Dissertationen zum Thema „Pathogenic“

Avian Pathogenic Escherichia coli (APEC) is an important pathogen of the poultry industry, responsible for 43% of condemnations of broiler birds at slaughter and annual losses of between 1-8% of a laying flock. APEC is also a public health concern; consumption of poultry meat has been linked with urinary tract infections (UTI) in humans, and also as a reservoir of potential antimicrobial resistance genes. In this study of strain 3770, a reproductive tract associated isolate of APEC, the isolate was characterised for typical APEC virulence phenotypes, and also underwent full-genome sequencing in order to further advance our understanding of the pathogenomics of reproductive tract associated APEC infections. Additionally, a population study examined the virulence gene profiles within a population of reproductive tract associated E. coli, with a particular focus on virulence factors associated with infections in avian species and UTI in humans. It was found that 3770 exhibited characteristic extra-intestinal pathogenic E. coli (ExPEC) virulence phenotypes; serum resistant, adhesive, and able to persist in the short term within phagocytic cells. It was also able to induce reproductive tract infections via the aerosol route. The genome sequence was 5.02Mb in size with a high number of virulence genes. The most closely related E. coli was an adherent-invasive E. coli isolate associated with Crohn's Disease. The population study of reproductive tract infections revealed a high level of variance within the population, and a higher prevalence of ibeA and K1 capsule genes than seen in other populations. These results provide further evidence that there is no one single APEC pathotype. It is likely that virulence in APEC isolates is a complex relationship between the virulence profile of the bacterium and the health status of the host.

Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly-pathogenic mouse-passaged amebae (Mp) and weakly-pathogenic axenically-grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium from Mp and Ax amebae were the presence of an activity band of approximately 58kDa and 100 kDa bands susceptible to the action of cysteine and metallopeptidase inhibitors, respectively. Further roles of the peptidases during the invasion process were examined by in vitro invasion assays in the presence of inhibitors and Cysteine and metallopeptidase inhibitors were found to greatly reduce invasion through the ECM. This study establishes a functional linkage of the expressed peptidases to the invasion process, and these peptidases may serve as a candidate target for therapeutic management of N. fowleri infection.

