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Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacterial cause of diarrhea. ETEC bacterial adherence to the small intestinal epithelial cells and delivery of enterotoxins cause diarrhea in children living in developing countries and international travelers. Currently, there are no vaccines licensed for ETEC associated children’s diarrhea and travelers’ diarrhea. Recently, toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] (toxoid fusion), adhesin MEFA (multiepitope fusion antigen) CFA/I/II/IV (CFA MEFA), and toxoid-adhesin MEFA CFA/I/II/IV-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] (CFA-toxoid MEFA) are demonstrated to induce neutralizing antitoxin and/or anti-adhesin antibodies in intraperitoneal (IP) or subcutaneous (SC) immunized mice, suggesting these antigens are potential candidates for ETEC subunit vaccines. However, these antigens have not been examined for immunogenicity using intradermal (ID) or intramuscular (IM) routes, the routes perhaps are more suitable for human vaccine administration. In this study, toxoid fusion 3xST[subscript aN12S]-mnLT[subscript R192G/L211A], CFA/I/II/IV MEFA, alone or combined, or toxoid-adhesin MEFA CFA-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] were ID or IM immunized to mice (8 mice per group) induced antigen-specific antibodies were titrated, and antibody neutralization activities were assessed in vitro. Data showed that mice ID or IM immunized with the toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] antigen developed anti-LT and anti-STa antibodies and mice immunized with the CFA/I/II/IV MEFA developed antibody responses to all seven adhesins (CFA/I, CS1-CS6). In addition, mice co-administered ID or IM with toxoid fusion 3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] and CFA/I/II/IV MEFA, or with toxoid-adhesin MEFA CFA-3xSTa[subscript N12S]-mnLT[subscript R192G/L211A] developed antibodies to both toxins and all seven adhesins. Antibody neutralization studies of the serum samples of the immunized mice showed that the induced antibodies neutralized enterotoxicity of LT and STa and/or inhibited adherence of ETEC or E. coli bacteria producing any of these seven adhesins. These data confirmed immunogenicity of these ETEC subunit vaccine target antigens and provide useful information for vaccine development against ETEC diarrhea.

Rotavirus is the major cause of infantile gastroenteritis. Each year more than 600,000 young children are estimated to die in developing countries throughout the world. Rotavirus infection can be either symptomatic or asymptomatic. But the genetic or molecular basis for rotavirus virulence is not yet clearly understood. NSP4, encoded by genome segment 10, is a multifunctional protein. It is identified as the first viral enterotoxin and is essential for virus morphogenesis and pathogenesis. Analysis of NSP4 from more than 175 strains failed to reveal any sequence motif or amino acid that segregated with the virulence phenotype of the virus. Further, a few studies indicated a lack of consistent correlation between virus virulence and diarrhea inducing ability of the cognate NSP4. To understand the basis for the inconsistency in the enterotoxigenic activity of a few NSP4s reported in a limited number of studies, comparative analysis of the biophysical, biochemical, and biological properties of NSP4ΔN72, which from SA11 and Hg18 was earlier shown to be highly diarrheagenic, from 17 different symptomatic and asymptomatic strains was carried out. To study structure-function relationship we used Thioflavin T fluorescence assay, gel filtration, CD spectroscopy, trypsin susceptibility and enterotoxin assay in newborn mice for all the proteins. Detailed comparative analysis of biochemical and biophysical properties and diarrheagenic activity of the recombinant ΔN72 peptides under identical conditions revealed wide differences among themselves in their resistance to trypsin cleavage, thoflavin T binding, multimerization and conformation without any correlation with their diarrhea inducing abilities. Since earlier studies showed that a secreted peptide (ΔN112) of SA11-NSP4 also induced diarrhea in newborn mice pups, we have generated NSP4ΔN112 deletions from six different strains and tested for their diarrhea inducing ability. The patterns of DD50 values of the ΔN112 peptides was similar to that for ΔN72 peptides, but were 1000-1200-fold less efficient than that of SA11ΔN72. NSP4 exists in multiple forms in the infected cells- as oligomers, higher molecular weight complexes and ER- and cytoplasmic membrane anchored forms. Previous studies suggest that the N-terminal boundary of the oligomerization domain could lie downstream to residue 94 from the N-terminus. A peptide from residue 112-175, secreted from rotavirus infected cells, was reported to induce dose-dependent diarrhea in suckling mice, suggesting that the N-terminal boundary of the enterotoxin activity could lie around residue 112. However, the precise N-terminal boundaries in NSP4 for oligomerization and diarrhea induction have not been identified. To address this question, a large number of deletion mutants C-terminal to residue 94 were generated and tested for their ability to induce diarrhea in newborn mouse pups. Our data suggest that while the deletions ∆N121 to ∆N131 failed to induce diarrhea, ΔN118 was diarrheagenic suggesting that the N-terminal boundary of the minimal diarrhea inducing domain lies between aa 118 and 121. Size exclusion chromatography revealed that residues 95 to 98 are critical and sufficient for oligomerization. Studies on oligomerization further revealed that NSP4ΔN94 exists in pentamers, tetramers and dimers, while deletion mutants C-terminal to aa 94 exist only as dimers. Our studies demonstrate for the first time that not only tetramers but pentamers as well as dimers possess enterotoxigenic properties. Most human rotavirus infections are caused by group A rotaviruses. Within this group, rotaviruses are further classified into subgroups based on the antigenic specificity associated with the protein product of the sixth gene, VP6. Previous studies have mapped SG I specificity to aa position 305 and the region between 296 and 299, and SG II specificity to residue 315 on VP6. However, the subgroup specific determinants on NSP4 have not been identified till date. In this study, we generated several amino acid substitution mutants in the SG I-specific SA11 NSP4∆N72 protein as in previous studies ∆N72 was found to efficiently bind DLPs. Using an enzyme linked immunosorbent assay method, the effect of the mutations in the C-terminal and N-terminal regions in ∆N72 on their binding ability to SG I and SG II DLPs was assayed. Residues at positions 85, 169, 174 and 175 and in the ISVD appear to collectively determine the specificity of binding to DLPs. While the conserved proline and glycines at positions 165, 168 and 162, respectively, are important for maintaining the required conformation for general recognition of DLP. The present study provides important insights towards understanding the determinants in NSP4 for SG-specific DLP interaction.

A relatively high percentage of porcine <i>Escherichia coli</i> isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding the adhesin involved in diffuse adherence I (AIDA-I). This gene and its corresponding protein were first identified and characterized in <i>E. coli</i> strain 2787 isolated from human infantile diarrhea. