Neste trabalho, esclarecemos tópicos da patogenicidade de EIEC que sustentam a sua menor virulência quando comparada à S. flexneri, e mostramos a importância das células dendríticas (CD) nesse processo. Estudou-se o comportamento de EIEC e S. flexneri quando em contato com células Caco-2, avaliando-se uma cinética de expressão dos genes envolvidos na invasão e disseminação bacteriana. Em geral, todos os genes foram menos expressos em EIEC, fato corroborado pelo fenótipo de disseminação bacteriana, onde EIEC foi menos eficiente do que Shigella. Também foi avaliada a modulação da resposta inflamatória de células dendríticas intestinais murinas pela produção de citocinas, expressão de moléculas co-estimulatórias e apresentação de antígenos, após desafio das células com as bactérias. Os resultados sugerem que EIEC induz a uma resposta protetora ao hospedeiro, enquanto que Shigella estaria \"driblando\" o sistema imune, além de provavelmente super-estimular o sistema imune adaptativo, fato que poderia levar a um agravamento da doença. As ações integradas das células Caco-2, células dendríticas e estímulos bacterianos foram estudadas em co-cultura celular. Observou-se que EIEC e suas proteínas secretadas induzem a migração das CDs ao compartimento apical da co-cultura; nada foi observado quando o desafio se deu com Shigella. Também foram avaliadas as concentrações de citocinas inflamatórias no microambiente infeccioso formado. A citocina TNF-α, bem como CCL20 e MCP-1 foram mais proeminentes após estímulo com EIEC, fato que poderia explicar parcialmente a migração das CDs ao lado apical da co-cultura após estímulo com EIEC e suas proteínas secretadas. Nossas evidências experimentais indicam que a doença desencadeada por EIEC é mais restrita a um determinado local da infecção, ou seja, não é capaz de se disseminar a ponto de estender a lesão tecidual de forma mais drástica, como Shigella. Esse fenômeno pode estar associado à menor expressão de seus dos fatores de virulência e à resposta imune inata induzida no sítio de infecção, o que levaria, fatalmente, à resolução da doença. / In this study, we clarify topics of pathogenicity from EIEC that support its lower level of virulence when it is compared to S. flexneri, and we have shown the importance of dendritic cells (DC) in this process. We studied the conduct of EIEC and S. flexneri when they were in contact with Caco-2 cells and we analyzed the kinetics of the genes expression that was involved in the spread and invasion of the bacteria. In general, all genes were expressed less in EIEC, as demonstrated by the phenotype of the bacterial spread, where EIEC was less efficient than Shigella. We also analyzed the modulation of the inflammatory response by the murine intestinal dendritic cells by the production of cytokine, expression of co-stimulators molecules and antigens presentation, after the interaction of the cells with the bacteria. The results showed that EIEC induces a response that protects the host while Shigella manipulate the host intestinal innate and adaptive immune system and it probably over-stimulates the adaptive immune system which could let the disease worse. The integrated actions of Caco-2 cells, dendritic cells and bacterial stimulus, were studied in a co-culture cell. We observed that EIEC and its secreted proteins induce the migration of the DCs to the apical compartment of the co-culture; nothing was observed related to Shigella. We also evaluated the concentrations of the inflammatory cytokines at the infective micro environment that was formed. The cytokine TNF-α, as CCL20 and MCP-1 were more prominent after been stimulated with EIEC, a fact that could partially explain the migration of DCs to the apical side of the co-culture after the stimulus with EIEC and its secreted proteins. Our experimental evidence shows that the disease triggered by the EIEC is more restricted at a definite infection place, which means that it is not capable of disseminating beyond a certain point to extend the tissue\'s injury and let it worsen, as Shigella do. This phenomenon can be associated with the lower level of expression of its virulence factors and to the immune response induced in the infection site, what could finally lead to the eradication of the disease.

