Onchocerca volvulus is a filarial parasite transmitted to humans by female Simulium spp. black flies. Infection with this parasite can cause blindness and severe skin disease among humans in Africa and the Americas. Enzyme-linked Immunosorbent Assay serological testing of OV-16 antigen is a diagnostic tool for determining effective elimination of the parasite. Programs typically rely on OV–16 ELISA to evaluate the progress towards interruption and/or elimination of disease by mass drug distribution of ivermectin and vector larvicidal control efforts. As elimination grows closer, monoclonal antibody positive controls for OV-16 ELISA become important to develop for Onchocerca testing due to the limited availability of pooled sera positive controls. Recent evaluation of laboratory designed OV-16 ELISA coating antigen by the Unnasch Lab (University of South Florida) showed that polymorphisms occurred which may alter the ability of the humanized monoclonal antibody to recognize the cognate antigen. With this development, it was important to evaluate these polymorphisms and isolate them for further testing against the standardized monoclonal antibody and positive sera to determine the effects antigenic polymorphisms could have on diagnostic testing. Upon evaluation, the polymorphisms did influence signaling when testing the monoclonal antibody. However, little effect on the recognition of the antigen was seen when different isoforms were evaluated against sera from O. volvulus infected individuals. Data suggest that the epitope recognized by the synthetically produced monoclonal antibody is not immuno-dominant in infected individuals.

Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo. We used an in vivo expression technology (IVET) strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 x 10(5) CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 x 10(9) CFU of wild-type S. Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, chi 9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae. Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity. IMPORTANCE Salmonella enterica is the leading cause of bacterial food-borne infection in the United States. S. Typhimurium is capable of producing up to 13 distinct surface structures called fimbriae that presumably mediate its adherence to surfaces. The roles of most of these fimbriae in disease are unknown. Identifying fimbriae produced during infection will provide important insights into how these bacterial structures contribute to disease and potentially induce protective immunity to Salmonella infection. We identified four fimbriae that are produced during infection. Deletion of all four of these fimbriae results in a significant reduction in virulence. We explored ways in which the expression of these fimbriae may be exploited for use in recombinant Salmonella vaccine strains and found that production of Saf and Stc fimbriae are important for generating a strong immune response against a vectored antigen. This work provides new insight into the role of fimbriae in disease and their potential for improving the efficacy of Salmonella-based vaccines.

Abstract Collagen molecules consist of three polypeptide chains that are coiled around each other to form a triple-helical structure. The formation of stable collagen triple helices requires the hydroxylation of proline residues catalyzed by collagen prolyl 4-hydroxylases (C-P4H). Vertebrate C-P4H is an ER-resident enzyme that consists of two catalytically active α subunits and two β subunits. Production of recombinant human collagen and gelatin could have numerous medical and industrial applications, but most recombinant systems lack the C-P4H activity. The yeast Pichia pastoris has been successfully engineered to produce stable human collagens and gelatins by co-expression of the collagen polypeptide chains with the two C-P4H subunits. This study examined the effect of deletion of the C-propeptide, or its replacement by a trimerizing foldon domain, on the assembly of type I and III collagen triple helices in P. pastoris. It was observed that the absence of the C-propeptide leads to inefficient collagen chain assembly whereas the replacement of C-propeptide with a foldon domain increased the assembly up to 3-fold. Moreover, the co-expression of α1(I) and α2(I) chains fused with foldon yielded heterotrimeric type I collagen molecules with a typical chain ratio of 2:1. As the foldon domain contains no information for collagen chain recognition, the present data indicate that the chain assembly is defined not only by the C-propeptides but also by other determinants present in the α chains. Another aspect studied here was the expression and secretion of gelatin fragments of varying size and conformation in P. pastoris. It was discovered that gelatin fragment size affects its secretion as the 90 kDa fragment was less efficiently secreted than the 45 kDa fragment. Secretion was also dependent on the fragment conformation as induction of the triple helix formation by either C-propeptide or foldon led to the accumulation of the fragments inside the yeast cells despite the presence of an efficient secretory signal. C-P4H was long assumed to exist as one type only but the cloning of several C-P4H α subunits raised questions concerning the specific roles of the C-P4H isoenzymes. The generation of mice lacking the type I C-P4H, which is regarded as the major C-P4H isoenzyme, indicated that this isoenzyme is essential for the embryonic development of the mouse. The embryos lacking type I C-P4H died at an early stage of their development due to the disruption of basement membranes. It was found that the basement membranes of the homozygous null embryos lacked type IV collagen whereas the fibrillar collagens were synthesized, although with altered morphology. The data reported here also demonstrate that the other C-P4H isoenzymes cannot compensate for the lack of type I isoenzyme.

