Shiga toxin-producing Escherichia coli (STEC) has been recognized as an important foodborne pathogen. Of this group, O91 is one of the common serogroups frequently isolated from patients and food in some countries, with O91:H21 being previously implicated in hemolytic uremic syndrome (HUS). Cattle are principle reservoirs for STEC, and studies examining STEC shedding in cattle often include experimental inoculation of strains of interest using antibiotic resistance markers for identifiable recovery. However, indigenous fecal microbes exhibiting similar resistance patterns can confound such studies. Such was the case in a study by our group when attempting to characterize shedding patterns of O91:H21 in calves, leading us to seek other, more effective, markers. Among our strategies was the development of a chromosomally integrated bioluminescence marker via transposon mutagenesis using a luxCDABE cassette from Photorhabdus luminescens and a plasmid borne GFP marker via transformation of the pGFP vector. The luxCDABE marker was inserted on host chromosome at a site that was 27 nucleotides before the stop codon of gene yihL and confirmed to have little impact on important virulence genes and growth rate with a very high stability. In contrast, plasmid borne GFP marker showed poor stability without the application of appropriate antibiotic selection pressure. For calves receiving luxCDABE-marked O91:H21, the fecal counts of the organismranged from 1.2 x 10 3 to 1.3 x 10 4CFU/g at two days post inoculation and decreased to 5.8 to 8.7 x 10 2 CFU/g or undetectable level after two weeks.Intestinal contents sampled from various positions at day 14 post inoculation indicated that cecum and descending colon may be the primary localization sites of this O91:H21 strain. Compared to antibiotic resistance markers, the use of bioluminescence markers does not require the restricted pre-inoculation screening of animals. The enumeration of luxCDABE-marked O91:H21 from feces and intestinal contents was easily accomplished and confirmed reliable by M-PCR analysis under the presence of indigenous bacteria which cannot be eliminated by antibiotic-supplemented selective plates. Therefore, the chromosomal integrated luxCDABE marker may be a better model for the study of STEC colonization and shedding in cattle.

La toxine de pertussis (PTX) est une A-B protéine considérée comme l’un des principaux facteurs de virulence de Bordetella pertussis, l’agent bactérien responsable de la coqueluche. Aujourd’hui, cette maladie représente encore un réel danger pour les nouveaux-nés et les nourrissons non ou partiellement immunisés. Actuellement, la coqueluche provoque encore la mort d’environ 350.000 individus par an. La toxicité de la PTX est liée à l’activité enzymatique de sa sous-unité A capable d’inhiber les voies de signalisation associées aux protéines Gi. La partie B, quant à elle, permet l’entrée de cette sous-unité A dans le cytoplasme des cellules cibles en se liant spécifiquement à son ou ses récepteurs membranaires toujours inconnus de nos jours. Des études réalisées chez la souris et chez l’homme ont montré que les vaccins anticoquelucheux combinés à différents antigènes vaccinaux étaient capables de moduler leurs réponses humorales spécifiques. Par ailleurs, la PTX est couramment qualifiée d’agent immunostimulant. En effet, des modèles murins de vaccination permirent d’identifier des propriétés adjuvantes de la PTX coadministrée avec des antigènes non relevants. Le travail développé dans ce manuscrit étudie les effets de la PTX sur 2 types cellulaires primordiaux sollicités lors d’une vaccination : la cellule dendritique (DC) et le lymphocyte T CD4+ naïf. Les DC sont les seules cellules présentatrices d’antigènes aptes à initier une réponse immune primaire. Dans un premier temps, nous avons montré que la PTX était capable d’activer des DC générées in vitro à partir de monocytes. En effet, elles acquièrent un phénotype mature caractérisé par une augmentation de l’expression membranaire des molécules costimulatrices et du CMH de classe II, démontrant un effet direct et spécifique de la PTX sur les DC myéloïdes. Parallèlement, ces DC produisent du TNF-a, de l’IL-12p40 et de l’IL-12p70 et activent NF-kappaB, un facteur de transcription essentiel au processus de maturation. Nous avons obtenu des résultats similaires avec une toxine génétiquement modifiée qui est enzymatiquement inactive. A partir de sang total incubé avec la PTX, nous avons par ailleurs observé que les DC circulantes du nouveau-né étaient déficientes dans leur maturation et leur sécrétion d’IL-12p70 comparées aux DC de l’adulte. D’autre part, il a été décrit précédemment que la PTX exerçait des effets mitogènes sur les lymphocytes T humains et murins. Cependant, le rôle qu’elle joue sur la population des lymphocytes T CD4 naïfs reste peu connu. A l’issue de notre second travail, nous pouvons dès lors affirmer que la PTX est également capable d’activer des lymphocytes T CD4+CD45RA+ naïfs isolés à partir des cellules mononuclées du sang périphérique, et ce indépendamment de son activité enzymatique. En effet, ces lymphocytes T CD4+ naïfs stimulés par la PTX prolifèrent, synthétisent des quantités non négligeables d'ARN messagers codant pour l’IL-2 et le TNF-a, augmentent l’expression membranaire des molécules CD40L, CD69 et CD25 et expriment la protéine Foxp3. Cette activation s’accompagne de la translocation nucléaire de NF-kappaB et NFAT. Parallèlement à l’adulte, la PTX active les lymphocytes T CD4 néonataux. Néanmoins, ceux-ci prolifèrent moins bien et expriment plus faiblement le CD40L à leur surface. Enfin, la PTX induit la sécrétion de taux importants d’IFN-g par des T CD4+CD45RA+ naïfs adultes mis en présence de DC autologues. Nous terminerons en proposant l’hypothèse suivante : La PTX pourrait exercer ses propriétés adjuvantes par l’intermédiaire de différents mécanismes comprenant notamment la maturation des DC d’origine myéloïde et l’activation des lymphocytes T CD4+CD45RA+ naïfs. Ces 2 populations cellulaires sont en effet les principaux protagonistes impliqués dans la réponse immune primaire.