La synthèse d’oligosides complexes reste difficilement réalisable par voie chimique. Le recours aux catalyseurs enzymatiques permettrait de pallier aux contraintes de la chimie mais les enzymes naturelles ne présentent pas toujours les propriétés adéquates et nécessitent d’être optimisées par ingénierie moléculaire. Le couplage de la chimie et de biocatalyseurs conçus « sur mesure », peut offrir une alternative prometteuse pour explorer de nouvelles voies de synthèse des sucres, notamment pour la mise au point de glycovaccins. L’objectif de cette thèse a ainsi visé à mettre en œuvre des stratégies d’ingénierie semi-rationnelles de l’amylosaccharase de Neisseria polysaccharea (ASNp), une α-transglucosylase utilisant le saccharose comme substrat, afin de concevoir de nouvelles spécificités de substrats et d’étendre le potentiel de cette enzyme à catalyser de nouvelles réactions, permettant ainsi d’aller bien au-delà de ce que la Nature peut offrir. Dans une première étude, une approche assistée par ordinateur a été suivie afin de remodeler le site actif de l’enzyme (sous-sites +1, +2 et +3) pour la reconnaissance et la glucosylation en α-1,4 d’un accepteur disaccharidique non-naturel (l’allyl 2-deoxy-2-N-trichloroacetyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranose). Le produit attendu, un trisaccharide, est un précurseur dans la synthèse chimio-enzymatique des oligosaccharides mimant les unités répétitives des lipopolysaccharides de Shigella flexneri, dont l’utilisation ultime est le développement de vaccins contre la Shigellose. Une approche computationnelle faisant appel à des outils dédiés au design automatisé de protéines et à une analyse des séquences a conduit au design d’une librairie d’environ 2.7x104 séquences, qui a ensuite été construite expérimentalement puis criblée. Au final, 55 variants actifs sur saccharose (le substrat donneur) ont été identifiés, et un mutant, appelé F3, a révélé sa capacité à glucosyler en α-1,4 le disaccharide cible. De manière étonnante, ce mutant possède 7 mutations au sein de son site actif, nécessaires au déploiement de sa nouvelle spécificité tout en maintenant son aptitude à utiliser le saccharose comme donneur d'unité glucosyle. Dans une deuxième étude, trois variants ont été identifiés lors du criblage de la librairie semi-rationnelle sur saccharose comme présentant de nouvelles spécificités de produits. Ces mutants ont été caractérisés plus en détails, ainsi que leurs produits, sur un plan biochimique et structural. Ces mutants, appelés 37G4, 39A8 et 47A10, contiennent entre 7 et 11 mutations dans leur site actif. Il a été montré qu’ils étaient capables de reconnaitre le saccharose et le maltose (un produit de la réaction avec le saccharose) comme donneur et accepteur pour synthétiser en quantités variables de l’erlose (α-D-Glucopyranosyl-(1→4)-α-D-Glucopyranosyl-(1→2)-β-D-Fructose) et du panose (α-D-Glucopyranosyl-(1→6)-α-D-Glucopyranosyl-(1→4)-α-D-glucose), des molécules non produites par l’enzyme sauvage. Des taux de production relativement élevés ont été obtenus pour ces molécules, dont les propriétés acariogènes et le pouvoir sucrant pourraient présenter un intérêt applicatif pour l’industrie alimentaire. Dans une dernière partie, un autre mutant, appelé 30H3, a été isolé lors du criblage primaire de la librairie de par son activité élevée sur saccharose (une amélioration d’un facteur 6.5 comparé à l’enzyme sauvage). Après caractérisation, le mutant s’est avéré synthétiser un profil unique de produits en comparaison de l’enzyme sauvage ASNp. Il s’est ainsi montré très efficace pour la synthèse de maltooligosaccharides solubles, de taille de chaînes contrôlée allant d’un DP 3 à 21, et de faible polydispersité. Aucun polymère insoluble n’a été identifié. La structure 3D du mutant résolue par cristallographie des rayons X a révélé un agrandissement de la poche catalytique en raison de la présence de 9 mutations introduites dans la première sphère.... / Chemical synthesis of complex oligosaccharides still remains critical. Enzymes have emerged as powerful tools to circumvent chemical boundaries of glycochemistry. However, natural enzymes do not necessarily display the required properties and need to be optimized by molecular engineering. Combined use of chemistry and tailored biocatalysts may thus be attractive for exploring novel synthetic routes, especially for glyco-based vaccines development. The objective of this thesis was thus to apply semi-rational engineering strategies to Neisseria polysaccharea amylosucrase (NpAS), a sucrose-utilizing α-transglucosylase, in order