Thesis advisor: Christina Faherty / Shigella, a Gram-negative bacterial pathogen, induces inflammation and diarrhea by invading the colonic epithelium. Annually, millions of Shigella infections occur globally, mainly in malnourished children. Despite extensive research, no effective vaccine exists. This work explores the mechanisms of Shigella proliferation before colonic infection, where an adverse environment is encountered, including bile salts exposure. One means of bile salts evasion is possibly lipopolysaccharide (LPS) modification. LPS—O-antigen, the polysaccharide core, and the lipid A—is a crucial outer membrane component for virulence. Transposon mutant analysis suggested a role of LPS in bile salts resistance; thus, the goal of this study was to define Shigella LPS modifications following bile salts exposure. LPS mutants were investigated to distinguish crucial components of the LPS structure for bile salts resistance. Mutants were analyzed relative to wild type for growth in bile salts and biofilm formation. The LPS from all strains was purified and analyzed by polyacrylamide gel electrophoresis. Stained gels show modifications in the Oag, lipid A, and core components. Key bands were sent for mass spectrophotometry sequencing. Results indicate that the O-antigen regulates Shigella bile salts resistance, as the complete O-antigen deletion mutant and partial deletion mutants exhibited slow growth in bile salts and failed to form a biofilm in the presence of bile salts. This work highlights the importance of bile salts exposure for Shigella in future targeted antibodies against the pathogen. / Thesis (BS) — Boston College, 2019. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Francisella tularensis is the causative agent of tularemia and is categorized by the CDC as a Tier 1 select agent. This gram-negative, facultative-intracellular bacterium infects macrophages by escaping the phagosome and replicating with high efficacy in the cytosol. Multiple virulence factors, including capsule and lipopolysaccharide (LPS), are expressed by F. tularensis. Biosynthesis of capsule and LPS O-antigen requires the same O-antigen biosynthesis gene cluster and, together, expression of capsule and O-antigen confer serum resistance. Mutations in the O-antigen biosynthesis gene cluster not only result in serum sensitivity, but also attenuate the ability to cause disease in vivo. In addition to changes in F. tularensis virulence, individual capsule and O-antigen mutants appear to have distinct intracellular phenotypes in macrophages. As previously shown by Lindemann et al. (2011), the capsule and O-antigen mutants FTT1236, FTT1237, and FTT1238 all replicated in human monocyte derived macrophages (MDMs) up to 16 hr and then ceased to replicate after that. This is hypothesized to be due to MDM cytotoxicity. In contrast, Raynaud et al. (2007) showed that the capsule and O-antigen mutant wbtA completely lacked replication in J774 macrophages, the reason for which has not been identified. A potential explanation for the loss of F. tularensis capsule and O-antigen mutant replication is capture and degradation by the host cell's autophagy pathway. Capture and degradation by autophagy is an accepted innate immune response to many intracellular pathogens. When small subpopulations of bacteria that normally replicate in membrane-bound vacuoles become cytosolic, such as Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium, they are targeted to forming autophagosomes through ubiquitination and binding of autophagy receptors. Pathogens have also developed methods to circumvent recognition and degradation by autophagy. Since F. tularensis replicates in the cytosol, it stands to reason that it has a means of evading detection by autophagy. We propose that expression of capsule and O-antigen acts as a mechanism used by F. tularensis to protect itself in an extracellular environment, as well as during intracellular infection. In this thesis we characterized nine different capsule and O-antigen mutants, and found different replication phenotypes in MDMs and varying degrees of MDM cytotoxicity. Also, only a subset of the mutants was detected by the autophagy marker, ubiquitin, supporting our hypothesis that different capsule and O-antigen mutants have diverse fates in MDMs. We also show that LVS and Schu S4 wbtA mutants had similar phenotypes. Upon further evaluation, we found that LVS wbtA more readily colocalized with ubiquitin, autophagy receptors, and the autophagy membrane protein LC3B, but not Beclin-1 or LAMP-1. This supports our hypothesis that capsule and O-antigen mutants are more susceptible to recognition by autophagy. Yet, because we did not observe LAMP-1 colocalization, there may be defects in the maturation of autophagosomes to degradative autolysosomes. Finally, we found that the fate of LVS wbtA in MDMs is dissimilar from J774 macrophages, suggesting macrophage species affect mutant fate. This thesis shows that different capsule and O-antigen mutants have multiple fates in MDMs, and suggest that F. tularensis capsule and O-antigen act as protective virulence factors that limit detection by autophagy.