From 1994, Helicobacter pylori was classified by WHO (World Health Organization) as a class I carcinogen and its infection has been associated to gastroduodenal disease. It colonizes more than half of worldwide population, with a prevalent infection rate in developed countries. In spite of the majority of infected people are asymptomatic, around 20% develop severe pathologies like peptic ulcers and the 1% lymphoma of the mucosa-associated lymphoid tissue (MALT) and stomach cancer. This significant epidemiological study both of the unique characteristics of H. pylori inspired many scientists, as bacteriologist, gastroenterologists, cancer and pharmaceutical scientists to understand physio-pathological aspects of this bacterium, and also microbiologist, taxonomist, microbial ecologist and molecular biologist, for a more detailed molecular approach. H. pylori, a Gram negative, microaerophilic bacteria that colonize human gastric mucosa. It is not an acidophilus bacterium and even if the stomach lumen presents inhospitable condition for most microbes, it is able to survive for a short period, sufficient to enter in the highly viscous mucosa, reach gastric epithelium, and colonize the gastro-enteric tract. H. pylori colonization is mediated by a predominant virulence factor, the flagellar motility associated to chemotaxis. To avoid its discharge in the intestinal tract by peristalsis, the bacteria establish a persistent infection inside the viscous gastric mucus film that covers the gastric epithelium. A nickel containing enzyme, the urease, hydrolyzes the urea present in the stomach to ammonia and CO2, buffering the pH of the periplasm. The most severe clinical outcomes are always associated to cag+ strains. cag-PAI is defined as the “Cytotoxic Associated Genes Pathogenicity Island” and it consists of a characteristic chromosome, flanked by transposable elements. Another important virulent factor is the vacuolating cytotoxin A, known as VacA, which induces the formation of large cytoplasmic vacuoles in gastric cultured cell lines. Moreover the iron and nickel acquisition is essential grow factors and a large number of genes are responsible of this mechanism. While the development of an efficient vaccine against H. pylori is now the aim of many researchers, the search for new specific antibiotics as a new pharmaceutical target is required for the complete eradication of H. pylori. In this thesis has been investigate the structural and function role of different pathogenic proteins involved in the H. pylori colonization of human gastric mucosa. These potential drug targets have been cloned, 8 out of 11 were expressed in a heterologous expression system, after purification, 2 of them generate protein crystals and only one was possible to characterize the molecular structure. In particular it has been elucidated a possible physiological role of CeuE (HP1561), a Class III SPB (Substrate Binding Protein), crystalized with Ni(His)2 complex and it was determined its affinity to the complex by an in vitro approach. The H. pylori flagella play a key role during infection allowing the bacterium to move through the mucous layer. The H. pylori hook scaffolding protein FlgD were cloned, expressed, purified and crystalized. A study of other purified pathogenic H. pylori factors belonging to flagellar component apparatus and transcriptional factors involved in cellular stress response has been reported. To obtain these results, different experimental approaches has been used. Bioinformatics analysis of target proteins has been performed to predict the best candidates for a crystallographic study and for genetic construction design. Molecular cloning in plasmid vectors has been performed from PCR amplification. The expression conditions were optimized and performed in E. coli, a heterologous system. The solubility of recombinant proteins were checked and obtained also with protein refolding methods. Different purification techniques were used in order to obtain pure protein. Target characterization was performed due analytical gel filtration, UV spectroscopy, DLS (Dynamic Light Scattering) and CD (Circular Dichroism). The proteins were concentrated to crystallization trials. The protein crystals obtained were analyzed at ESRF synchrotron (Grenoble, France). Functional in vitro approaches were performed using fluorescence spectroscopy, SPR (Surface Plasmon Resonance) and Mass spectroscopy. In the second chapter is described the three dimensional structure of a H. pylori pathogenic protein crystalized in presence with its possible physiological substrate. HP1561 (CeuE) is a H. pylori protein predicted to be an ABC transporter component, periplasmic iron-bind transporter. Recently it was published that CeuE and fecDE genes of H. mustelae encode for a nickel and cobalt acquisition system. In Gram-negative bacteria, nickel uptake is guaranteed by multiple and complex systems that operate at the membrane and periplasmic level. H. pylori employs other yet uncharacterized systems to import the nickel required for the maturation of key enzymes, such as urease and hydrogenase. To understand this contradiction of the data about Ni2+ acquisition system in H. pylori CeuE was cloned, expressed, purified, crystallized and its structure determined. Identity between the sequences of the two Helicobacter is 44%. The two Histidine residues (H103 and H197), potentially involved in Siderophores/Ni2+ binding coordination in H. pylori CeuE, are partially conserved. The His corresponding to H. pylori position 103 is conserved, whilst His197 is replaced by a Leucine. In order to check, if this substitution influence the binding of siderophores/Ni2+, the mutant of H. pylori CeuE H197L was than produced and purified. The crystal structure of H. pylori CeuE has been determined at 1.65Å resolution using the SAD method, in Apo-form and in complex with Ni(His)2. It comprises two structurally similar globular domains, each consisting of a central five-stranded β-sheet surrounded by α-helices, an arrangement commonly classified as a Rossmann-like fold. Structurally, H. pylori CeuE belongs to the class III periplasmic substrate-binding protein. Crystallographic data, fluorescence binding assays and SPR analysis allow to exclude a role of the protein in the transport of VitB12, heme, enterobactin and isolated Ni2+ ions. On the contrary, the crystal structure of the protein/Ni(L-His)2 complex and dissociation constant obtained by SPR technique suggests that H. pylori CeuE binds and transport nickel in vivo thanks to the formation of a Ni2+/histidine complex or to some ligand that mimics it. In the third chapter is presented the study of FlgD, a flagellar component involved in the formation the extracellular complex, the flagellar hook. The motility of H. pylori is considered a colonization factor, due the fact that less motile strains are less able to colonize or survive in the host than full motile strains. In the flagellum machinery are involved more than 50 genomic genes for regulation and assembly. The three major components are the filament, the hook and the basal body. FlgD is not present when the flagellum is completed, but plays a key role during the assembly. Therefore, it has been classified as the hook-scaffolding protein, considering it also as the hook capping protein, interacting with FlgL and FlgK and the basal body rod – modification protein. In H. pylori G27 strain FlgD correspond to the gene hp0858 that was amplified from purified genomic DNA and cloned in an expression plasmid vector. The protein was produced in E. coli BL21 in reach medium ad it resulted to a soluble protein. DLS and analytical gel filtration confirm the oligomeric state of FlgD that resulted to be a tetramer in solution. The protein was concentrated to 30g/l and crystalized after a couple of month of incubation. The crystals had diffracted at 2.7Å of maximum resolution. For molecular replacement approach was used homology modeling. Different molecular models were built to fit experimental diffraction data. The secondary structure of the generated models was fitted with experimental CD spectra, where FlgD resulted to have around 12% of helices and 45% of β-sheets (190-260nm). Crystallographic statistics do not properly converged to a positive molecular refinement with the tested models. To solve FlgD structure are necessary crystals of recombinant Selenomethionine FlgD that was expressed, purified and crystalized. In the fourth chapter are reported H. pylori pathogenic proteins that had been characterized. These proteins could be divided in two groups, the first one of flagellar proteins and the second of cellular stress response factors, in collaboration with Professor V. Scarlato of the department of Biology of Bologna University. FliN is a cytosolic protein, localized in the C ring of the flagellar basal body. It interacts with the other two components FliM and FliG. Missense or mutation of fliN had been associated to non-motile strains. It has been reported that regulates the clockwise/counterclockwise switching of flagella. H. pylori FliN was cloned, expressed and purified from the inclusion body after refolding. Oligomerization after refolding was tested by DLS and analytical gel filtration. The protein resulted to be poly-disperse in solution and no protein crystals have been obtained. FliD is the filament capping protein and it was observed that interact with FliT that is not only a flagellar type III substrate specific export chaperone but also inhibits the expression of fliD thought its specific interaction with the master regulator FlhD4C2 complex. In order to analyze possible structure of the co-crystalized FliD-FliT, it was plan to co-express these proteins. Both were cloned with a different affinity purification system, but only FliT was possible to express and purify from inclusion bodies. The CD spectra presented a strong β-sheet component in the secondary structure. DLS and analytical gel filtration revealed that this protein is poly-disperse in solution and no protein crystals were be obtained. FlgN is a type III secretion chaperone and it has been reported to interact with the two hook junction protein FlgK and FlgL preventing the protein proteolysis when the flagellum is not assembled. These proteins have been cloned in different type of plasmid vectors for a co-expression experiment, but only FlgN was properly expressed in E. coli. Recombinant FlgN was purified by Ni-IMAC and resulted to be soluble in solution. The protein was characterized by analytical gel filtration, DLS and CD. The protein resulted to be a monomer in solution with a 30% of not defined secondary structure (190-260nm). FlgN was concentrated and different crystallization conditions were tested. In the latter group there are three proteins related to Heat shock response, produced when bacteria encounter stress such as the elevated temperatures, ethanol, H2O2 and acid. It was demonstrated that H. pylori Hsps play an important role during the host infection. HrcA and HspR are negative repressor of groESL and dnaK machinery. HrcA activity depends by the presence of HspR, because it is demonstrated that HrcA is not able to bind DNA in absence of HspR. These two proteins were expressed in E. coli and purified by Ni-IMAC affinity. During the concentration step, these proteins present a solubility limit influenced by the concentration. Mutagenesis of a Cys in HspR and detergent solubility screening with HrcA has been performed, but no suitable protein for crystallization trials has been obtained. Hp1026 is a gene present in the same operon of HspR (hp1025). The function of this gene has not been reported. From sequence homology was possible to identify a helicase domain and ATP-binding domain. This protein, ORF, has been expressed in E. coli and purified by Ni-IMAC affinity. Analytical gel filtration and CD has been performed to characterize this protein. The protein was a dimer in solution with a 35% of α-helices component. Crystallization trials have been performed at different protein concentrations and also in presence of its possible cofactor, ATPγS. No crystals have been obtained in tested condition. Appendix: Structural and functional study on a human protease S1P/ SKI1 The study of human S1P/SKI1 protease was performed in collaboration with Professor S. Kunz of the Institute of Microbiology, University Hospital Center and university of Lausanne, Switzerland. S1P/SKI-1 is a serine protease that belongs to the mammalian family of Proprotein Convertases (PC). The aim of this family member is to mediate the activation of different important substrates for cell live. Among these proteases, S1P has been shown to have unique substrate specificity, preferring cleavage after non-basic amino acids. Known S1P cellular targets are SREBP-2, involved in the biosynthesis and uptake of lipids and cholesterol, BDNF, ATF-6 and the surface glycoprotein of viruses belonging to the family of Arenaviridae. S1P is 118 kDa multi-domain protein; two regions of S1P have been investigated, the "Prodomain", involved in the regulation of S1P catalytic activity, and the so called "catalytic domain", which include the residues responsible for the cleavage reaction itself. Moreover it was analyzed an inactive mutant of cS1P: H249A. Also for ProD was chosen one constructs (ProD_AB and ProD_AC) involved in the affinity of the protease substrate. Hence, the sequences corresponding to the domains were synthesized as optimized genes for the expression in E. coli and sub-cloned in expression plasmids in order to obtain C-term His-tagged fusion proteins. These constructs have been expressed in E. coli, purified by Ni-IMAC and positive fractions have been collected and concentrated in order to perform crystallization trials. Unfortunately no protein crystals have been obtained in tested condition. To elucidate the role of a mutated variant of the cleavage site “C” of Pro Domain, it was performed a mass spectrometry analysis. Secreted S1P/SKI1 mutant C was purified from culture medium of HEK293 cell line was isolated by IMAC-Co. The sample, loaded in RP-HPLC, was denatured in 6 M Guanidine-HCl. The chromatographic fractions corresponding to the major HPLC peaks were dried out in a speed-vac concentrator and directly injected in the ESI source. Mass measurements were performed with a quadrupole-TOF spectrometer. Analysis of mass spectra, compared with wild-type form of S1P, allows generating a preliminary Pro Domain auto-processing profile.
Dal 1994 il batterio Helicobacter pylori è stato classificato come organismo cancerogeneno di prima classe e la sua infezione è associata a patologie gastroduodenali. Più di metà della popolazione mondiale ne è infettata con una maggiore prevalenza nei paesi sviluppati. Nonostante la maggior parte dei casi le infezioni sono asintomatiche, il 20% sviluppa gravi patologie come ulcere peptiche e nell’1% dei casi genera linfomi e gastro carcinomi. L’incidenza e le caratteristiche di questo batterio hanno ispirato batteriologi, gastroenterologi, oncologi e farmacologi per indagare gli aspetti fisiopatologici legati all’infezione, così come microbiologi, ecologi, biologi molecolari hanno cercato i fattori di virulenza coinvolti in nell’infezione. H. pylori è un batterio microaerofilico Gram negativo che colonizza la mucosa gastrica. Non è un batterio acidofilo, anche se è in grado di sopravvivere nel lume dello stomaco per un breve periodo necessario per raggiungere le cellule epiteliali spostandosi attraverso la mucosa gastrica. La colonizzazione è mediata da fattori di virulenza predominanti come la motilità flagellare associata alla chemiotassi. Per evitare che sia espulso dal tratto intestinale dalla peristalsi, il batterio H. pylori stabilisce un’infezione cronica. L’ureasi, che è un enzima nickel dipendente, che idrolizza l’urea presente in ammoniaca e CO2 tamponando il pH acido dello stomaco. I casi più gravi sono associati ai ceppi che esprimono l’isola di patogenicità cag-PAI, che consiste in un cromosoma delimitato da elementi trasponibili. Un altro importante fattore di virulenza è la tossina vacuolizzante VacA, che induce la formazione di vacuoli citoplasmatici. Anche il meccanismo di acquisizione di ferro e nickel è fondamentale per la colonizzazione batterica e dunque finemente regolata da un gran numero di geni. Lo sviluppo di un vaccino e nuovi antibiotici nutrono una costante ricerca di nuovi possibili bersagli farmacologici, necessari per completa ed efficiente eradicazione del batterio H. pylori. In questa tesi sono stati analizzati il ruolo e la struttura di alcune proteine patogenetiche del H. pylori. Questi potenziali target farmacologici sono stati clonati, otto su undici sono stati espressi in un sistema eterologo, due proteine di quelle purificate hanno generato cristalli e di una sola ne è stata definita la struttura molecolare. In particolare è stato definito un possibile ruolo della proteina CeuE (HP1561), appartenete alla famiglia delle proteine che legano un substrato, cristallizzata in presenza del complesso Ni(His)2 e definita l’affinità con lo stesso in vitro. Del flagello, che svolge un ruolo chiave durante l’infezione, ne è stata studiata la proteina coinvolta nella formazione dell’uncino FlgD che è stata clonata, espressa, purificata e cristallizzata. Inoltre è stato riportato anche uno studio di altri fattori del flagello e di alcune proteine coinvolte nella risposta allo stress cellulare. Per ottenere tali risultati sono stati utilizzati approcci differenti. Per individuare le migliori proteine candidate per uno studio cristallografico e progettare costrutti funzionali sono state effettuate predizioni bioinformatiche. Gli amplificati di PCR sono stati clonati in vettori plasmidici. Le condizioni di espressione sono state ottimizzate e fatte in E. coli, un sistema di espressione eterologo. La solubilità delle proteine ricombinanti è stata analizzata e ottenuta anche mediante refolding. Sono stati usati diversi sistemi di purificazione per ottenere un buon grado di purezza. Per la caratterizzazione proteica sono state usate come tecniche la gel filtrazione analitica, spettroscopia UV, DLS (Dynamic Light Scattering) e dicroismo circolare. Le proteine sono state concentrate e sottoposte a esperimenti di cristallizzazione. I cristalli sono stati analizzati al sincrotrone ESRF (Grenoble, France). Spettroscopia di fluorescenza, SPR (surface plasmon resonance) e spettroscopia di massa sono le tecniche utilizzate per la caratterizzazione In Vitro. Nel secondo capitolo viene decritta la struttura tridimensionale di una proteina patogenetica di H. pylori, cristallizzata in presenza del suo possibile substrato fisiologico. HP1561 (CeuE) è una proteina di H. pylori annotata come componente periplasmatico di un trasportatore ABC che lega e trasporta il ferro. Recentemente è stato pubblicato chele ceuE e fecDE di H. mustelae codificano per proteine coinvolte nel acquisizione del nickel e cobalto. Nei Gram negativi, l’acquisizione del nickel è garantita da sistemi di proteine che operano a livello di membrana e periplasmatico. Per l’acquisizione del nickel, l’ H. pylori integra diversi sistemi non ancora caratterizzati, necessari per la maturazione di enzimi chiave come l’ureasi e l’idrogenasi. Per chiarire tale contraddizione nel sistema di acquisizione del nickel nell’H. pylori, CeuE è stata clonata, espressa, purificata, cristallizzata e la sua struttura è stata risolta. L’identità di sequenza tra i due Helicobacter (pylori e mustelae) è del 44%. Le due Istidine (H103 e H197), potenzialmente coinvolte nel legame di coordinazione del sistema sideroforo/Ni2+ nel H. pylori CeuE, risultano essere parzialmente conservate. L’His corrispondente alla His103 di H. pylori è conservata, mentre His197 è sostituita da una Leucina. Al fine d’identificare se tale mutazione possa influenzare il legame sideroforo/Ni2+, è stato prodotto e purificato il mutante H. pylori CeuE H197L. La struttura molecolare di H. pylori CeuE è stata determinata con una risoluzione di 1.65 Å mediante metodo SAD, sia nella forma apo, che in complesso col Ni(His)2. Essa è costituita da due domini globulari simili, ognuno costituito da cinque foglietti-β circondati da α-eliche, comunemente classificato come Rossman fold. Strutturalmente H. pylori CeuE appartiene alla Classe III della famiglia di proteine che legano un substrato specifico (SBPs). Dati cristallografici, saggi di fluorescenza e analisi all’SPR ci permettono di escludere il coinvolgimento della proteina nel trasporto della VitB12, eme, entrobactina, e ioni Ni2+ isolati. Al contrario la struttura della proteina/complesso Ni(His)2 e le costanti di dissociazione ottenute mediante SPR suggeriscono che H. pylori CeuE lega e trasporta il nickel in vivo mediante il complesso Ni2+/His o altro ligando che lo mima. Nel terzo capitolo viene presentato lo studio su FlgD, una proteina flagellare fondamentale nella formazione di un complesso extracellulare, l’uncino del flagello. La motilità dell’H. pylori è considerata un fattore di colonizzazione, attraverso il quale ceppi meno motili hanno minori possibilità di colonizzare e sopravvivere nell’ospite di ceppi più motili. Per la formazione del flagello sono coinvolti più di 50 geni per la regolazione e l’assemblaggio delle varie componenti. Le tre componenti principali sono il filamento, l’uncino e il corpo basale. FlgD non è presente quando il flagello è maturo, ma ha un ruolo chiave durante l’assemblaggio. Perciò, è stato classificato come proteina necessaria per l’impalcatura dell’uncino (hook scaffolding protein), considerata anche proteina di testa dell’uncino (capping protein) in quanto interagisce con FlgL, FlgK e le proteine del corpo basale. Nel ceppo H. pylori G27, FlgD corrisponde al gene hp0858 che è stato amplificato dal DNA genomico purificato e clonato in un vettore plasmidico. La proteina è stata prodotta in E. coli BL21 e la proteina è risultata essere solubile. Gel filtrazione analitica e misure al DLS confermano il suo stato di oligomerizzazione, che risulta essere un tetramero in soluzione. La proteina è stata concentrata fino a 30 g/l e cristallizzata dopo un paio di mesi d’incubazione. I cristalli hanno diffratto a una risoluzione massima di 2.7 Å. Per la sostituzione molecolare è stata usata la tecnica del homology modelling. Sono stati costruiti diversi modelli molecolari per fittare i dati sperimentali. La struttura secondaria dei modelli generati è stata comparata con gli spettri di dicroismo circolare, dove FlgD è risultata essere composta da un 12% di eliche e complessivamente da un 45% di foglietti beta (190-260nm). Le statistiche cristallografiche non hanno dato convergenza positiva negli esperimenti di sostituzione molecolare con i modelli testati. Per risolvere la struttura di FlgD sono necessari cristalli di FlgD derivatizzata con Selenometionine, che è stata espressa, purificata e cristallizzata. Nel quarto capitolo sono riportate le proteine patogenetiche di H. pylori che sono state caratterizzate in questa tesi. Queste proteine possono essere divise in due gruppi, il primo delle proteine flagellarli ed il secondo delle proteine coinvolte nella risposta allo stress cellulare in collaborazione con il Prof. V. Scarlato del dipartimento di Biologia dell’università di Bologna. FliN è una proteina citosolica localizzata nell’anello C del corpo basale del flagello ed interagisce con altri due componenti FliM e FliG. Mutazioni missenso di fliN sono state associate a ceppi non-motili ed è stato riportato che regola la rotazione oraria/antioraria del flagello. H. pylori FliN è stata clonata, espresso e purificata dai corpi d’inclusione dopo refolding. Lo grado di oligomerizzazione è stato analizzato mediante DLS e gel filtrazione analitica. La proteina è risultata essere polidispersa i soluzione e non sono stati ottenuti cristalli di proteina. FliD è la proteina “capping” del filamento cellulare ed è stato osservato che interagisce con FliT, che non è solo un chaperon substrato specifico del sistema III di esporto flagellare, ma inibisce anche l’espressione di fliD attraverso l’interazione con il complesso FlhD4C2. Al fine di analizzare la struttura del complesso FliD-FliT, è stata pianificata la co-espressione di queste proteine. Entrambe sono state clonate con un sistema di purificazione differente, ma solo la purificazione di FliT è stata possibile dai corpi d’inclusione. Lo spettro di dicroismo circolare ha rivelato una forte componente di foglietti-β nella struttura secondaria. Secondo le misure di DLS e gel filtrazione analitica FliT è polidispersa in soluzione e perciò non stati ottenuti cristalli della stessa. FlgN è una proteina del sistema secrezione tipo III ed è stato osservato che interagisce in maniera specifica con le proteine di giunzione dell’uncino con il filamento FlgK ed FlgL, prevenendone la proteolizzazione prima della maturazione del flagello. Queste proteine sono state clonate in differenti tipi di vettori plasmidici, ma solo FlgN è stata efficacemente espressa in E. coli. FlgN ricombinante è stata purificata mediante Ni-IMAC è risultata essere solubile. La proteina è stata caratterizzata con gel filtrazione analitica, DLS e CD. La proteina è un monomero in soluzione con un 30% di struttura secondaria non definita (190-260 nm). FlgN è stata concentrata e sottoposta a test di cristallizzazione. Nell’ultimo gruppo ci sono tre proteine HSPs (Heat Shock Response), prodotte dal batterio quando incontra stress come elevate temperature, etanolo, H2O2 e acidi. E’ stato accurato che le HSPs di H. pylori svolgono un ruolo importante durante l’infezione dell’ospite. HrcA e HspR reprimono la trascrizione di groESL e dnaK. L’attività di HrcA è influenzata dalla presenza di HspR, in quanto è stato dimostrato che HrcA non è in grado di legare il DNA in assenza di HspR. Queste due proteine sono state espresse in E. coli e purificate con Ni-IMAC. Durante le fasi di concentrazione hanno mostrato un limite di solubilità. Mutagenesi mirata sul costrutto di HspR e screening di detergenti su HrcA sono hanno migliorato il sistema, senza però riuscire ad ottenere una condizione ottimale per la formazione di cristalli di proteina. HP1026 (ORF) è un gene presente nello stesso operone di HspR (hp1025), ma con funzione non nota. Dall’analisi della sequenza è stato identificato un dominio con attività elicasica ed un dominio legante l’ATP. La proteina è stata espressa in E. coli e purificata con Ni-IMAC. Per la caratterizzazione sono state effettuate gel filtrazione analitica e dicroismo circolare. La proteina risulta essere un dimero in soluzione con un 35% di α-elica. I test di cristallizzazione son stati effettuati scrinando diverse concentrazioni e anche in presenza del possibile cofattore, ATPγS in forma non idrolizzabile. Nessun cristallo è stato ottenuto dalle condizioni testate. Appendice: Studio strutturale e funzionale della proteasi umana S1P/SKI1 Lo studio di questa proteasi umana è stato effettuato in collaborazione con il Prof. S. Kunz dell’Istituto di Microbiologia, del Centro Universitario Ospedaliero e dall’ Univ. Di Lausanne, Svizzera. S1P/SKI1 è una serina proteasi della famiglia delle Proprotein Convertasi (PCs). Lo scopo di membri di questa famiglia è quello di mediare l’attivazione di diversi importanti substrati per la vita cellulare. Tra queste proteasi, S1P presenta una specificità di substrato, con un sito di taglio dopo un residuo non basico. Tra i target cellulari di S1P sono stati identificati SREBP-2, coinvolto nella biosintesi dei lipidi e del colesterolo, BDNF, ATF-6 e glicoproteine superficiali di virus appartenenti alla famiglia delle Arenaviridae. S1P pesa 118kDa ed è una proteina multidominio; quindi 2 regioni di S1P sono state studiate, il “Prodomain” (ProD) che regola l’attività catalitica, ed il “cathalytic domain” (cS1P) che include i residui responsabili per la reazione proteasica. Inoltre è stato analizzato un mutante inattivo (cS1P_H249A) e due costrutti per il dominio di regolazione (ProD_AB e ProD_AC). Le sequenze nucleotidiche dei corrispettivi costrutti sono state sintetizzate come geni ottimizzati per l’espressione in E. coli e subclonati in vettori plasmidici per l’espressione ottenendo proteine in fusione con una coda di 6-His. Questi costrutti sono stati espressi in E. coli, purificati con Ni-IMAC e le frazioni positive sono state raccolte e concentrate per test di cristallizzazione. Sfortunatamente non sono stati ottenuti cristalli di proteina nelle condizioni testate. Per chiarire il ruolo di una variante mutata nel sito di taglio “C” del dominio di regolazione è stata effettuata una analisi di spettrometria di massa. La proteina secreta S1P mut C (sS1P_MutC, 116kDa) è stata purificata dal medium di coltura di una linea di HEK293 trasfettate e isolata con Co-IMAC. Il campione è stato denaturato in Guanidinio 6M e caricato in HPLC. Le frazioni corrispondenti ai picchi predominanti sono stati essiccati ed iniettati in spettrometro di massa (ESI-TOF). L’analisi delle masse, confrontate con la forma nativa (sS1P_WT) ha permesso di generare un profilo preliminare del pattern di processamento del dominio di regolazione (ProD) with a quadrupole-TOF spectrometer. Analysis of mass spectra, compared with wild-type form of S1P, allows generating a Pro Domain auto-processing profile.