Abstract Collagen prolyl 4-hydroxylase (C-P4H) plays a central role in the biosynthesis of collagens by hydroxylating proline residues. The enzyme has been a subject of intense interest as a target enzyme for drug development. The recombinant expression of human C-P4H in prokaryotes has not yet been described. This work reports on the development of an expression system for human C-P4H in E. coli. The vertebrate C-P4H enzymes are α2β2 tetramers, consisting of two β subunits which are identical to protein disulphide isomerase (PDI), aside from the two α subunits which have the catalytic activity. The function of PDI is to keep the α subunit in a soluble and active state. Therefore, the expression system should assure the expression of the β subunit in the cell before the α subunit by using two different promoters. An active C-P4H tetramer was obtained in the periplasm of E. coli. However, further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human C-P4H tetramer. The exchange of four rare E. coli codons of the pdi gene and the optimized distance between ribosome binding site and translation initiation, resulted in 50-fold P4H-activity and 25 mg/l purified enzyme. Comparison of the expression level of mRNA from the α and β subunits by Sandwich hybridization identified single induction with anhydrotetracycline in fed-batch fermentations as a limiting parameter. This caused an insufficient expression level of mRNA and thereby a low yield of C-P4H. A maximum yield was obtained by repeated addition of anhydrotetracycline that led to higher mRNA levels and increased productivity. A newly developed stochastic simulation model of translational ribosome traffic in bacteria assesses the effect of codon usage to ribosome traffic and to the overall translation rate and mRNA stability. Using human PDI, it was shown that substitution of four 5' codons of the human PDI sequence that are rare in E. coli sequences, by synonymous codons preferred in E. coli led to a 2-fold increase of total PDI amount and even to a 10-fold increase of soluble PDI amount.

La thèse présentée porte sur la préparation de dérivés de polypeptides recombinant à base de motifs élastine (ELPs) ainsi que sur l'étude de leurs propriétés physicochimiques et biologiques. Des ELPs contenant des résidus méthionine ont été modifiés de manière chimiosélective soit en utilisant des halogénures d'alkyle ou différents époxydes, soit par oxydation des résidus méthionine. La caractérisation par RMN et par spectrométrie de masse des composés obtenus a permis de confirmer leur fonctionnalisation quantitative. L'étude de la réponse en température de ces dérivés d'ELP par des mesures de turbidité ou par des mesures de diffusion de la lumière a montré le fort impact des modifications entreprises sur la température de transition (TI) des ELPs. Il a également été montré que la n peut être modifiée par échange des contre-ions des dérivés cationiques. Enfin, des monosaccharides ont été conjugué aux ELPs contenant des groupements alcyne par cycloaddition de Huisg en afin d'obtenir des glycopolypeptides. Les propriétés thermosensibles ainsi que les propriétés biologiques de ces conjugués ont été testées et ces dernières ont permis de montrer leur capacité à se lier sélectivement à des lectines. Leur utilisation pour trier des protéines d'intérêt et les redisperser est finalement évaluée de façon préliminaire. / This thesis describes the preparation of elastin-like polypeptides (ELPs) derivatives and the study of their physico-chemical and biological properties. Methionine-containing ELPs were chemoselectively modified using either alkyl halides or epoxides or by oxidation of their methionine residues. The successful functionalization was assessed by NMR and mass spectrometry analysis of the resulting compounds. The thermoresponsive properties of these ELP derivatives were evaluated either by light scattering or by turbidity measurements showing the strong effect of these modifications on the ELPs transition temperature (TI). The counterion affect on the thermosensitivity of the polycationic derivatives was also studied. The synthesis of ELP glycopolypeptides was finally achieved by conjugating monosaccharides to the ELP alkyne derivatives through Huisgens cycloaddition. Along with the thermoresponsive properties, the bioactivity of the ELP glycoconjugates was studied and proved their ability to specifically bind lectins. Their use for protein sorting and release was preliminary evidenced.