Francisella tularensis is the etiological agent of tularemia, a severe and potentially fatal disease in humans. It is extremely infectious by the aerosol route, being thought to cause disease in humans with an infectious dose as small as one to ten organisms, which led to its weaponization by several nations and classification as a category A select agent by the Centers for Disease Control and Prevention. An intracellular pathogen, relatively little is known about the mechanisms by which Francisella is capable of successfully modulating host cell processes to escape its phagosome and replicate within the cytosol and what genes beyond the Francisella pathogenicity island are required. Furthermore, in the context of aerosol exposure, it is unknown what cells F. tularensis initially interacts with and the overall contribution of those interactions to inhalational tularemia. I initiated this study by generating an in vitro model system to study interactions of F. tularensis with epithelial cell lines in tissue culture. Utilizing this system, I determined that F. tularensis LVS was capable of adherence to human epithelial cell lines of alveolar (A549), bronchial airway (HBE), and cervical carcinoma (HEp-2) origin. Furthermore, LVS was capable of invading these cell lines and growing productively within them. In order to detect genes important for virulence in this system, I generated a ~15,000 member transposon library in virulent strain Schu S4 that was could be screened in a high-throughput manner by transposon site hybridization. As uptake in the in vitro epithelial cell line system was relatively inefficient, I screened this library through human primary macrophages. Results of the screen implicated 207 genes as negatively selected in the human macrophage model. Of these, I generated mutants in genes residing in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238, to determine their virulence phenotypes. Mutants in these genes demonstrated significant vulnerability to complement-mediated lysis as compared with wild type Schu S4. Analysis of purified LPS and capsule from these mutants further showed that they had marked defects in O-antigen and capsular polysaccharide biosynthesis. Complementation of these mutants restored surface polysaccharide biosynthesis and further determined that FTT1236 and FTT1237 compose an operon, as a mutation in FTT1236 is polar onto FTT1237. Characterization of the intracellular defect of these mutants in the absence of active complement demonstrated that they were taken up more efficiently by primary human macrophages than wild type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic analysis of macrophages infected with mutant bacteria revealed that, as early as 16 hpi, these macrophages exhibited signs of cell death. In contrast, cells infected with Schu S4 exhibited a healthy, spread morphology as late as 32 h, despite significantly more extensive F. tularensis cytosolic replication. Quantitation of cell death by the release of lactate dehydrogenase, signifying membrane permeability, confirmed that mutants in FTT1236, FTT1237, and FTT1238 induced early cell death in infected macrophages as compared with wild type Schu S4. Together, this work contributes to our understanding of the factors, such as O-antigen and capsule, required for and genes involved in Francisella's lifecycle as an intracellular pathogen.