La investigació descrita en aquesta tesi es centra en la determinació de la influència de les pràctiques de maneig del cultiu, processament i les condicions d'emmagatzematge en la qualitat microbiològica de l'enciam mínimament processat i en l'estudi de les estratègies de mitigació per millorar la seva seguretat. S'ha estudiat l'efecte del sistema de producció, orgànica o convencional, de la qualitat microbiològica d'enciam fresc. Es va avaluar la transferència i la persistència de L. innocua i E. coli O157:H7 en els fulls d'enciam i en el sòl durant la tardor i la primavera utilitzant diferents mètodes de reg contaminats artificialment i el compost. La capacitat de L. monocytogenes, Salmonella i E. coli O157:H7 per créixer en enciam envasat en tres condicions d'atmosfera diferents creades per mitjà de la utilització de pel•lícules de diferents permeabilitats a dues temperatures d'emmagatzematge va ser evaluada. Es va examinar el potencial patogènic de dues soques de S. Typhimurium DT104, amb l'objectiu de mesurar la seva capacitat per sobreviure al tracte gastrointestinal simulat i d'adherir i envair cèl•lules diferenciades Caco-2, després d’una incubació seqüencial al sòl, l'enciam i enciam tallat emmagatzemat en condicions de MAP. En aquesta tesi l'efecte de millorar la microbiota d'enciam sotmesa a les diferents etapes de pre-condicionament en la supervivència de L. monocytogenes i E. coli O157:H7 com a mètode bioconservant també va ser avaluat. Finalment, s'ha estudiat l'ús de l'addició de bioconservants com a mètode per a controlar o reduir PTA en enciam mínimament processada.
La investigación descrita en esta tesis se enfoca en la determinación de la influencia de las prácticas de manejo del cultivo, procesamiento y condiciones de almacenamiento en la calidad microbiológica de la lechuga mínimamente procesada y en el estudio de las estrategias de mitigación para mejorar su seguridad. Se ha estudiado el efecto de los sistemas de producción, orgánico o convencional, en la calidad microbiológica de lechuga fresca. Se evaluó la transferencia y la persistencia de L. innocua y E. coli O157:H7 en las hojas de lechuga y en el suelo durante el otoño y la primavera, utilizando diferentes métodos de riego contaminado artificialmente y el compost. La capacidad de L. monocytogenes, Salmonella y E. coli O157:H7 para crecer en lechuga mínimamente procesada y envasada en tres diferentes condiciones de atmósfera, creada por la utilización de películas con diferentes permeabilidades a dos temperaturas de almacenamiento fue evaluada. Se examinó el potencial patogénico de dos cepas de S. Typhimurium DT104, con el objetivo de medir su capacidad para sobrevivir al tracto gastrointestinal simulado y de adherirse e invadir a las células diferenciadas Caco-2, después de la incubación secuencial en el suelo, lechuga y lechuga cortada almacenada en MAP. En esta tesis también se evaluó el efecto de aumentar la microbiota de lechuga sometida a las diferentes etapas de pre-acondicionamiento en la supervivencia de L. monocytogenes y E. coli O157:H7 como método bioconservante. Por último, se ha estudiado el uso de la adición de bioconservantes como método para controlar o reducir los PTA en lechuga mínimamente procesada.
The research described in this thesis is focused on the determination of the influence of field management practices, processing and storage conditions on the microbial quality of fresh-cut lettuce and on the study of mitigation strategies to enhance its safety. The effect of production system, organic or conventional, on the microbiological quality of fresh lettuce was studied. The transfer and persistence of L. innocua and E. coli O157:H7 on lettuce leaves and in soil during fall and spring using different artificially contaminated irrigation methods and compost was evaluated. The ability of L. monocytogenes, Salmonella and E. coli O157:H7 to grow on shredded lettuce packaged in three different atmosphere conditions created by means of using different permeability films at two storage temperatures was evaluated. The pathogenic potential of two S. Typhimurium DT104 strains was examined, with the aim to measure their capability to survive a simulated gastrointestinal tract system and to adhere to and invade differentiated Caco-2 cells, after sequential incubation into soil, lettuce and cut lettuce stored under MAP conditions. In this thesis the effect of enhancing native microbiota of lettuce submitted to different pre-conditioning steps on survival of L. monocytogenes and E. coli O157:H7 as a biopreservative method was also evaluated. Finally, the use of adding biopreservatives as a method to control or reduce FBP in fresh-cut lettuce was studied.