Cytoplasmic dynein is a molecular motor that moves cargos along microtubules. Dynein, together with its large co-factor dynactin, is responsible for the vast majority of traffic towards the centre of the cell. The largest subunit of the dynein complex is called the dynein heavy chain (DHC). The DHC includes a C-terminal motor domain, which converts ATP hydrolysis into mechanical force, an N-terminal tail domain, and a flexible linker domain to join the two together. An intermediate chain (DIC) and light intermediate chain (DLIC) bind directly to the DHC tail, while light chains (DLCs) bind to the DIC. This tail complex is important for both cargo binding as well as homodimerisation of the DHC, which is necessary for processive movement. Previous studies suggest that the DLCs play an important role in homodimerisation, but it remains unclear how else the DHCs are held together. Using S. cerevisiae as a model system, I co-expressed all four dynein subunits and purified functional dynein motors. In this background, I found that truncating the DHC to include only the first 1004 residues (out of the total 4092) eliminates the motor domain as well as the flexible linker domain, while preserving binding to the DIC, DLIC and DLC. However, truncating just another 50 residues off of the C-terminus led to a loss of all accessory subunits. I developed a protocol for expressing and purifying large quantities of the 1004 residue construct, thus I provide the first description of a recombinant dynein tail domain. Using negative stain electron microscopy (EM), I also present the first 3D structural information for the tail region of the cytoplasmic dynein motor. I then describe a construct including only the first 557 residues of the DHC, which dimerises despite not being able to bind any of the other subunits. I present a crystal structure of this smaller DHC fragment, which shows that the N-terminal 180 residues of the DHC constitute an intricate dimerisation domain made up of a β-sheet sandwiched between α-helices. Not only is this the first crystal structure of any part of the DHC N-terminus, but it reveals a previously undocumented dimerisation domain within the DHC itself. Furthermore, information garnered from this crystal structure allowed for interpretation of a recent cryo-EM structure of a triple complex containing the dynein tail, dynactin and the cargo adaptor BICD2 (TDB) that was solved by my colleagues in the Carter group. Only by docking the DHC N-terminus crystal structure within the TDB EM density did it become clear that the N-terminus of the DHC is responsible for the majority of the contacts the dynein tail makes with both dynactin and BICD2. Therefore the work that I present here sheds new light on the unexpected importance of the DHC N-terminus and allows two important conclusions to be made. First, the N-terminal 180 residues of the DHC constitute a dimerisation domain of its own. Second, the next ~400 residues of the DHC form a domain that plays a key role in the complex interface between dynein, dynactin and BICD2.

No description available.

Les sols qui ne rencontrent pas les normes d’ingénierie civile doivent êtres soumis à des améliorations géotechniques car les vibrations causées par les tremblements de terre ou par la surcharge sur des infrastructures en hauteur peuvent mener à la liquéfaction partielle ou totale des sols saturés en eau. Ceci peut donc entrainer des dommages importants aux structures construites sur ces sols. Certaines méthodes existent pour remédier à ce problème, mais elles demeurent couteuses et parfois toxiques car elles utilisent de l’acrylamide et des lignosulfates. La bio-précipitation in situ de calcite dans les sols représente une méthode alternative. Le tout se fait avec des bactéries qui démontrent une activité uréolytique. La présente étude s’est intéressée à l’activité uréolytique des souches Escherichia coli, Sporosarcina ureae, Bacillus pasteurii, Lysinibacillus sphaericus, Bacillus subtilis et Bacillus megaterium. Les résultats démontrent que l’urée est seulement dégradée par les souches S. ureae et S. pasteurii. L’incubation de S. ureae en présence de Ni2+ (0.1-1 ppm) et Fe2+ (1-10 ppm) a toutefois permis d’augmenter l’activité catalytique de la souche, ce qui démontre l’importance des éléments nutritifs lors de l’hydrolyse de l’urée. Afin de tester l’activité uréolytique des autres souches, nous avons introduit un système d’expression uréase dans la souche E. coli en substituant des amino-acides dans la structure primaire des protéines. Suite à cette modification, l’activité uréolytique de E. coli s’est améliorée et est devenue comparable à celle des souches S. ureae et S. pasteurii. L’injection de S. ureae et du mutant E. coli dans des sables non-consolidés a permis de cimenter de façon significative (p < 0.05) le matériel par rapport à des sables non inoculés, et ce après seulement 48 heures. Le transfert du système recombinant de E coli vers S. ureae est présentement en cours. Ces résultats prometteurs indiquent qu’il est possible de stimuler la précipitation in situ de calcite en utilisant des bactéries et de stabiliser les sols prônes à la liquéfaction. === Soils often do not satisfy functional requirements for civil engineering projects and as a result geotechnical improvements to soils are often made. Dynamic shaking during earthquakes or static overloading by overlying structures may still result in liquefaction in partially or fully water saturated soils. These have little bearing capacity for structures. Severe damages can result. Moreover, preventative soil grouting strategies are expensive, toxic, and permanent due to acrylamides, lignosulfonates, and otherwise harmful compounds present therein. Alternative methods of strength enhancement are advisable. Microbial induced calcite precipitation (MICP) was assessed in this investigation to consolidate loose, sandy soils. Ureolytic activty of Escherichia coli, Sporosarcina ureae, Bacillus pasteurii, Lysinibacillus sphaericus, Bacillus subtilis and Bacillus megaterium were assessed. Urea was readily degraded foremost by S. ureae and next by S. pasteurii with no significant (p <0.05) activity in other strains. Incubation of S. ureae with 0.1 - 1ppm Ni2+ and 1-10ppm Fe2+ was shown to improve catalytic activity, suggesting their importance as a dietary source for urea hydrolysis. A urease expression system was established in E. coli and particular amino acid substitutions in protein primary structure made. Enhanced ureolytic activity was observed in these E. coli mutants, comparable to native S. ureae activity. Application of wild type S. ureae and recombinant E. coli for MICP in a model sand showed significant (p < 0.05) improvements compared to controls after 48 hours. Transfer of the recombinant system in E. coli to S. ureae is currently underway. These results provide valuable insight affirming that a practical system for the application of MICP may be feasible in the field for the strength enhancement of native and construction-laid loose, sandy soils.

