and memory. Behavioral deficits in mice without Srr were exacerbated by MK-801 and rescued by treatment with D-serine or clozapine. A genetically-induced loss of DAO function in mice resulted in the elevation of brain D-serine levels, and produced improvements in spatial reversal memory and extinction of a learned response in the Morris water maze, consistent with the effects of exogenous D-serine application in wild-type mice. Thus, deficiencies in NMDAR glycine site function and D-serine availability produce behavioral disturbances that are relevant to the negative and cognitive symptoms of schizophrenia. Activation of the NMDAR glycine site by D-serine, GlyT-1 inhibition, or diminished DAO activity may be beneficial for the treatment of schizophrenia and other psychopathologies involving cognitive dysfunction and persistent repetitive behaviors.

The O-acetylation of peptidoglycan (PG) is currently known to occur in greater than 50 eubacterial species, including numerous pathogens. This modification, which occurs at the C-6 hydroxyl of the N-acetylmuramoyl residues within the heteropolymer’s glycan backbone, serves as a cell wall autolytic regulatory mechanism, and contributes to pathogenesis and persistence within a host. Despite these significant physiological and pathobiological roles however, the identity of the pathway(s) responsible for the modification was only recently elucidated, for which two unrelated systems were identified, viz., the O-acetylpeptidoglycan (OAP) cluster-encoded multi-component system typical of Gram-negative species and the singular OatA of Gram-positives. As part of the OAP PG O-acetylation system, our group previously identified O-acetylpeptidoglycan esterase (Ape) as an enzyme responsible for the removal of the modification, permitting the continued metabolism of the PG sacculus. Herein, studies were performed to assess the postulated viability of this class of enzyme as a novel antibacterial target. Specifically, recombinant Ape1 from Neisseria gonorrhoeae was purified to homogeneity and the inhibitory effect of purpurin, a natural product identified as such, evaluated in detail. Kinetic analysis demonstrated that the compound elicited a competitive mode of inhibition (Kic ~3.7 μM), while the in vivo treatment of an array of environmental and pathogenic species was found to result in growth arrest for those cells containing both O-acetylPG and Ape. Evaluation of modification levels, cell wall morphology, and viability indicated a bacteriostatic effect. Taken together these data provide proof of principle that this class of enzyme presents a worthy therapeutic target. In addition to the presence of an Ape, the OAP system further differs from that of OatA through the use of two PG O-acetyltransferases. While purported to be mutually exclusive and evolutionarily divergent, in silico genomic analyses indicated their potential copresence in Bacillus anthracis and other closely related organisms. Indeed, purpurin-mediated differential growth inhibition between several such isolates and other bacilli indicated Ape activity therein. To investigate this possibility, the hypothetical Ape3 protein from Bacillus cereus ATCC 10987 was overproduced, purified, and its function assessed. Data from activity assays involving natural and synthetic substrates indicated that the protein possesses basal esterase activity in vitro. Phenotypic analysis of B. anthracis mutants deficient in each of the organism’s putative integral membrane PG O-acetyltranslocases subsequently indicated that Ape3 preferentially functions as a PG O-acetyltransferase (Pat) in vivo and that the OAP-mediated system is required for the separation of daughter cells following division. In addition, the presence of an Oat homologue was also confirmed. Thus, this is the first report of a bacterium known to possess both types of PG O-acetylation systems. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Macrophages are active participants in many important biological processes, including antimicrobial activity, tumour surveillance, apoptotic cell clearance, homeostasis and wound healing. The activity of all cells is under the direct influence of their genetic makeup and macrophages are no exception. Natural resistance-associated macrophage protein 1 (Nramp1, also known as SLC11A1) is a macrophage-restricted gene that confers resistance to intracellular pathogens in mice. Mitogen activated protein kinase activated protein kinase 2 (MAPKAPK-2 or MK2), a substrate of p38 MAPK, is known to influence the activation of macrophages in response to stressors, including the Toll-like receptor (TLR)-4 ligand LPS. Like NRAMP1, MK2 has also been shown to influence the efficiency of the antibacterial response. The present study evaluates the role of NRAMP1 and MK2 in TLR-mediated cytokine induction and their role in cutaneous wound healing. Mice lacking NRAMP1 are severely impaired in their rate of cutaneous wound healing. Nramp1 gene ablation has been associated with lower levels of SLPI, a protein previously demonstrated to influence the rate of wound healing in a non-redundant fashion. Macrophages derived from Nramp1-null mice are less efficient in activating p38 MAPK signaling, which results in lower levels of MK2 phosphorylation. The reduced level of p38 MAPK and MK2 activation in Nramp1-null macrophages also correlates with decreased cytokine induction in response to TLR7 ligand stimulation of these cells. Using p38 MAPK inhibitor and MK2-deficient macrophages, we demonstrate that TLR7- and TLR9-mediated cytokine induction is directly under the control of this signaling pathway. Furthermore, cytokine induction is regulated by MK2 at the post-transcriptional level. Macrophage-induced cytokines play an important role in cutaneous wound healing. Since MK2-deficient macrophages are severely impaired in their ability to induce cytokines following activation, we next evaluated the role of MK2 in cutaneous wound healing. Our results demonstrate that the rate of wound healing is significantly delayed in the absence of MK2. The level of cytokine expression in the wounds is impaired and macrophages are major players in cutaneous wound healing. Our data also show that intradermal transfer of macrophages with intact MK2 significantly improved wound healing kinetics. Overall, the studies presented in this dissertation demonstrate the importance of NRAMP1 and MK2 in the modulation of macrophage gene expression, and their important role in the control of cutaneous wound healing.

Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and characterisation of a serine oligopeptidase from Trypanosoma brucei brucei and from T. congolense. Serine peptidase activity has previously been described for T. b. brucei although the responsible enzyme was not purified to electrophoretic homogeneity. In the present study this enzyme was purified from bloodstream-form T. b. brucei by a combination of three-phase partitioning, ion-exchange, affinity and molecular exclusion chromatography. Characterisation of the enzyme revealed that it closely resembled a bacterial serine oligopeptidase, Escherichia coli oligopeptidase B, in terms of cleavage-site specificity, inhibition characteristics and molecular mass. Its overall properties indicate that it is probably a serine oligopeptidase and we have called it OP-Tb (oligopeptidase from Trypanosoma brucei). Antibodies to OP-Tb were prepared in chickens. These antibodies were used in the purification of a similar enzyme, designated OP-Tc, from T. congolense. OP-Tc closely resembled OP-Tb in its enzymatic properties. OP-Tb appears to be monomeric, with an apparent molecular mass of 80 kDa. Activity is optimal between pH 8.0 and 10.0, and is enhanced in the presence of reducing agents. Inhibition by 4-(2-aminoethyl)benzenesulfonylfluoride, 3,4-dichloroisocoumarin and diisopropylfluorophosphate indicates that the enzyme may be classified as a serine protease. While various natural and synthetic fluorogenic peptide substrates were hydrolysed by OP-Tb, larger potential substrates (proteins) were not. Studies of the digestion of naturally occurring bioactive peptides suggested that substrates were restricted to peptides smaller than approximately 4 or 5 kDa. These peptides were cleaved at the carboxy side of basic amino acid residues such as arginine and lysine. This is characteristic of a trypsin-like specificity. Because the enzyme is known to be readily released from the parasites, and because it was possible to detect OP-Tb-like activity in the blood of T. b. brucei-infected mammalian hosts, it appears that the enzyme is released into the host bloodstream where it remains uninhibited by endogenous protease inhibitors. Indeed, OP-Tb was not inhibited by mammalian plasma serpins or 012-macroglobulin in vitro. This, and the degradation of host peptide regulatory hormones in vitro, suggests that OP-Tb may have secondary, but important, extracellular roles in the pathogenesis of African trypanosomiasis. A variety of serine protease inhibitors, including inhibitors of OP-Tb were tested for their potential as trypanocidal agents. The results from both in vitro and in vivo studies, suggest that inhibitors of trypanosome oligopeptidases are promising new lead targets for drug development. Furthermore, data presented here also shows that OP-Tb is efficiently inhibited by several of the currently employed trypanocidal drugs. Thus, OP-Tb may already be a cellular target for trypanocidal drugs. If correct, this may represent an important step towards understanding the biochemical mechanisms of the trypanocidal activity of these drugs, as well as providing valuable clues as to how to improve their efficacy. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

A procedure for the preparation of spinach leaf mitochondria was developed. The procedure combines differential centrifugation, partition in dextran- polyethyleneglycol two-phase system and Percoli density gradient centri- fugation. The different steps separate the material mainly according to size, surface properties and density, respectively. No chlorophyll was present in the final mitochondrial preparation and the mitochondria were also markedly enriched relative to peroxisomes and microsomes as esti­mated from the recovery of marker enzymes. The latency of enzyme activities was used to study the apparent intactness of the mitochondrial membranes. These measurements showed that both the inner and outer mitochondrial membranes were more than 90 % intact. The mitochondria were also functionally intact since the coupling between respiration and oxidative phosphorylation was retained. The purity of the preparation made it possible to study cytochromes from leaf mitochondria. The cytochrome content of stalk and leaf mitochondria was measured in order to compare mitochondria from photosynthesizing and non-photosynthesizing tissue. The measurements were performed by difference spectroscopy both at room temperature and at liquid nitrogen temperature. Qualitatively the cytochrome content in mitochondria from stalks and leaves was identical. Quantiatively leaf mitochondria contained,on a protein basis, only half