Transfer RNAs (tRNA) function as adaptor molecules in the translation of mRNA into protein. These adaptor molecules require modifications of a subset of their nucleosides for optimal function. The most frequently modified nucleoside in tRNA is position 34 (wobble position), and especially uridines present at this position. Modified nucleosides at the wobble position are important in the decoding process of mRNA, i.e., restriction or improvement of codon-anticodon interactions. This thesis addresses the functional aspects of the wobble uridine modifications. The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes; elongation of RNA polymerase II transcription, regulation of polarized exocytosis, and formation of modified wobble nucleosides in tRNA. In Paper I, we show that the phenotypes of Elongator deficient cells linking the complex to transcription and exocytosis are counteracted by increased level of and . These tRNAs requires the Elongator complex for formation of the 5-methoxycarbonylmethyl (mcmlnGUUGsmcm25tRNALysUUUsmcm25tRNA5) group of their modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Our results therefore indicate that the relevant function of the Elongator complex is in formation of modified nucleosides in tRNAs and the defects observed in exocytosis and transcription are indirectly caused by inefficient translation of mRNAs encoding gene products important for these processes. The lack of defined mutants in eukaryotes has led to limited understanding about the role of the wobble uridine modifications in this domain of life. In Paper II, we utilized recently characterized mutants lacking the 2-thio (s2) or 5-carbamoylmethyl (ncm5) and mcm5 groups to address the in vivo function of eukaryotic wobble uridine modifications. We show that ncm5 and mcm5 side-chains promote reading of G-ending codons, and that presence of a mcm5 and an s2 group cooperatively improves reading of both A- and G-ending codons. Previous studies revealed that a S. cerevisiae strain deleted for any of the six Elongator subunit genes shows resistance towards a toxin (zymocin) secreted by the dairy yeast Kluyveromyces lactis. In Paper III, we show that the cytotoxic γ subunit of zymocin is a tRNA endonuclease that target the anticodon of mcm5s2U34 containing tRNAs and that the wobble mcm5 modification is required for efficient cleavage. This explains the γ-toxin resistant phenotype of Elongator mutants which are defective in the synthesis of the mcm5 group.

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.

Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.

Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.

Shiga toxin-producing <i>E. coli</i> (STEC) is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a type III secretion system (T3SS), which forms attaching and effacing lesions (A/E). The necessary effectors which form A/E by manipulation of host signalling and actin nucleation are present on a pathogenicity island called the Locus of Enterocyte Effacement (LEE).<p> It has been reported that vaccination of cattle with Type III-secreted proteins (T3SPs) from STEC O157 resulted in decreased shedding. In order to extend this to non-O157 STEC serotypes, we examined the serological cross-reactivity of T3SPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Groups of cattle were vaccinated with T3SPs produced from each of the serotypes and the magnitude and specificity of the responses were measured resulting in limited cross reactivity. Overall, results suggest that vaccination of cattle with T3SPs as a means of reducing the risk of STEC transmission to humans will induce protection that is serotype specific.<p> To pursue the possibility of a cross-protective vaccine, we investigated the protective properties of a chimeric Tir protein against STEC serotypes. Several studies have reported that Tir is highly immunogenic and capable of producing high antibody titers. Potter and colleagues also demonstrated that the vaccination of cattle with ∆tir STEC O157 strain did not protect as well as the wildtype strain. We constructed thirty-mer peptides to the entire STEC O157 Tir protein, as well as to the intimin binding domain of the Tir protein from STEC serotype O26, O103 and O111. Using sera raised against STEC O157 and non-O157 T3SPs, we identified a number of immunogenic peptides containing epitopes unique to a particular serotype. Two different chimeric Tir proteins were constructed containing the STEC O157 Tir protein fused with six STEC non-O157 peptides with or without the Leukotoxin produced by <i>Mannheimia haemolytica</i>. However, the vaccination of mice with the chimeric protein did not protect against challenge with STEC O157 or STEC O111. These results suggest that to achieve cross protection against STEC serotypes using a recombinant protein vaccine, other immunogenic and protective antigens must also be included.<p> In order to identify other immunogenic and cross-protective antigens we cloned and expressed the genes coding for 66 effectors and purified each as histidine-tagged proteins. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with T3SPs from four STEC serotypes, experimentally infected cattle and human sera from 6 HUS patients. A total of 20 proteins were recognized by at least one of the STEC T3SP- vaccinated rabbits using Western blots. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA and NleA were recognized by the majority of the samples tested. Overall, proteins such as Tir, EspB, EspD, NleA and EspA were highly immunogenic for both vaccinated and naturally infected subjects.<p> Based on the above results, two different mixtures of secreted proteins (5 proteins and 9 proteins) were used to vaccinate mice and test the level of shedding following challenge with STEC O157. Overall, the cocktail vaccine containing 9 immunogenic effectors including Tir, EspB, EspD, NleA and EspA was capable of reducing shedding as effectively as the current STEC T3SPs vaccine, Econiche®.