Les pneumopathies d'hypersensibilité sont des maladies respiratoires liées à l'inhalation répétée de substances antigéniques. Le diagnostic nécessite la présence d'un ensemble d'arguments cliniques, radiologiques, fonctionnels et immunologiques car les symptômes sont peu spécifiques. La mise en évidence d'immunoglobulines G (IgG) spécifiques des agents étiologiques est un élément essentiel dans la prise en charge diagnostique et thérapeutique. L'objectif de ce travail de thèse était d'identifier des protéines bactériennes et fongiques reconnues par les IgG des patients atteints de la maladie du poumon de fermier (PDF) et du poumon de mécanicien (FDM), et de produire ces protéines de façon recombinante afin de développer des tests sérologiques standardisés de type ELISA. Deux micro-organismes impliqués dans le PDF (Saccharopolyspora rectivirgula et Eurotium amstelodami, un Aspergillus), et un micro-organisme impliqué dans le PDM (Mycobacterium immunogenum) ont été étudiés. L'approche immunoprotéomique développée a permis de sélectionner les protéines d'intérêt, puis de produire les antigènes recombinants correspondants par génie génétique. Des sérums de patients PDF, PDM et de témoins exposés ont été recueillis dans le cadre de protocoles cliniques multicentriques. Les performances (sensibilité, spécificité, aire sous la courbe) des tests ELISA-IgG utilisant les antigènes recombinants produits ont été évaluées par l'analyse en courbe ROV (Receiver operating characteristics). A partir des trois micro-organismes étudiés, 71 protéines immuno-réactives ont été identifiées et 28 protéines recombinantes ont été produites et testées en ELISA. Pour le diagnostic du PDM, deux antigènes recombinants, la dihydrolipoyl déshydrogénase and Facyl-CoA déshydrogénase, étaient particulièrement performants avec une sensibilité de 90% lorsqu'ils étaient utilisés en combinaison. Pour le diagnostic du PDF, deux antigènes recombinants à'Aspergillus, la Glu/Leu/Phe/Val déshydrogénase et la glucose-6-phosphate isomérase, ont permis d'obtenir une sensibilité de 89% et une spécificité de 84%. L'utilisation de trois antigènes recombinants de S. rectivirgula,SRl¥A (protéine de fonction inconnue), SRI? (catalase) et SR22 (kétol acide réducto-isomérase), ont permis d'obtenir une sensibilité et spécificité optimales de 83% et 77%. Les protéines identifiées étaient majoritairement des protéines enzymatiques, dont certaines ont été mis en évidence comme facteurs de virulence dans d'autres pathologies. Des études complémentaires, sur des modèles animaux et sur des modèles cellulaires, devront être mennées pour explorer l'implication de ces protéines dans l'induction de la maladie. Ce travail a permis d'améliorer les connaissances sur les protéines bactériennes et fongiques reconnues par les anticorps des patients atteints de PDF et de PDM, de développer des antigènes recombinants, et de mettre au point des tests sérologiques standardisés et performants. Ces tests pourront faire l'objet d'une valorisation vers l'industrie. En effet, les sérologies pour le diagnostic des PHS sont des demandes courantes dans les laboratoires d'analyse médicale. / The hypersensitivity pneumonitis is a pulmonary disease caused by repetitive inhalation of antigens. The diagnosis requires clinical, radiological, functional and immunological features because of unspecific symptoms. The identification of specifie antibodies to causative antigens is an essential way for the diagnosis and therapeutic management.The aims of this thesis work were to identify bacterial and fungal immunogenic proteins specifie to patient with farmer's lung (FL) and machine operator's lung (MOL) diseases, to synthesize specifie recombinant antigens for the development of a standardized ELISA. Two microorganisms involved in FL (Saccharopolyspora rectivirgula and Eurotium amstelodami (Aspergillus sp)), and one involved in MOL had been srudied. The immunoproteomics approach has permit to select interesting proteins and to synthesize recombinant antigens by genomics techniques. The sera from FL patients (n=52), MOL patients (n=16) and sera from exposed control subjects were recruited. ELISA-IgG using recombinant antigens efficacies were evaluated by Receiver operating characteristics analysis (sensitivity, specifïcity, area under the curve).From the three srudied microorganisms, 71 immunogenic proteins were identified and 28 recombinant antigens were produced and tested by ELISA. For the MOL diagnosis, the recombinants dihydrolipoyl dehydrogenase and acyl-CoA dehydrogenase were useful with a sensitivity of 90% when used in combination. For the FL diagnosis, two recombinant proteins from Aspergillus, Glu/Leu/Phe/Val dehydrogenase and glucose-6-phosphate isomerase had a sensitivity of 89% and a specifïcity of 84%. A combination of the three recombinant antigens from S. rectivirgula, SRI FA (unknown function), SRI 7 (catalase) and SR22 (ketol acid reductoisomerase) allowed to obtain a sensitivity of 83% and a specificity of 77%. The immunogenic proteins were mainly enzymes, and some of these have been implicated in important functions for survival or the virulence of others pathogens. Further studies are required to determine the role of these proteins in immunological or virulence processes by animal and cellular model application. This present work has improved our knowledge about bacterial and fungal proteins recognized by FL and MOL patient's antibodies, and to develop useful standardized serological tests with new recombinant antigens. These tests could be exported to the health management in industry. Indeed, hypersensitivity pneumonitis serodiagnosis tests are common requests in medical analysis laboratories