to conceive novel substrate specificities and extend the potential of this enzyme to catalyze novel reactions, going beyond what nature has to offer. In a first study, a computer aided-approach was followed to reshape the active site of the enzyme (subsites +1, +2 and +3) for the recognition and α-1,4 glucosylation of a non-natural disaccharide acceptor molecule (allyl 2-deoxy-2-N-trichloroacetyl-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranose). The trisaccharide product is a building block for the chemo-enzymatic synthesis of oligosaccharides mimicking the repetitive units of the Shigella flexneri lipopolysaccharides, and ultimately, for the production of a vaccine against Shigellosis disease. Using computational tools dedicated to the automated protein design, combined with sequence analysis, a library of about 2.7x104 sequences was designed and experimentally constructed and screened. Altogether, 55 mutants were identified to be active on sucrose (the donor substrate), and one, called mutant F3, was subsequently found able to catalyze the α-1,4 glucosylation of the target disaccharide. Impressively, this mutant contained seven mutations in the first shell of the active site leading to a drastic reshaping of the catalytic pocket without significantly perturbing the original specificity for sucrose donor substrate. In a second study, three variants were identified from the screening of the semi-rational library on sole sucrose as displaying totally novel product specificities. They were further characterized, as well as their products, at both biochemical and structural level. These mutants, called 37G4, 39A8 and 47A10, contained between 7 and 11 mutations into their active site. They were found able to use sucrose and maltose (a reaction product from sucrose) as both donor and acceptor substrates to produce in varying amounts erlose (α-D-Glucopyranosyl-(1→4)-α-D-Glucopyranosyl-(1→2)-β-D-Fructose) and panose (α-D-Glucopyranosyl-(1→6)-α-D-Glucopyranosyl-(1→4)-α-D-glucose) trisaccharides, which are not produced at all by parental wild-type enzyme. Relatively high yields were obtained for the production of these molecules, which are known to have acariogenic and sweetening properties and could be of interest for food applications. In a last part, another mutant 30H3 was isolated due to its high activity on sucrose (6.5-fold improvement compared to wild-type activity) from primary screening of the library. When characterized, the mutant revealed a singular product profile compared to that of wild-type NpAS. It appeared highly efficient for the synthesis of soluble maltooligosaccharides of controlled size chains, from DP 3 to 21, and with a low polydispersity. No formation of insoluble polymer was found. The X-ray structure of the mutant was determined and revealed the opening of the catalytic pocket due to the presence of 9 mutations in the first sphere. Molecular dynamics simulations suggested a role of mutations onto flexibility of domain B’ that might interfere with oligosaccharide binding and explain product specificity of the mutant.

Das Typ III Sekretionssystem (T3SS) ist ein Proteinkomplex den Gramnegative Bakterien nutzen um in einem Schritt Effektorproteine (Effektoren) aus dem Zytosol über die Doppelmembran zu sekretieren. Für viele Bakterien ist das T3SS ein essenzieller Virulenzfaktor, der es ihnen erlaubt mit ihrem Wirt zu interagieren und diesen zu manipulieren. Charakteristisch für das T3SS ist die strukturelle Komponente, der Nadelkomplex. Dieser ähnelt strukturell einer Spritze, deren Basalkörper die bakteriellen Membranen und das Periplasma durchspannt und einer Nadel, die vom Basalkörper aus dem Bakterium ragt. Basierend auf dem Modell einer Spritze wird angenommen, dass Effektoren entfaltet und anschließend durch Basalkörper und Nadelkanal sekretiert werden. Trotz der kontinuierlichen Forschung an T3SS entbehrt dieses Modell einer experimentellen Grundlage und der Mechanismus ist nicht vollständig erklärt. Ziel der Arbeit war es, eine experimentelle Basis für den Sekretionsmechanismus des T3SS zu schaffen. Um zu verstehen, wie das T3SS Effektoren sekretiert, wurden zunächst Fusionsproteine konstruiert, welche aus einem Effektor und einem stabil gefalteten Knotenprotein bestehen. Aufgrund des Knotens in der Fusion ist davon auszugehen, dass dieser während der Sekretion nicht entfalten kann. Die Effektordomäne wird sekretiert während der Knoten im Kanal verbleibt und diesen verstopft. Nach unseremWissen ist diese Arbeit die erste Visualisierung von Effektorfusionen an isolierten Nadelkomplexen. Die