The formation of the Salmonella extracellular matrix is a multicellular behavior important for environmental persistence. It is comprised of uniquely but ill-defined assembled thin aggregative fimbriae (Tafi), cellulose and uncharacterized polysaccharides. Consequently, investigations were launched into further clarifying Tafi assembly and the polysaccharide constituents of the extracellular matrix. In the Salmonella agfBAC Tafi operon, the transcription and role of agfC has been elusive. In this study using the clinical isolate, Salmonella Enteritidis 27655-3b, agfBAC transcripts were detected using a reverse transcriptase and transcription was not enhanced by replacement of a stem-loop structure immediately preceding agfC. AgfChis was purified, localized to the periplasm, and found to specifically bind noncrystalline cellulose suggesting an association with the extracellular matrix. An inframe ΔagfC mutant displayed an abundance of 20 nm fibers, which could be complemented with agfC in trans, in addition to Tafi and an increase in cell hydrophobicity. Depolymerization of purified 20 nm fibers required exceptionally stringent conditions to release what proved to be AgfA subunits revealing the 20 nm fibers as AgfA assemblages of unique morphology. The role of AgfC in Tafi assembly was investigated further via a novel, quantitative antibody-capture assay of in-frame agf mutants. A soluble antibody-accessible form of AgfA was captured in wt, ΔagfB and ΔagfF strains in support of the extracellular nucleation-precipitation pathway of Tafi assembly, but not in ΔagfC or ΔagfE mutants. These results suggest that AgfC and AgfE are required for AgfA’s extracellular assembly and thus may act as atypical AgfAspecific chaperones which facilitate Tafi assembly. The implications of these results are presented in an assembly model for Tafi. Additional investigations revealed that Salmonella produces an O-Antigen capsule co-regulated with the extracellular matrix. Structural analysis of purified extracellular polysaccharides (EPS) yielded a repeating oligosaccharide unit similar to iv that of lipopolysaccharide O-Antigen with modifications. Putative carbohydrate transport and regulatory operons important for capsule expression, designated emcA-H and emcIJ, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various in-frame Δemc mutants where emcG and emcE were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that, AgfD differentially regulated the emc operons in coordination with extracellular matrix genes. Survival assays demonstrated the capsule is important for desiccation tolerance. The emc genes were found to be conserved in Salmonellae and thus, the O-Antigen extracellular matrix capsule may be a conserved survival strategy important for environmental persistence. Finally, a compositionally unique acidic EPS was found associated with the extracellular matrix. In-frame ΔbcsA, ΔemcG and ΔagfA mutants but neither ΔagfAΔbcsA nor ΔagfD mutants bound calcofluor, a β-glucan binding fluorescent agent, suggesting that multicellular behavior itself and not necessarily AgfD alone was influencing EPS expression. A transposon library was screened by ELISA using serum generated against purified EPS. This identified mutations inactivating genes involved in quorum sensing AI-2 degradation, flagella repression and Tafi and TolA expression. All mutations resulted in the loss of multicellular behavior and immunologically decreased levels of Tafi. This is the first report that implicates quorum sensing AI-2 degradation and flagella repression as part of the regulatory circuit for Tafi expression. Together, the results reveal Tafi uses assembly factors to facilitate extracellular polymerization which likely assists the formation of a network of branched, amorphous fimbriae. Tafi together with EPS form the extracellular matrix: Tafi stabilizes the EPS on the microbial communities; EPS imparts it with physical properties such as hydration, charge and diffusion barriers that protect it from adverse environmental conditions such as desiccation and antimicrobials. This probably contributes to Salmonella survival in the environment and facilitates its cyclic lifestyle.