The Zur (zinc uptake regulator) regulon of a range of pathogenic and non-pathogenic E. coli strains was explored by combining in silico Zur binding site predictions with experimental approaches. Initially, novel Zur-binding sites were predicted in the genomes of K-12 MG1655, 0157:H7 str. Sakai, H10407 and CFT073 using patser. The effect of both zinc depletion and zur deletion was probed in each strain by transcriptomics analysis, in order to identify direct Zur targets. Known Zur targets, including zinT, an auxiliary component of the znuABC zinc uptake system, and ykgM, an alternative non-zinc-binding ribosomal protein were up-regulated in all strains under both zinc depletion and zur deletion conditions. In contrast, up-regulation of the high affinity zinc uptake system znuABC, was more modest and variable. Transcription of pUG (ycgK), encoding a periplasmic G-type lysozyme inhibitor, was up-regulated under zinc depletion conditions and upon zur deletion in all strains, and also possessed a high-scoring predicted Zur binding site within its promoter region, leading us to believe that it is a novel member of the conserved Zur regulon. Additionally, a novel Zurregulated operon, c1265-7 was predicted in the CFT073 genome, and validated by microarray data and the use of a promoter-lux fusion. C1265 appears to be a TonBdependent heme/hemoglobin receptor, whilst C1266 and C1267 showed significant homology to components of metal uptake systems. Deletion of c1265J 7, even in the presence of a znuABC deletion, showed no clear phenotypic outcome under a range of conditions tested. C1265-7 was found to be almost exclusively distributed in ExPEC strains suggesting it is of importance to extra-intestinal survival. Additionally, the severe growth defect of CFT073 D.zur, even in zinc-deplete media suggests the CFT073 zur regulon may include extra genes involved in zinc uptake. iii ... \ ...