Effektorfusion wird N-terminal voran durch den Kanal sekretiert, wobei der Kanal das Substrat umschließt und gegen Proteasen und chemische Modifikationen abschirmt. Die Ergebnisse dieser Arbeit untermauern eine Grundidee der Funktionsweise des T3SS und liefern eine vielversprechende Strategie für in situ-Strukturanalysen. Dieser Ansatz lässt sich auch auf andere Proteinsekretionssysteme übertragen, bei welchen Substrate vor dem Transport entfaltet werden müssen. / The Type III Secretion System (T3SS) is a complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. This work aimed to provide an experimental basis for the model of the T3SS mechanism. In order to elucidate details of the effector secretion mechanism, fusion proteins consisting of an effector and a bulky protein containing a knotted motif were generated. It is assumed that the knot cannot be unfolded during secretion of the chimera. Consequently, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. This is, to our best knowledge, the first time effector fusions have been visualized together with isolated NCs and it demonstrates that effector proteins are secreted directly through the channel with their N-terminus first. The channel encloses the substrate and shields it from a protease and chemical modifications. These results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.

Bei neun von vierzehn Serogruppen der Subspezies E. coli Flexneri (SF) konnte ein pHS-2-Plasmid nachgewiesen werden. Es besteht eine positive Korrelation zwischen pHS-2-positiven SF und dem Auftreten einer Reaktiven Arthritis, eine Komplikation der bakteriellen Ruhr. Verschiedene Autoren führen das gehäufte Auftreten der Reaktiven Arthritis bei pHS-2-positiven SF auf eine erhöhte Serumresistenz zurück, die den Bakterien durch das cld(pHS-2)-Gen vermittelt wird. Das cld(pHS-2)-Gen als längstes aller 18 "open reading frames" auf dem pHS-2-Plasmid kodiert für ein 35 kD großes Protein, das aufgrund seiner Funktion als "chain length determinant" (Cld(pHS-2)) bezeichnet wurde. Cld(pHS-2) ist maßgeblich an der Produktion von Lipopolysacchariden vom VL-Typ beteiligt. LPS vom VL-Typ produzieren O-Seitenketten mit mehr als 70 bis 80 Oligosaccharideinheiten und sollen die bakterielle Membran vor einer Komplementbindung und Serum-Antikörpern schützen, wodurch den Bakterien eine erhöhte Serumresistenz vermittelt werde. In dieser Arbeit wurde cld(pHS-2) in einen pHS-2-negativen Stamm der SF Serogruppe 6 integriert, um nachzuweisen, dass cld(pHS-2) das LPS-Muster pHS-2-negativer SF in charakteristischer Weise modifiziert. Während der Expressionsnachweis von cld(pHS-2) als Einzelprotein misslang, war eine Expression von cld(pHS-2) als Fusionsprotein in SF Serogruppe 6 erfolgreich. Im Vergleich zu pHS-2-negativen SF war unsere SF Serogruppe 6-Mutante nun in der Lage, LPS vom VL-Typ in Form einer zusätzlichen Bande mit ca. 80 Oligosaccharideinheiten zu produzieren. Diese phänotypische Veränderung war zwar nur gering ausgeprägt, konnte jedoch den spezifischen Einfluss von cld(pHS-2) als Fusionsprotein auf das LPS-Muster pHS-2-negativer SF nachweisen. / In nine out of fourteen serogroups of E. Coli Flexneri (SF) a pHS-2 plasmid has been isolated. There is a positive correlation between pHS-2-positive SF and the occurence of reactive arthritis (ReA) which is a complication of bacterial dysentery. Different authors reduce the widespread appearance of ReA by pHS-2-positive SF to an increased serum resistance caused by a cld(pHS-2)-gene. The cld(pHS-2)-gene ist the longest of 18 open reading frames in pHS-2 and it is the coding for a 35 kD protein which has been identified as a chain length determinant because of its function. Cld(pHS-2) plays a decisive role in the production of lipopolysaccharides (LPS) of the VL-type. LPS of the VL-type produce O antigen with more than 70-80 repeat units, and protect the bacterial outer membrane from complement factors and serum antibodies by a higher serum resistance. In this research the cld(pHS-2)-gene was integrated in a pHS-2-negative SF serogroup 6 to prove that cld(pHS-2) is able to modify the LPS pattern (destribution) of pHS-2-negative SF in a characteristic manner. The expression of cld(pHS-2) in SF serogroup 6 as an isolated protein failed, but the expression of cld(pHS-2) as a fusion protein was successful. In contrast to the pHS-2-negative SF our SF serogroup 6 mutant was able to produce LPS of the VL-type with 80-90 repeat units. Despite the slight effect, a specific influence of cld(pHS-2) as a fusion protein on LPS pattern and the bacterial phaenotype has to be confirmed.

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Automated charge state deconvolution, spectral subtraction and spectral mirroring were used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarkers were discovered based on their unique mass, retention time and relative intensity. These markers were implemented to differentiate closely related strain types, (e.g. two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. Notable, was the distinction of multiple serotypes of enterohaemorrhagic E. coli which cannot be distinguished by clinical manifestation alone. Additionally, speciation of Shigella was achieved, a task for which no commercial real-time polymerase chain reaction (PCR) primers exist. This method was subsequently applied to two pathogenic Clostridium species: C. difficile and C. perfringens. Due to the increased difficulty during lysis, two new lysis protocols were developed, and each extracted a distinct set of proteins (by both mass and retention time). Extracts from both lysis procedures were utilized to discover biomarkers useful for identification and characterization at the species and strain levels. These biomarkers were successfully implemented to identify unknowns during a blind study and would enhance serological and genetic approaches by serving as new targets for detection. Two sets of the C. perfringens isolates that were deemed 100% similar by the gold standard for strain differentiation, pulsed-field gel electrophoresis (PFGE), were distinguished using LC/MS, demonstrating the high specificity of this approach. The final part of this work demonstrated the application of ultra performance liquid chromatography (UPLC) to this project to improve the throughput of the method. Given that numerous small molecule applications of UPLC have been published, efforts were made to examine the potential of UPLC to enhance the separation of intact proteins. Beginning with typically employed conditions, column temperature and organic solvent were optimized followed by an HPLC vs. UPLC comparison. When applied to a mixture of ten protein standards, the optimized UPLC method yielded improved chromatographic resolution, enhanced sensitivity, and a three-fold increase in throughput. Application of this method to cell lysate analysis demonstrated no compromise in chromatographic or mass spectral data quality; a reduction in run time from 75 minutes to 25 minutes was achieved.

Abstract not available

The aromatic beta-glucosides of plant origin, salicin and arbutin, serve as carbon sources for the sustenance of bacteria when ‘preferred’ sugars are absent in the environment. In the family Enterobacteriaceae, there are varied patterns for utilization of these beta-glucosides, wherein, in some members the ability to utilize salicin or arbutin is cryptic while in others it is completely absent. Escherichia coli harbors silent or cryptic genetic systems for the utilization of arbutin and salicin, which are activated by spontaneous mutation(s). Of these systems, the bgl operon of E.coli has been used as a paradigm for silent genes and extensive studies have been carried out to understand its silencing and activating mechanisms. Mutational activation of the wild type bgl operon in E.coli leads to the acquisition of the ability to utilize both arbutin and salicin. Preliminary studies have shown that aromatic beta-glucoside utilization in Shigella sonnei, which is evolutionarily related to E.coli, shows a two-step activation process wherein the wild type strain first becomes Arb+, which subsequently mutates to Sal+. The genetic systems responsible for beta-glucoside utilization, including the bgl operon, are conserved in S.sonnei to a large extent. A major difference is that the bglB gene encoding the phosphor-β-glucosidase B is insertionally inactivated in S.sonnei. As a result, activation of the bgl operon in the first stage leads to expression of the permease, BglF, which along with the phosphor-β-glucosidase A expressed from an unlinked constitutive gene, bglA, confers an Arb+phenotype. Salicin is not a substrate for the enzyme BglA and therefore a second mutational event is required for the acquisition of the Sal+ phenotype. Interestingly, the insertion within bglB is retained in AK102, the Sal+ second step mutant of S.sonnei. Therefore, the locus involved in conferring salicin utilization ability is unknown. However, S.sonnei is not amenable to routine genetic echniques and an E.coli bglB model was generated by creating an insertion in the bglB gene to identify the locus involved in conferring the Sal+ phenotype. Like S.sonnei, this E.coli strain, SD-1.3, also showed a two-step activation process for the utilization of salicin. Utilization of salicin in the Sal+ second step mutant of SD-1.3 could require activation of other silent genetic systems such as the asc operon and the chb operon or mutation in loci such as bglB or bglA. Linkage analysis by P1 transduction showed that activation of the asc operon is required for conferring a Sal+ phenotype in the second step mutant. The asc operon comprises of two genes, ascF encoding a PTS permease and ascB encoding a phosphor-β-glucosidaseB.The Precise mechanism of activation of the asc operon is not known but, it has been speculated that AscG, encoded by an upstream gene, acts as a repressor. Results presented in this thesis show that BglF is responsible for the transport of salicin and AscB provides the phosphor-β-glucosidase B in the Sal+ second step mutant of the E.coli strain SD-1.3. Analysis of the expression of the ascFB operon by measuring the transcripts as well as the activity of phosphor-β-glucosidase B showed that it is enhanced in the Sal+ second step mutant of SD-1.3 in the presence of the inducer. The expression of the ascFB operon is also increased constitutively when ascG is replaced by an antibiotic cassette in the parent strain SD-1.3 and the Arb+ first step mutant, indicating that AscG acts as a repressor for the asc operon. Moreover, inactivation of ascG in the parent leads to utilization of salicin in a single step by the activation of the bgl operon to provide the transport function, indicating that the inactivation of ascG is sufficient to activate the expression of ascB. Similarly, loss of AscG–mediated repression of the asc operon confers salicin utilization ability to the Arb+ first step mutant of SD-1.3. Interestingly, measurement of phosphor-β-glucosidase B activity in a Sal+ second step mutant derivative deleted for ascG showed a constitutive increase in the expression of the ascFB operon. Thus, AscG mediates the induction of the asc operon in response to salicin. In order to study the mechanism of activation of the asc operon, the ascB gene was cloned from the Arb+ first step mutant and the Sal+ second step mutant of SD-1.3 in a low copy number vector. Both these constructs were able to confer a Sal+ phenotype to the Arb+ first step mutant indicating absence of any genetic change in ascB in the Sal+ second step mutant. This was also confirmed by sequencing of ascB gene from the strains that showed no changes in the nucleotide sequence. Absence of any insertions within ascG showed that activation of the ascoperon is not achieved through disruption of ascG in the Sal+ second step mutants analyzed. AscG belongs to the GalR family of repressors in which some members require a mutation to enable the binding of sugar to mediate induction. Nucleotide sequence analysis showed that there was no change in the ascG gene in the Sal+ mutants analyzed. However, when the upstream regulatory region of the ascFB operon was analyzed a mutation was found in the -10 sequence of the putative promoter of the ascFB genes. This change leads to a stronger promoter as it brings the -10 sequence closer to the consensus sequence. Therefore, salicin utilization is achieved in the Sal+ second step mutant analyzed by an increase in expression of the asc operon by a promoter-up mutation. The negative effect of binding of AscG on expression of the ascFB operon is relieved in presence of the inducer, salicin. The possible role of the asc operon in salicin utilization in S.sonnei was tested by replacing the ascB gene by anantibiotic cassette in AK102, the Sal+ second step mutant of S. sonnei. This did not lead to loss of salicin utilization. By gene targeting approach it was also found that none of the phosphor-β-glucosidases known in E.coli are involved in degradation of salicin in AK102. A search of the S. sonnei genome database indicated the presence of two putative phosphor-β-glucosidases encoded by glvG and SSO1595. Replacement of glvG gene by anantibiotic cassette in AK102 did not lead to loss of salicin utilization. However, a similar replacement of SSO1595 in AK102 resulted in a Sal+ phenotype indicating that SSO1595 provides the phosphor-β-glucosidase in the Sal+ second step mutant of S. sonnei. A homolog of this enzyme is not present in E.coliorinany of the other members of the Shigella genus. Transcription alanalysis as well as measurement of phosphor-β-glucosidase