The Shigella species are Gram-negative enteropathogens that produce severe diarrhea, cramping, and dehydration in millions of people annually. The pathogens most commonly infect children under the age of 5 years in developing nations, where the rise of multidrug-resistant species is increasingly problematic. Despite several attempts to develop a vaccine against these pathogens, no successful vaccine has been produced. In order to achieve this goal, several characteristics of Shigella must be further elucidated. Namely, we must better understand the mechanisms Shigella employs in order to circumvent the immune response. A key way in which Shigella circumvents the innate defenses of the host is through resistance to bile salts, the principal component of bile, a substance found in the small intestine that is required for digestion. One such bile salt resistance mechanism of Shigella involves lipopolysaccharide (LPS), an extracellular structure composed of three regions: a transmembrane lipid, a polysaccharide core, and an O-antigen. LPS and LPS modifications have been implicated in bile salts resistance in other enteropathogens. Thus, the goal of this study was to build from preliminary findings to understand the role of LPS in conferring bile salts resistance in Shigella. Two Shigella flexneri mutants were studied to understand the roles of the polysaccharide core and O-antigen on bacterial growth and LPS modifications during exposure to bile salts. Growth comparisons of the mutants relative to wild type bacteria in the presence of bile salts were performed, including analysis of growth with exposure to bile salts and with varying levels of environmental glucose. Additionally, LPS was extracted from wild type and mutant bacteria grown in these conditions for analysis by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The growth curves demonstrated that both the O-antigen and polysaccharide core mutants exhibited slow growth with exposure to bile salts, while the SDS-PAGE analyses revealed changes in the LPS profile of wild type and both LPS mutants when grown in bile salts. These data indicate that the O-antigen likely has an important role in conferring bile salts resistance and that the polysaccharide core may also facilitate resistance. This study allows us to better understand how LPS contributes to bile salts resistance in S. flexneri, which may enhance efforts to develop an effective vaccine against this pathogen. / 2023-12-10T00:00:00Z

Kohlenhydrate stellen aufgrund der strukturellen Vielfalt und ihrer oft exponierten Lage auf Zelloberflächen wichtige Erkennungsstrukturen dar. Die Wechselwirkungen von Proteinen mit diesen Kohlenhydraten vermitteln einen spezifischen Informationsaustausch. Protein-Kohlenhydrat-Interaktionen und ihre Triebkräfte sind bislang nur teilweise verstanden, da nur wenig strukturelle Daten von Proteinen im Komplex mit vorwiegend kleinen Kohlenhydraten erhältlich sind. Mit der vorliegenden Promotionsarbeit soll ein Beitrag zum Verständnis von Protein-Kohlenhydrat-Wechselwirkungen durch Analysen struktureller Thermodynamik geleistet werden, um zukünftig Vorhersagen mit zuverlässigen Algorithmen zu erlauben. Als Modellsystem zur Erkennung komplexer Kohlenhydrate diente dabei das Tailspike Protein (TSP) aus dem Bakteriophagen HK620. Dieser Phage erkennt spezifisch seinen E. coli-Wirt anhand der Oberflächenzucker, der sogenannten O-Antigene. Dabei binden die TSP des Phagen das O-Antigen des Lipopolysaccharids (LPS) und weisen zudem eine hydrolytische Aktivität gegenüber dem Polysaccharid (PS) auf. Anhand von isolierten Oligosacchariden des Antigens (Typ O18A1) wurde die Bindung an HK620TSP und verschiedener Varianten davon systematisch analysiert. Die Bindung der komplexen Kohlenhydrate durch HK620TSP zeichnet sich durch große Interaktionsflächen aus. Durch einzelne Aminosäureaustausche im aktiven Zentrum wurden Varianten generiert, die eine tausendfach erhöhte Affinität (KD ~ 100 nM) im Vergleich zum Wildtyp-Protein (KD ~ 130 μM) aufweisen. Dabei zeichnet sich das System dadurch aus, dass die Bindung bei Raumtemperatur nicht nur enthalpisch, sondern auch entropisch getrieben wird. Ursache für den günstigen Entropiebeitrag ist die große Anzahl an Wassermolekülen, die bei der Bindung des Hexasaccharids verdrängt werden. Röntgenstrukturanalysen zeigten für alle TSP-Komplexe außer für Variante D339N unabhängig von der Hexasaccharid-Affinität analoge Protein- und Kohlenhydrat-Konformationen. Dabei kann die Bindestelle in zwei Regionen unterteilt werden: Zum einen befindet sich am reduzierenden Ende eine hydrophobe Tasche mit geringen Beiträgen zur Affinitätsgenerierung. Der Zugang zu dieser Tasche kann ohne große Affinitätseinbuße durch einen einzelnen Aminosäureaustausch (D339N) blockiert werden. In der zweiten Region kann durch den Austausch eines Glutamats durch ein Glutamin (E372Q) eine Bindestelle für ein zusätzliches Wassermolekül generiert werden. Die Rotation einiger Aminosäuren bei Kohlenhydratbindung führt zur Desolvatisierung und zur Ausbildung von zusätzlichen Wasserstoffbrücken, wodurch ein starker Affinitätsgewinn erzielt wird. HK620TSP ist nicht nur spezifisch für das O18A1-Antigen, sondern erkennt zudem das um eine Glucose verkürzte Oligosaccharid