the amount of the different cytochromes as compared to stalk mitochondria. The relative content of the different cytochromes was, however, similar suggesting that the composition of the respiratory chain was the same. The photorespiratory conversion of glycine to serine takes place in the mitochondria and involves oxidative decarboxylation of glycine. The ability to oxidize glycine via the respiratory chain was present in spinach leaf mitochondria, but absent in mitochondria prepared from roots, stalks and leaf veins from the same plants. This confirmed the specific localization of the glycine oxidizing activity to photosyntheticaliy active tissue, as suggested by studies with other plant material. The conversion of glycine to serine is a complex reaction depending on the combined action of two enzymes: glycine decarboxylase and serine hydroxymethyltransferase. The effect of inhibitors on the serine hydroxy­methyl transferase activity and the rate of the glycine bicarbonate exchange reaction associated with glycine decarboxylase was studied. These reactions represent partial steps in the conversion of glycine to serine and the aim was to investigate the site of inhibition for the different inhibitors, namely, isonicotinyl hydrazide (a pyridoxa!phosphate antagonist), amino- acetonitrile, glycinehydroxamate (glycine analogues) and cyanide. The results showed that these inhibitors had a complex pattern of inhibition. The same inhibitor affected more than one site and often with an apparently different mechanism. It was, however, found that aminoacetonitrile at low concentrations specifically inhibited glycine decarboxylase and that cyanide specifically inhibited serine hydroxymethyltransferase. / digitalisering@umu

Bacterial resistance to antibiotics is an emerging epidemic throughout the world and there is a desperate need for new antibiotics and new strategies to maintain the effectiveness of current agents. ??-Lactams, such as the penicillins and cephalosporins, have been the most important class of antibiotic for several decades and represent half of the global antibacterial market, but the continued use of ??-lactams is threatened by ??-lactamases, enzymes that efficiently inactivate ??-lactams through hydrolysis. Class A, C, and D ??-lactamases use an active-site serine residue for hydrolysis and achieve turnover through an acylenzyme intermediate while the class B metallo-??-lactamases (MBLs) use a zinc-bound hydroxide as the active-site nucleophile. Two successful approaches to combat ??-lactamase-mediated resistance have involved the development of ??-lactam antibiotics which bind poorly to ??-lactamases and the combination of ??-lactams with ??-lactamase inhibitors. These strategies have been effective for overcoming resistance due to class A ??-lactamases, but the ever-increasing prevalence of extended-spectrum ??-lactamases (ESBLs), metallo-??-lactamases, and carbapenemases compromises the effectiveness of current penicillins, cephalosporins, carbapenems, and mechanism-based ??-lactamase inhibitors. Cyclobutanone analogues of ??-lactam antibiotics were explored in the early 1980s as potential inhibitors of ??-lactamases and D-Ala-D-Ala transpeptidases, but simple analogues showed only weak inhibitory activity and this approach was subsequently abandoned. The increasing threat of multidrug-resistant ??-lactamase-producing organisms in recent years, however, has inspired a re-evaluation of these inhibitors since cyclobutanones have the potential to exhibit broad-spectrum inhibition of both serine- and metallo-??-lactamases through the formation of enzyme-bound hemiketals or hydrates. 7,7-Dichloro-2-thia-bicyclo[3.2.0]heptan-6-one-4-carboxylic acid (65), a dichlorocyclobutanone that had shown modest inhibition of the class B and D ??-lactamases IMP-1 and OXA-10 in earlier work in this laboratory, was prepared in an efficient seven-step sequence from triethyl phosphonoacetate (103) with an overall yield of 28%. Initial efforts to improve upon the potency of the cyclobutanones involved functionalization at C3 and a highly stereoselective chlorination with sulfuryl chloride provided the 3??-chloro derivative 117?? in nearly quantitative yield. Elimination of HCl from 117?? was achieved under a variety of conditions and 3-alkoxy derivatives were prepared from 117?? through diastereoselective substitution reactions with alcohols. Cyclobutanones with 3??-OR substituents were found to favour an endo envelope conformation while the 3??