Immune dysfunction in spondyloarthritis (SpA) causes significant morbidity to a large number of patients. An understanding of the underlying pathological processes involved in causing the disease is essential for the development and better targeting of therapies. I have shown for the first time in patients with SpA an overall expansion of peripheral blood and synovial fluid T cells producing GM-CSF and an expansion of GM-CSF positive Th17 cells. In addition, I have shown GM-CSF to be a major effector cytokine of joint-derived innate lymphoid cells. Surface phenotyping and transcriptional analysis of the IL-17A/GM-CSF double positive cells showed that whilst they are related to classic Th17 cells, they also have a uniquely activated transcriptional profile. Additionally, I have shown IL-7 to be a promoter of GM-CSF production by CD4 T lymphocytes in vitro. However, my data shows that polymorphisms of the IL7R, which have been shown to be associated with AS in genome-wide association studies, play a functional role through cell surface expression of the IL7R on CD14 monocytes. This genotypic expression of the IL7R on monocytes is only seen after activation with LPS or TNFa. I further show CD14 monocytes expressing IL7R to be present in SpA joints. My data shows that ROR?t, the master transcription factor of Th17 cells, can be therapeutically targeted using small molecule inhibitors in-vitro. These inhibitors lead to a specific suppression of both polyfunctional and IL-17A single positive Th17 cells. The work in this thesis has highlighted several areas of novel immune biology with potential therapeutic applications in SpA and across the spectrum of related inflammatory diseases.

The ability to mount an efficient immune response should be an important life-history trait as parasitism can impact upon an individual's fecundity and survival prospects, and hence its fitness. However, immune function is likely to be costly as resources must be divided between many important traits. Whilst many studies have examined host resistance to particular parasite types, fewer have considered general immune responses. Studies that have considered general immune responses tend to do so in vertebrate models. However, the complexity of the vertebrate immune system makes the examination of evolutionary aspects of immune function difficult. Using larvae of the genus Spodoptera (Lepidoptera: Noctuidae) as a model system, this study examines' genetic and phenotypic aspects of innate immunity. The aims were to assess the levels of additive genetic variation maintained in immune traits, to consider possible costs that could maintain this variation, and to assess the role of phenotypic plasticity in ameliorating those costs. A key finding of this study was that high levels of additive genetic variation were maintained in all of the measured Immune traits. Analysis of the genetic correlations between traits revealed potential trade-offs within the immune system and between immune components and body condition. In addition, it was shown that larvae living at high densities invest more in immune function than those living in solitary conditions, suggesting that larvae can minimise the costs of immune function by employing them only when the risk of pathogenesis is high.

Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.