B activity showed that expression of SSO1595 is enhanced constitutively in AK102. To study the mechanism of mutational activation for achieving salicin utilization in S. sonnei, SSO1595 was cloned from AK101, theArb+ first step mutant and AK102, the Sal+ second step mutant in a low copy numbe rvector. Both these constructs were able to confer a Sal+ phenotype to AK101 indicating an absence of genetic change in SSO1595 in AK102. This was also confirmed by sequencing of SSO1595 gene from the strains. Analysis of the upstream regulatory region of SSO1595 in AK102 indicated a deletion of around 1.0kbp sequence. This was also confirmed by nucleotide sequencing of the region. By primer extension analysis it was found that a new transcriptional start site is generated upstream to the deletion in the Sal+ second stepmutant of S.sonnei. Acquisition of the Sal+ phenotype in AK102 is therefore the resultof the SSO1595 gene being brought under a new promoter as a result of a DNA rearrangement. Overall, this study suggests that a high degree of similarity at the genomic level between organisms does not always ensure similarity in genetic mechanisms as two distinct pathways are responsible for conferring utilization of salicinin S. sonnei and E.coli.

Shigella flexneri attacks cells of the intestinal tract, causing over 1 million deaths annually from bacterial dysentery. A type III secretion system (T3SS) initiates the host-pathogen interaction and transports virulence factors directly into host cells via a needle complex (NC) comprising an extracellular needle and membrane-spanning basal body. Rings formed by the single-pass membrane proteins MxiG and MxiJ are arranged concentrically within the inner membrane ring (IMR) of the NC. The Neterminal domain of MxiG (MxiG-N) is the predominant IMR cytoplasmic structure, however it was structurally and functionally uncharacterised. Determination of the solution structure of MxiG-N in this study revealed it to be a forkhead associated (FHA) domain, although subsequent analyses of conserved residues suggested it does not have the canonical role in cell-signalling via phospho-threonine recognition. Subsequent positioning of the structure in the electron microscopy (EM) density for the S. flexneri NC supported models with 24-fold symmetry in the IMR. Both MxiG and MxiJ also have significant periplasmic domains, which were purified to homogeneity in this study, facilitating preliminary characterisation of their structures and intermolecular interactions. In addition, the entire IMR within the context of intact basal bodies was isolated and visualised in vitro by EM. The essential function of MxiG-N could be to localise the putative cytoplasmic ring (Cering) at the base of the T3SS. Although absolutely required for secretion, the Csring component, Spa33, was structurally uncharacterised. The crystal structure of the Cvterminal domain of Spa33 (Spa33-C) was determined in this study, showing an intertwined dimer that aligned with homologous structures and exhibited a novel interaction with the N-terminus of the ATPase regulator, MxiN. Subsequently, Spa33-C was identified as an altemative translation product of spa33 that formed a 2: 1 complex with Spa33 in vitro. This complex oligomerised further, demonstrating for the first time that Spa33 has the propensity to form the ordered, high molecular weight assemblies that would be required for C-ring formation in S. flexneri.

MSc (Chemistry) / Department of Chemistry

Understanding local perceptions of disease causation could help public health officials improve strategies to prevent bloody diarrhoea. A cross-sectional survey was conducted in Dhaka, Bangladesh to elicit community beliefs about the causes of and prevention strategies for bloody diarrhoea. Between March and June 2003, we interviewed 541 randomly selected respondents. Overall, 507 (93%) respondents perceived that a vaccine could prevent bloody diarrhoea. If a vaccine provided lifetime protection, 445 (83%) respondents stated that they would opt to get the vaccine and would pay a median of $0·05 (range U.S.$0·01-0·15) for it, equivalent to <1% of their median weekly income. There was almost universal perception that an effective vaccine to prevent bloody diarrhoea was highly beneficial and acceptable. While respondents valued a vaccine for prevention of bloody diarrhoea, they were only willing to pay minimally for it. Therefore, achieving a high rate of Shigella vaccine coverage may require subsidy of vaccine purchase.