des Typs O18A und hydrolysiert polymere Strukturen davon. Studien zur Bindung von O18A-Pentasaccharid zeigten, dass sich die Triebkräfte der Bindung im Vergleich zu dem zuvor beschriebenen O18A1-Hexasaccharid verschoben haben. Durch Fehlen der Seitenkettenglucose ist die Bindung im Vergleich zu dem O18A1-Hexasaccharid weniger stark entropisch getrieben (Δ(-TΔS) ~ 10 kJ/mol), während der Enthalpiebeitrag zu der Bindung günstiger ist (ΔΔH ~ -10 kJ/mol). Insgesamt gleichen sich diese Effekte aus, wodurch sehr ähnliche Affinitäten der TSP-Varianten zu O18A1-Hexasaccharid und O18A-Pentasaccharid gemessen wurden. Durch die Bindung der Glucose werden aus einer hydrophoben Tasche vier Wassermoleküle verdrängt, was entropisch stark begünstigt ist. Unter enthalpischen Aspekten ist dies ebenso wie einige Kontakte zwischen der Glucose und einigen Resten in der Tasche eher ungünstig. Die Bindung der Glucose in die hydrophobe Tasche an HK620TSP trägt somit nicht zur Affinitätsgenerierung bei und es bleibt zu vermuten, dass sich das O18A1-Antigen-bindende HK620TSP aus einem O18A-Antigen-bindenden TSP evolutionär herleitet. In dem dritten Teilprojekt der Dissertation wurde der Infektionsmechanismus des Phagen HK620 untersucht. Es konnte gezeigt werden, dass analog zu dem verwandten Phagen P22 die Ejektion der DNA aus HK620 allein durch das Lipopolysaccharid (LPS) des Wirts in vitro induziert werden kann. Die Morphologie und Kettenlänge des LPS sowie die Aktivität von HK620TSP gegenüber dem LPS erwiesen sich dabei als essentiell. So konnte die DNA-Ejektion in vitro auch durch LPS aus Bakterien der Serogruppe O18A induziert werden, welches ebenfalls von dem TSP des Phagen gebunden und hydrolysiert wird. Diese Ergebnisse betonen die Rolle von TSP für die Erkennung der LPS-Rezeptoren als wichtigen Schritt für die Infektion durch die Podoviren HK620 und P22. / Carbohydrates are important for recognition events because of their diverse structure and their exposition on cell surfaces. Interactions between proteins and carbohydrates mediate a specific exchange of information crucial for manifold biological functions. The energetics of protein-carbohydrate-interactions are not very well understood so far due to the lack of structural data of proteins in complex with extensive oligosaccharides consisting of more than two building blocks. This dissertation improves the understanding of how proteins recognize complex carbohydrates by analysis of structural thermodynamics, which might lead to reliable algorithms for predictions of protein-carbohydrate-interactions. As model system for this work the tailspike protein (TSP) from coliphage HK620 was used. This phage recognizes specifically the surface O-antigen of its E. coli host by its TSP. HK620TSP does not only bind the O-antigen of host lipopolysaccharide (LPS), but also cleaves the polysaccharide (PS) by its endo-N-acetylglusaminidase activity. HK620TSP binds hexasaccharide fragments of this PS with low affinity (KD ~ 130 μM). However, single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants (KD ~ 100 nM). Strikingly, at room temperature association is driven by enthalpic and entropic contributions emphasizing major solvent rearrangements upon complex formation. Regardless of their affinity towards hexasaccharide the TSP complexes showed only minor conformational differences in crystal structure analysis accept of mutant D339N. The extended sugar binding site can be subdivided into two regions: Firstly, there is a hydrophobic pocket at the reducing end with minor affinity contributions. Surprisingly, access to this site is blocked by a single exchange of aspartate to asparagine (D339N) without major loss in hexasaccharide affinity. Secondly, there is a region where specific exchange of glutamate for glutamine (E372Q) creates a site for an additional water molecule. Upon sugar binding side chain rearrangements lead to displacement of this water molecule and additional hydrogen bonding. Thereby this region of the binding site is defined as the high affinity scaffold. HK620TSP is not only specific for the O18A1-antigen, but also the lacking of the branching glucose in the O18A1-antigen can be tolerated so that the accordant O18A PS can be bound and cleaved by HK620TSP as well. Surprisingly, in binding studies with the smallest O-antigen units of these PS the O18A pentasaccharide was bound by TSP variants with nearly the same affinity or even a slightly increased one compared to the O18A1 hexasaccharide. However, there is a change in thermodynamic contributions to binding: the lack of the glucose moiety leads to a less entropically favored binding compared to binding of O18A1-hexasaccharide (Δ (-TΔS) ~ 10 kJ/mol). In contrast the