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.

Shiga toxin-producing <i>E. coli</i> (STEC) is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a type III secretion system (T3SS), which forms attaching and effacing lesions (A/E). The necessary effectors which form A/E by manipulation of host signalling and actin nucleation are present on a pathogenicity island called the Locus of Enterocyte Effacement (LEE).<p> It has been reported that vaccination of cattle with Type III-secreted proteins (T3SPs) from STEC O157 resulted in decreased shedding. In order to extend this to non-O157 STEC serotypes, we examined the serological cross-reactivity of T3SPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Groups of cattle were vaccinated with T3SPs produced from each of the serotypes and the magnitude and specificity of the responses were measured resulting in limited cross reactivity. Overall, results suggest that vaccination of cattle with T3SPs as a means of reducing the risk of STEC transmission to humans will induce protection that is serotype specific.<p> To pursue the possibility of a cross-protective vaccine, we investigated the protective properties of a chimeric Tir protein against STEC serotypes. Several studies have reported that Tir is highly immunogenic and capable of producing high antibody titers. Potter and colleagues also demonstrated that the vaccination of cattle with ∆tir STEC O157 strain did not protect as well as the wildtype strain. We constructed thirty-mer peptides to the entire STEC O157 Tir protein, as well as to the intimin binding domain of the Tir protein from STEC serotype O26, O103 and O111. Using sera raised against STEC O157 and non-O157 T3SPs, we identified a number of immunogenic peptides containing epitopes unique to a particular serotype. Two different chimeric Tir proteins were constructed containing the STEC O157 Tir protein fused with six STEC non-O157 peptides with or without the Leukotoxin produced by <i>Mannheimia haemolytica</i>. However, the vaccination of mice with the chimeric protein did not protect against challenge with STEC O157 or STEC O111. These results suggest that to achieve cross protection against STEC serotypes using a recombinant protein vaccine, other immunogenic and protective antigens must also be included.<p> In order to identify other immunogenic and cross-protective antigens we cloned and expressed the genes coding for 66 effectors and purified each as histidine-tagged proteins. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with T3SPs from four STEC serotypes, experimentally infected cattle and human sera from 6 HUS patients. A total of 20 proteins were recognized by at least one of the STEC T3SP- vaccinated rabbits using Western blots. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA and NleA were recognized by the majority of the samples tested. Overall, proteins such as Tir, EspB, EspD, NleA and EspA were highly immunogenic for both vaccinated and naturally infected subjects.<p> Based on the above results, two different mixtures of secreted proteins (5 proteins and 9 proteins) were used to vaccinate mice and test the level of shedding following challenge with STEC O157. Overall, the cocktail vaccine containing 9 immunogenic effectors including Tir, EspB, EspD, NleA and EspA was capable of reducing shedding as effectively as the current STEC T3SPs vaccine, Econiche®.

Some yeast strains secrete extracellular polypeptide toxins known to have potential growth inhibitory activity on sensitive yeast cells. These yeast strains are known as killer yeasts and their toxins are named as killer toxins or killer proteins. Yeast killer proteins are found inhibitory to Gram-positive bacteria in several studies which were based on microbial interactions of the producer strains tested with sensitive strains. K5 type yeast killer protein produced by Pichia anomala NCYC 434 was previously purified and characterized in our laboratory. The protein is glycosilated and has a pI value of 3,7 and molecular mass of 49 kDa, with exo &amp / #946 / -1,3-glucanase activity. Antibacterial activity of the pure K5 type yeast killer protein was tested against 19 clinical isolates of gram-positive bacteria causing skin infections and 2 quality control strains and found to have inhibitory activity on the isolates of Methicillin-sensitive Staphylococcus aureus (MSSA) and Enterococcus faecium. Toxin MIC and MBC ranges were 32 - 256 &micro / g/ml and 64 - &gt / 512 &micro / g/ml respectively. Cell killing analysis revealed that toxin has a bacteriostatic activity and the inhibitory effect starts between 8. and 12. hours. Regrowth of the bacteria is retarded with the increased dose of the toxin. K5 type yeast killer protein might be used as a topical antibacterial agent with its bacteriostatic activity for skin and wound infections caused by MSSA and Enterococcus faecium with appropriate formulation studies upon the antibacterial spectrum determination of the toxin in this study.