Several expression systems for recombinant protein production, essentially cells or whole organisms are currently in use today. Recently, research into recombinant protein production revealed a more attractive expression system based on the microalgae, C. reinhardtii, for significant savings in cost and production of correctly folded recombinant proteins. However, protein yield in the microalgae remain very low, non-predictable and whether this was due to limitations in the system was unclear. Using the expression of E. coli β-glucuronidase (gus) in C. reinhardtii chloroplast, the overall aim of the project was to address if the low recombinant gus yield in C. reinhardtii was due to limitations that affect growth and protein production, and if the fluxes for recombinant gus production were suboptimal (limiting). The finding was used to implement a strategy for a more predictable recombinant protein yield in C. reinhardtii. The research involved a range of experiments, analysis, and Flux Balance Analysis (FBA) modelling. The growth of C. reinhardtii cultures were characterized in autotrophic, heterotrophic and mixotrophic conditions to identify factors that limit growth and recombinant gus yields. These factors were availability of light, carbon and nitrogen substrates, pH changes, protein burden and energetic limitation (ATP). The highest biomass was obtained in autotrophic and mixotrophic cultures (>1 g/litre), the lowest biomass was in heterotrophic cultures (~0.4 g/litre). The recombinant gus yields on the basis of dry cell weight were: mixotrophic cultures (0.038%), autotrophic cultures (0.032%), heterotrophic cultures (0.026%). No detectable protein burden was observed for expression of recombinant gus in autotrophic and mixotrophic conditions, but protein burden was significant in heterotrophic condition (15 – 18% reduction in growth rate). A strategy that significantly increased growth and cell productivity (>3 fold) in heterotrophic condition was identified. FBA was used to identify suboptimal amino acid steady state fluxes (bottlenecks) that limited the gus yield. Using FBA modelling, model verifications and corrections, a strategy that significantly increased the yield of recombinant gus in each cell (~2 fold) was identified. Put together, the total increase represents a 6 fold increase in recombinant gus yield. Furthermore, this research presented a framework for identifying, analysing and understanding the effect of the uptake of individual amino acid towards recombinant protein yield.