enthalpic contribution to the binding is more favorable (ΔΔH ~ -10 kJ/mol) for the binding of O18A pentasaccharide. The side-chain glucose contributes to entropy by the release of four water molecules out of a hydrophobic pocket. The binding of this branching glucose is paid by an enthalpic penalty because of the breakup of hydrogen bonding of displaced water molecules and destabilizing contacts between sugar and protein in this hydrophobic pocket. Therefore the binding of the glucose in this pocket does not account for generating affinity and an evolutionary relation of HK620TSP to an O18A-antigen binding protein is presumed. Finally, the infection mechanism of phage HK620 was studied as well. In analogy to the related phage P22 the DNA-ejection could be triggered by incubation of HK620 with the host LPS in vitro. The morphology and chain length of the LPS as well as the activity of HK620TSP towards the LPS are crucial for this in vitro DNA-ejection. Thus, the DNA-ejection could also be induced by LPS from bacteria of serogroup O18A which can be bound and hydrolyzed by HK620TSP. These results stress the role of TSP for the recognition of host LPS-receptors as a crucial step of infection by podoviruses P22 and HK620.

Carbohydrates are essential biomolecules that decorate cell membranes and proteins in organisms. They are important both as structural elements and as identification markers. Many biological and pathogenic processes rely on the identification of carbohydrates by proteins, thereby making them attractive as molecular blueprints for drugs. This thesis describes how NMR spectroscopy can be utilized to study carbohydrates in solution at a molecular level. This versatile technique facilitates for investigations of (i) shapes, (ii) motions and (iii) interactions. A conformational study of an E. coli O-antigen was performed by calculating atomic distances from NMR NOESY experiments. The acquired data was utilized to validate MD simulations of the LPS embedded in a membrane. The agreement between experimental and calculated data was good and deviations were proven to arise from spin-diffusion. In another study presented herein, both the conformation and the dynamic behavior of amide side-chains linked to derivatives of D-Fucp3N, a sugar found in the O-antigen of bacteria, were investigated. J-couplings facilitated a conformational analysis and 13C saturation transfer NMR experiments were utilized to measure rate constants of amide cis-trans isomerizations. 13C NMR relaxation and 1H PFG diffusion measurements were carried out to explore and describe the molecular motion of mannofullerenes. The dominating motions of the mannofullerene spectral density were found to be related to pulsating motions of the linkers rather than global rotational diffusion. The promising inhibition of Ebola viruses identified for a larger mannofullerene can thus be explained by an efficient rebinding mechanism that arises from the observed flexibility in the linker. Molecular interactions between sugars and caffeine in water were studied by monitoring chemical shift displacements in titrations. The magnitude of the chemical shift displacements indicate that the binding occurs by a face to face stacking of the aromatic plane of caffeine to the ring plane of the sugar, and that the interaction is at least partly driven by solvation effects. Also, the binding of a Shigella flexneri serotype Y octasaccharide to a bacteriophage Sf6 tail spike protein was investigated. This interaction was studied by 1H STD NMR and trNOESY experiments. A quantitative analysis of the STD data was performed employing a newly developed method, CORCEMA-ST-CSD, that is able to simulate STD data more accurately since the line broadening of protein resonances are accounted for in the calculations. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.</p>

<p>In order to understand biological processes, to treat and diagnose diseases, find appropriate vaccines and to prevent the outbreak of epidemics, it is essential to obtain more knowledge about carbohydrate structures. This thesis deals with structure and conformation of carbohydrates, analysed by NMR spectroscopy and MD simulations.In the first two papers, the structures of O-antigen polysaccharides (PS) from two different <i>E. coli</i> bacteria were determined using NMR spectroscopy. The O-antigenic PS from <i>E. coli</i> O152 (paper I) consists of branched pentasaccharide repeating units, built up of three different carbohydrate residues and a phosphodiester, whilst the repeating unit of the O-antigen from <i>E. coli</i> O176 (paper II) is built up of a linear tetrasaccharide consisting of two different monosaccharides.