-OR derivatives adopt the exo envelope conformation. Evidence from X-ray crystal structures and ab initio molecular orbital calculations suggests that an anomeric effect contributes to the large conformational preference of the tetrahydrothiophene ring that favours the 3-alkoxy substituent in an axial orientation. In addition, the conformation of the bicyclic system was found to have a dramatic effect on the tendency of the cyclobutanone to undergo hemiketal formation. Cyclobutanone analogues of penicillins, including 3-alkoxy derivatives, and cyclobutanone analogues of penems were evaluated against class A, B, C, and D ??-lactamases and found to be moderate inhibitors of KPC-2, IMP-1, GC1, and OXA-10. The cyclobutanones found to be most potent were those which are hydrated to a larger extent in aqueous solution. Dichlorocyclobutanones were found to be better inhibitors than dechlorinated cyclobutanones and a 3??-methoxy derivative 152??, which favours the exo envelope conformation in which the C4 carboxylate is equatorial, was found to be a better inhibitor than cyclobutanones that favour the endo envelope conformation. A 3,4-unsaturated penem analogue, 153, showed comparable potency to that of 152?? and molecular models of enzyme-inhibitor complexes indicate that an equatorial carboxylate is required for binding to ??-lactamases. An X-ray crystal structure of 152?? bound to the class D ??-lactamase OXA-10 confirms that a serine hemiketal is formed in the active site and that the inhibitor adopts the exo envelope. The biochemical data described above demonstrate that cyclobutanones can indeed act as inhibitors of serine- and metallo-??-lactamases and these cyclobutanones represent the first class of reversible inhibitors to show moderate inhibition of all four classes of ??-lactamase. Although the inhibitory potency of these compounds is modest (low micromolar IC50 values), penem analogue 153 was able to enhance the potency of meropenem against carbapenem-resistant MBL-producing clinical isolates of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia and the synergy demonstrated in these antimicrobial assays is encouraging. Synthetic studies toward other C3-alkyl and C3-thioalkyl-substituted inhibitors are described and the design and synthesis of C7-monochloro- and 7??-hydroxymethyl-7??-chloro cyclobutanone derivatives is presented.

Mast cells are innate immune cells, historically involved in allergy responses involving IgE. Through this, they have earned a reputation as a fairly detrimental cell type. Their beneficial roles remain somewhat enigmatic although they clearly have the ability to modulate the immune system. This is due to their ability to synthesise many cytokines and chemokines as well as immediately release potent granule-stored mediators. One such mediator is a serine protease, chymase, which has been targeted by pharmaceutical companies developing inhibitors for use in inflammatory conditions. In order to address roles of the proteases, information regarding their cleavage specificity using substrate phage display can help find potential in vivo substrates.  The human chymase cleaves substrates with aromatic amino acids in the P1 position and has a preference for negatively charged amino acids in the P2’ position. The molecular interactions mediating this P2’ preference was investigated by site-directed mutagenesis, where Arg143 and Lys192 had a clear effect in this selectivity. As humans express one chymase and rodents express multiple chymases, extrapolating data between species is difficult. Here, the crab-eating macaque was characterised, which showed many similarities to the human chymase including a near identical extended cleavage specificity and effects of human chymase inhibitors.  Appropriate models are needed when developing human inhibitors for therapeutic use in inflammatory conditions. The effects of five specific chymase inhibitors in development were also tested. The selectivity of inhibitors was dependent on both Arg143 and Lys192, with a greater effect of Lys192. Identification of residues involved in specific inhibitor interactions is important for selective inhibitor development. Another innate cell type, the NK cell, is important in virus and tumour defence. In the channel catfish, a serine protease from an NK-like cell, granzyme-like I, was characterised. A strict preference for Met in the P1 position was seen, and caspase 6 was identified as a potential in vivo target. This may highlight a novel apoptosis-inducing mechanism from a similar cell type has been conserved for approximately 400 myr. Here, important residues mediating chymases’ specificity and interactions with inhibitors has been addressed, as well as finding a new animal model for providing ways to combat their roles in pathological settings.