Acanthamoeba are free-living and opportunistic protozoa that are ubiquitous in nature. Human infection due to Acanthamoeba can involve the brain, skin, lung and eyes. However, eye infection, Acanthamoeba keratitis, is the most common infection. Acanthamoeba keratitis is a sight threatening corneal disease producing a progressive, blinding infection of the corneal surface. Several species of Acanthamoeba have been identified as human pathogens, however, current laboratory techniques for identification and speciation of Acanthamoeba are time consuming and laborious. The purpose of this study was to develop and improve current methods for the detection and differentiation of Acanthamoeha spp. Initially a number of isolates and species were obtained and their pathogenicity determined using animal model studies and cytopathic effect assays. Based on these data Acanthamoeba were classified in pathogenic and non-pathogenic groups. Using this information a variety of techniques were developed to differentiate these groups. Simple plating assays were used to differentiate pathogenic Acanthamoeba. We successfully differentiated pathogenic and non-pathogenic Acanthamoeba on the basis of physical characteristics such as growth at 37°C and ability to grow in the presence of 1 M mannitol. Morphological characteristics were observed using scanning electron microscopy. Although these were useful, not all Acanthamoeba tested could be differentiated. Due to the increased analytical sensitivity and and specificity, PCR based assays were used to identify and speciate Acanthamoeba spp. A new DNA extraction method was developed which can be directly used with the given specimen without the prior need of cultivation of parasites. This method together with Acanthamoeba specific primers makes an ideal assay for the detection of Acanthamoeba. Using this assay we were able to identify Acanthamoeba up to 5 cells. Ribosomal DNA sequencing was performed to aid in differentiation of pathogenic and non-pathogenic Acanthamoeba. Acanthamoeba isoates from different sources (environmental and eye infections) were clustered into distinct genotypes according to their rONA sequence. All tested pathogenic Acanthamoeba fell into one group indicating that virulence may be associated with specific cluster of cladistic groups of Acanthamoeba. Owing to simplicity and popularity of immunoassays as diagnostic methods we isolated antibody fragments from a phage display antibody library which can be potentially used for the clinical diagnosis of Acanthamoeba spp. These antibody fragments were tested against a range of different organisms and cell types for their specificities. In a recent study by Cao et al. (1998), a correlation between proteases and rabbit primary corneal epithelial cell damage has been shownbut the study is limited as only one isolate of Acanthamoeba was used. In our study we have shown clear differentiation between pathogenic and non-pathogenic species of Acanthamoeba on the basis of extracellular protease activity. In addition we have also observed a contact-dependent protease when whole pathogenic Acanthamoeba were added to primary corneal epithelial cell monolayers. These data will help in furthering our understanding of the basic mechanisms of Acanthamoeba pathogenicity and thus to prevent the development of infection.

Doctor of Philosophy
This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

Doctor of Philosophy
This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.

Pseudomonas aeruginosa (PA) infections in Cystic Fibrosis (CF) patients are not easily cleared due to the conversion from a nonmucoid to a mucoid phenotype. Alginate is an acetylated exopolysaccharide produced by mucoid PA that is responsible for increased resistance to antibiotics, host phagocytic killing, and propagating biofilm formation. Understanding the interaction between PA and host cells is critical to understanding chronic infection and inflammation in CF. In order to investigate this, we used A549 pulmonary epithelial cells and murine alveolar macrophages (MH-S) to examine host response to nonmucoid versus mucoid PA infection. Adhesion assays in A549 pulmonary epithelial cells revealed that mucoid PA mutants adhere poorly compared to their nonmucoid counterparts. Similarly, phagocytosis assays using MH-S infected with PA revealed that mucoid PA are increasingly resistant to phagocytosis. The alginate acetylation mutant FRD1175 is more susceptible to phagocytic killing than alginate+ FRD1. Adherence and phagocytosis of mucoid FRD1 was increased by increasing the multiplicity of infection (MOI) from 50:1 to 500:1. Furthermore, confocal microscopy revealed that mucoid PA are inherently less inflammatory than nonmucoid strains in both A549 and MH-S. Increasing the MOI of mucoid FRD1 from 50:1 to 500:1 significantly increased caspase-1 activation in MH-S but not in A549, revealing that intensity of inflammatory signaling by epithelial cells is likely independent of increased adherence. FRD1175 infection in both A549 and MH-S revealed that alginate acetylation plays a significant role in reducing inflammasome activation. Western analysis revealed that PA does not actively induce TGF-β secretion by A549 epithelial cells. Similarly, NF-κB expression was reduced in both A549 and MH-S when infected with mucoid FRD strains, but not PA from the PAO background, suggesting FRD strains have accumulated additional mutations facilitating escape of inflammation. MH-S treated with cytochalasin D to block phagocytosis were still able to activate NF-κB signaling, suggesting NF-κB activation is adherence but not phagocytosis dependent. These data increase our understanding of the various mechanisms in which mucoid PA is able to evade host immune defenses and provides insight into potential therapies to treat PA infections.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by neurofibrillay tangles and deposition of amyloid-β (Aβ) peptides. Several mutations in the gene for amyloid precursor protein (APP) cause familial AD and affect APP processing leading to increased levels of Aβ42. However, the Arctic Alzheimer mutation (APP E693G) reduces Aβ levels. Instead, the increased tendency of Arctic Aβ peptides to form Aβ protofibrils is thought to contribute to the pathogenesis.

In this thesis, the pathogenic mechanisms of the Arctic mutation were further investigated, specifically addressing if and how the mutation affects APP processing. Evidence of a shift towards β-secretase cleavage of Arctic APP was demonstrated. Arctic APP did not appear to be an inferior substrate for α-secretase, but the availability of Arctic APP for α-secretase cleavage was reduced, with diminished levels of cell surface APP in Arctic cells. Interestingly, administration of the fatty acid docosahexaenoic acid (DHA) stimulated α-secretase cleavage and partly reversed the effects of the Arctic mutation on APP processing.

In contrast to previous findings, the Arctic mutation generated enhanced total Aβ levels suggesting increased Aβ production. Importantly, this thesis illustrates and explains why measures of both Arctic and wild type Aβ levels are highly dependent upon the Aβ assay used, with enzyme-linked immunosorbent assay (ELISA) and Western blot generating different results. It was shown that these differences were due to inefficient detection of Aβ oligomers by ELISA leading to an underestimation of total Aβ levels.

In conclusion, the Arctic APP mutation leads to AD by multiple mechanisms. It facilitates protofibril formation, but it also alters trafficking and processing of APP which leads to increased steady state levels of total Aβ, in particular at intracellular locations. Importantly, these studies highlight mechanisms, other than enhanced production of Aβ peptide monomers, which could be implicated in sporadic AD.

The United States currently provides a health care system that is neither efficient nor equitable. Despite outspending the world on health care, over three-fourths of developed countries produce better health outcomes (Auerbach et al., 2000). Simultaneously, the "Ecological School of Thought" has documented the large impact that social, economic, and environmental circumstances play in health outcomes. Unfortunately, these 'ecological" studies are frequently conducted without theoretical justification, and rely solely on a cross-sectional research design and a myriad of unrelated variables. This study represents an important step towards the development of a true theory of "ecology". More specifically, we argue that the adversity associated with socio-economic disadvantage, social disorganization, and a lack of health care resources, leads to adverse health outcomes, represented by sentinel health events. This research employs both a cross-sectional (2000) and longitudinal designs (1990 – 2000) to assess the antecedents of sentinel health events in 309 United States counties. Structural Equation Modeling was the statistical technique employed in the study. Findings revealed that socioeconomic disadvantage remains a primary contributor to sentinel health. Indeed the economic growth between 1990 and 2000 was associated with increased rates of sentinel health events. Social disorganization was identified as a primary contributor to sentinel health events at a specific time point (2000), but was not significant over time (1990 -2000). Conversely, the inadequacy of health care resources was non-significant in the cross-sectional model (2000), but significant in the longitudinal model (1990 -2000). In both models, racial characteristics were fundamentally linked to ecological predictors of health We found support for the notion that sentinel health events would be reduced through economic equity and the development of healthy environments where community ties are reinforced. Less support is found for saturating given geographical areas with health care resources in order to reduce sentinel health events. Future research should be directed by the theoretical advancements made by this study. More specifically, future studies should examine independent cross-level effects, that is, through the inclusion of behavior variables as mediating factors for ecological constructs.
Ph.D.
Other
Health and Public Affairs
Public Affairs PhD

Plant roots generate electrical fields in the rhizosphere as a consequence of their ion transport activities. In this thesis it is shown that zoospores of Phytophthora palmivora and Pythium aphanidermatum exhibit electrotaxis in electrical fields ≤ 50 mV/cm, comparable in size to the physiological fields around plant roots. P.palmivora zoospores showed anodotaxis and P.aphanidermatum zoospores cathodotaxis. An experimental protocol for applying weak electrical fields is described. In this sytem zoospores suspensions are isolated from the electrodes and their products using agarose bridges. Therefore, electrotaxis was not due to movement or trapping of zoospores in chemical, oxygen, pH or inhibitor gradients established by electrolysis. The electrophoretic and electroosmotic mobilities of zoospores were measured. These forces did not influence the distribution of zoospores in electrotactic experiments at physiological field strengths. Applied electrical fields elicited topotaxis and klinokinesis of zoospores of P.palmivora. These responses may be one mechanism whereby zoospores accumulate preferentially at specific areas on plant roots. Electro-topotaxis is likely to be the result of differential charges on the anterior and posterior flagella of zoospores, causing electrophoretic orientation in an electrical field.

Microscopical examination of Candida albicans grown on contoured artificial surfaces provided evidence that hyphae responded thigmotropically to features of the growth substrate. Hyphae of C. albicans followed grooves and ridges on various artificial membranes and penetrated pores of Nucleopore filters. The thigmotropic response in C. albicans was attenuated by gadolinium ions and by verapamil suggesting that calcium uptake may be involved in thigmotropic regulation. Thigmotropism was also observed for the first time in three genera of dermatophytic fungi (Epidermophyton, Trichophyton and Microsporum) and two saprophytic fungi (Mucor mucedo and Neurospora crassa). Therefore thigmotropism may be a general feature of fungal hyphae that must forage for nutrients on surfaces and within solid materials. Since Ca²⁺ appears to be involved in the regulation of thigmotropism attempts were made to construct strains expressing the Ca²⁺ sensitive photoprotein aequorin. The apoaequorin d gene was cloned in to C. albicans and S. cerevisiae using the YPB-ADHpt expression vector. Southern analysis indicated low copy number of the plasmid in C. albicans as compared with S. cerevisiae. Aequorin was reconstituted in protein extracts of C. albicans and S. cerevisiae by supplementing them with coelenterazine. The level for C. albicans was ten times higher than for Neurospora crassa, the only filamentous fungus to be transformed with this gene so far. Aequorin was successfully reconstituted in transformed living cells, and the luminescence levels were sufficiently high to be detected when external Ca²⁺ was added to the growth medium. Transformed C. albicans cells undergoing the dimorphic transition from yeast-to-hyphal form exhibited higher resting levels of luminescence indicating that cells induced to form hyphae have higher [Ca²⁺] than yeast cells. The work presented in this thesis presents first evidence of construction of strains expressing the luminescence photoprotein aequorin in a pathogenic fungus. This method provides a non-toxic, non-invasive method for monitoring [Ca²⁺] in C. albicans.

Strains of Legionella pneumophila which were virulent for guinea pigs were passaged on laboratory media such that they became avirulent. Virulent/avirulent pairs of strains were compared in an attempt to identify potential virulence or pathogenic factors. Virulent forms multiplied within guinea pig alveolar macrophages maintained in tissue culture over 48 hours and phagosome lysosome fusion was not inhibited in these macrophages. Avirulent strains did not multiply but those produced by passage on supplemented Mueller Hinton agar (MHIH) maintained viability whereas those produced by passage on buffered charcoal yeast extract agar (BCYE) were killed. Virulent legionellae produced more flagella than their paired avirulent forms but it was considered unlikely that this was related to pulmonary virulence. Minor differences in outer membrane proteins and reactivity with various antisera were observed between virulent/avirulent pairs but these were not consistent between the L.pneumophila strains and no virulence-associated proteins were found. The lipopolysaccharide and extracellular enzyme activities of the pairs of strains were indistinguishable. The Corby strain of L.pneumophila (CV) was isolated from its intracellular in vivo environment and these bacteria compared with the same strain grown on BCYE. The in vivo-grown CV showed no change in uptake by, or intracellular replication within, alveolar macrophages but, when Western blotted and incubated with various antisera, the two types of bacteria reacted differently. There was a reduced reaction by the CV grown in vivo, suggesting a change in the number and/or type of proteins expressed when exposed to an intracellular environment. The cellular responses in the lungs of guinea pigs immediately following aerosol challenge with virulent and avirulent forms of the Corby strain were compared. The avirulent forms evoked little change in the alveolar cell populations whereas an inflammatory response to CV occurred. Polymorphonuclear Ieucocytes (PMNLs) were the principal cell type involved and the initial increase in numbers of this cell type corresponded with a transient decrease in the viablility of CV, providing evidence that virulent L.pneumophila are killed by PMNLs. The multiplication of CV which followed suggested a developing resistance to the killing. Numbers of avirulent Corby decreased rapidly and, for the MHIH passaged form (CA), this was in contrast to its intracellular survival in vitro. An attempt was made to follow the changes in cellular responses using immunolabelling techniques and flow cytometry. Difficulties with obtaining consistent, sequential samples did not allow a full interpretation of the results but the technique showed promise for such studies. The tissue destructive protease (TDP) of L.pneumophila was shown to degrade or inactivate gamma interferon, IgG and possibly interleukin 2, proteins of possible significance to the host in protection against infection with L.pneumophila. Inhibition of TDP by the protease inhibitor a2 macroglobulin was demonstrated and, prophylactic treatment of guinea pigs with the inhibitor resulted in prolonged survival (compared with untreated controls) following aerosol challenge with CV. Intracellular production of TDP by L.pneumophila multiplying within guinea pig alveolar macrophages was demonstrated by ELISA and immunogold labelling and functional activity of the enzyme purified from infected guinea pig lungs was shown. A mutant of L.pneumophila, deficient in TDP production, was compared in the guinea pig model with its TDP-producing parent and CV. The mutant and parent were considerably less virulent than CV but, despite the deficiency in TDP production, the mutant was lethal for guinea pigs. The lung damage caused by the mutant was less severe than that caused by the parent or CV and it was suggested that progression of disease and cause of death was not typical of Legionnaires' disease. An immunocompromised mouse model of Legionnaires' disease was investigated using aerosol infection of severe combined immune deficient (SeID) mice. SCID mice did not become ill when given a potentially lethal aerosol of L.pneumophila and alveolar macrophages of these mice did not support the growth of L.pneumophila in vitro. SCID mice which had been reconstituted with human leukocytes were susceptible to aerosol challenge and developed lung lesions similar to those seen in guinea pigs and humans.