</p><p>In papers III and IV, the conformational analysis of different disaccharides is described. Conformational analysis was performed using NMR spectroscopy and MD simulations (paper IV). In paper III four different glucobiosides were studied using coupling constants and Karplus-type relationships. By use of specific ¹³C isotopically labelled derivatives, additional coupling constants were obtained and the number of possible torsion angles was reduced by half. In paper IV, we examine the conformations of two disaccharides that are part of an epitope of malignant cells. From NOE and T-ROE experiments, short proton-proton distances around the glycosidic linkage were estimated. Furthermore, interpretation of the extracted coupling constants using Kaplus relationships gave the values of the torsion angles. As in paper III, isotopically labelled compounds were synthesised in order to enhance the sensitivity of the analysis. Finally, MD simulations were performed and the results were compared with results from NMR data.</p>

In order to understand biological processes, to treat and diagnose diseases, find appropriate vaccines and to prevent the outbreak of epidemics, it is essential to obtain more knowledge about carbohydrate structures. This thesis deals with structure and conformation of carbohydrates, analysed by NMR spectroscopy and MD simulations.In the first two papers, the structures of O-antigen polysaccharides (PS) from two different E. coli bacteria were determined using NMR spectroscopy. The O-antigenic PS from E. coli O152 (paper I) consists of branched pentasaccharide repeating units, built up of three different carbohydrate residues and a phosphodiester, whilst the repeating unit of the O-antigen from E. coli O176 (paper II) is built up of a linear tetrasaccharide consisting of two different monosaccharides. In papers III and IV, the conformational analysis of different disaccharides is described. Conformational analysis was performed using NMR spectroscopy and MD simulations (paper IV). In paper III four different glucobiosides were studied using coupling constants and Karplus-type relationships. By use of specific 13C isotopically labelled derivatives, additional coupling constants were obtained and the number of possible torsion angles was reduced by half. In paper IV, we examine the conformations of two disaccharides that are part of an epitope of malignant cells. From NOE and T-ROE experiments, short proton-proton distances around the glycosidic linkage were estimated. Furthermore, interpretation of the extracted coupling constants using Kaplus relationships gave the values of the torsion angles. As in paper III, isotopically labelled compounds were synthesised in order to enhance the sensitivity of the analysis. Finally, MD simulations were performed and the results were compared with results from NMR data.

The Escherichia coli O9a and O8 antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. Their O-antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a variable repeat-unit domain, and a non-glycan terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid acceptor, undecaprenyl pyrophosphoryl-β-GlcNAc, due to the sequential activities of two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The work reported in this doctoral thesis establishes a model for biosynthesis of the O8 and O9a antigens using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two α-(1,3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat-unit domains. WbdAO9a polymerizes a tetrasaccharide repeat unit containing two α-(1,3)- and two α-(1,2)-linked mannose residues, while WbdAO8 polymerizes trisaccharide repeat units containing single α-(1,3), α-(1,2), and β-(1,2)-mannoses. Consistent with the multifunctional nature of the WbdA mannosyltransferases, two separable domains were identified in WbdAO9a and three in WbdAO8. Results from mutation of the catalytic site motifs of WbdAO9a and in vitro assays with synthetic acceptors demonstrated that the N-terminal domain of WbdAO9a possesses α-(1,2)-mannosyltransferase activity. Therefore, these studies form a framework to investigate the hypothesis that each domain of WbdA is a catalytically active mannosyltransferase module, possessing one of the activities associated with the enzyme. The O8 and O9a systems provide examples where a unique combination of single domain mannosyltransferases, one of which is capable of adding two mannose residues in succession, and a multidomain polymerizing mannosyltransferase is exploited to build a single glycan. The information gained from this project is expected to extend to other bacteria that utilize similar pathways for biogenesis of cell surface glycopolymers. / Natural Sciences and Engineering Research Council of Canada