Thesis (MScMedSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The current anti-TB drugs have had success in decreasing the number of deaths caused by TB, however, this success is limited by the emergence of drug resistant TB strains. Therefore, a novel TB therapy that limits the development of resistance has become necessary in an attempt to effectively control TB. The anti-TB drugs directly target mycobacterial enzymes, and potentiate the development of this resistance, and have therefore provided the rationale for this study. The aim was therefore to identify host macrophage genes that affect M. tb intracellular survival. The proposed alternative anti-TB therapy potentially involves the application of RNA interference (RNAi) and RNA activation (RNAa) biological processes that will target host genes, thereby inducing an indirect bactericidal effect. We hypothesized that macrophage genes that are differentially expressed by pathogenic and non-pathogenic mycobacterial species may be important in the regulation of M. tb intracellular survival. The lipid-rich mycobacterial cell wall is implicated in the excessive clumping of the mycobacterial cells in liquid culture. In order to minimize this, Tween 80 detergent was supplemented (mycobacteriaT). However, due to substantial evidence emphasising the detrimental effects of Tween 80 on the mycobacterial cell wall, mycobacteria were also cultured without Tween 80 (mycobacteriaNT), in order to investigate if the perturbed mycobacterial cell wall induced by Tween 80 affects the transcriptional response of macrophages. We endeavoured to develop a new method to culture mycobacteria without Tween 80 that will still generate single cells. We further hypothesized that the macrophage gene expression profile induced by mycobateriaNT differs from the response induced by mycobacteriaT. Differentiated THP-1 (dTHP-1) cells were infected with pathogenic and non-pathogenic mycobacteria (for 3 h, 24 h and 48 h with M. tb and M. bovis BCG, and 3 h and 8 h with M. smegmatis) cultured in the presence or absence of Tween 80. The expression of 12 macrophage genes, selected based on their involvement in the phagocytic pathway and autophagy, as well as their general involvement in the immune response, was determined by qRT-PCR and further analysed on the REST programme. The expression of each target gene was normalised relative to the expression of the reference gene (Beta actin). We observed that out of the 12 genes, TLR7 and VAMP7 were consistently downregulated in dTHP-1 cells infected with M. tbNT and upregulated in dTHP-1 cells infected with M. smegmatisNT. Their response to M. bovis BCG was inconsistent and not significantly different, and therefore could not be interpreted. Furthermore, CCL1 was upregulated by all the mycobacterial species. However, its expression was more pronounced in response to mycobacteriaNT, when compared to mycobacteriaT. Differential gene expression of TLR7 and VAMP7 in response to pathogenic and non-pathogenic mycobacteriaNT suggests that these 2 genes may be potential targets for RNAa-based anti-TB therapy, even though we could not conclude whether their response was specific to macrophages. In addition, the observed difference in the expression of CCL1 induced by mycobacteriaNT, compared to mycobacteriaT suggests that the perturbation caused by Tween 80 on the mycobacterial cell wall most likely affected the response of macrophages to infection with mycobacteria. Furthermore, this study has demonstrated a feasible method by filtration to generate single cells from mycobacteriaNT, which should be considered for future mycobacterial infection studies.
AFRIKAANSE OPSOMMING: Die huidige anti-tuberkulose middels se sukses lê daarin dat dit die aantal sterftes verminder maar hierdie sukses word weer beperk met die ontstaan van middel-weerstandige M.tb stamme. Daarom is nuwe middels nodig wat die ontwikkeling van middel-weerstandigheid beperk in ʼn poging om effektiewe TB behandeling te bewerkstellig. Anti-tuberkulose middels teiken hoofsaaklik mycobakteriële ensiemsisteme en ontlok sodoende weerstandigheid in M.tb stamme en dit vorm die rasionale vir hierdie studie. Die doel was om gasheer makrofaag gene te identifiseer wat M.tb oorlewing intrasellulêr bewerkstellig. Die voorgestelde alternatiewe anti-TB behandeling sal dan behels die toepassing van RNA intervensie (RNAi) en RNA aktivering (RNAa) tegnologie wat gasheer selgene teiken (inaktiveer) en sodoende ʼn bakterisidiese respons induseer. Die kanse is skraal dat mycobakterieë weerstandigheid sal kan ontwikkel onder hierdie omstandighede. Ons hipotetiseer dus dat makrofaag gene wat differensieel uitgedruk word deur patogeniese en nie-patologiese mycobakteriële spesies belangrik mag wees vir die oorlewing van M.tb intrasellulêr. Die lipiedryke selwand van mycobakterieë word geïmpliseer in die oormatige sameklomping van die bakterieë in vloeistofkulture. Om hierdie effek te minimaliseer word Tween 80 normaalweg tot die medium gevoeg (mycobakterieëT). Maar weens genoegsame bewyse dat Tween-80 die selwand van bakterieë nadelig beïnvloed, is mycobakterieë ook in die afwesigheid van Tween 80 gekultureer (mycobakterieëNT) om te bepaal of die nadelige effek van Tween 80 op die selwand die transkripsionele respons in makrofage beïnvloed post-infeksie. Dit was daarom ook ons doelstelling om ʼn nuwe tegniek te ontwikkel om mycobakterieë te kultureer in die afwesigheid van Tween 80 wat ook enkelselle sal genereer vir beter gekontroleerde makrofaag infeksie. Ons hipotetiseer ook verder dat makrofaag geenuitdrukking-profiele verskil afhangende of infeksie gedoen is met mycobakterieë wat in die afwesigheid of teenwoordigheid van Tween 80 gekultureer is. Gedifferensieerde THP-1 (dTHP-1) was geïnfekteer met patogeniese en nie-patogeniese mycobakterieë (vir 3 h, 24 h en 48 h met M.tb en M.bovis BCG, en 3 h en 8 h met M.smegmatis) gekultureer in die teenwoordigheid en afwesigheid van Tween 80. Die uitdrukking van 12 makrofaag gene, geselekteer op grond van hul betrokkenheid in die fagositose meganisme en in outofagie asook hul betrokkenheid in die immuunrespons, is gekwantifiseer met qRT-PCR en daaropvolgens geanaliseer met die REST-program. Die uitdrukking van elke geen is genormaliseer relatief tot die uitdrukking van die verwysingsgeen (Beta actin). Daar is bevind dat van die 12 gene, TLR7 en VAMP7 deurlopend afgereguleer was in dTHP-1 selle geïnfekteer met M.tbNT en opgereguleer was in dTHP selle geïnfekteer met M.smegmatisNT. Selrespons met M.bovis BCG was onbeduidend en derhalwe kon geen gevolgtrekking hier gemaak word nie. Ook, CCL1 was opgereguleer met infeksie deur enige van die mycobakteriële spesies, maar CCL1 se uitdrukking was groter in respons tot mycobakterieëNT wanneer vergelyk word met respons tot mycobakterieëT. Differensiële geenuitdrukking van TLR7 en VAMP7 in respons tot patogeniese en nie-patogeniese mycobakterieëNT impliseer dat hierdie twee gene potensiële teikens kan wees vir RNAa-gebaseerde anti-TB behandeling, alhoewel ons nie kon beslis of hierdie respons spesifiek vir makrofage was nie. Ook, die verskille waargeneem in die uitdrukking van CCL1 geïnduseer deur mycobakterieëNT, vergeleke met mycobakterieëT, impliseer dat die steuring in die selwand veroorsaak deur Tween 80, heelwaarskynlik die respons van die makrofaag beïnvloed het. Hierdie studie beskryf ook ʼn filtrasiemetode om enkele mycobakteriële selle te genereer wat oorweeg moet word by toekomstige mycobakteriële infeksiestudies.

Planktothricoides raciborskii and Roseofilum reptotaenium are physiologically similar, yet ecologically distinct organisms found in a hot spring outflow and coral black band disease (BBD), respectively. The aim of this study was to elucidate the relationship between R. reptotaenium and sulfide in BBD, to compare microcystin (MC) production in response to environmental factors, and to determine chemotactic responses to MC and sulfide by the two organisms. Results showed that the pathogenicity of R. reptotaenium in BBD is dependent on sulfate-reducing bacteria as secondary pathogens. Roseofilum reptotaenium produced significantly more MC than P. raciborskii, as measured using ELISA. Roseofilum reptotaenium was negatively chemotactic to sulfide, determined using horizontal and vertical gradients in agar, while P. raciborskii was not affected. Neither cyanobacterium was chemotactic to MC in the agar assays. The ecophysiology of P. raciborskii and R. reptotaenium in relation to MC production and response to sulfide reflected their pathogenic versus non-pathogenic status.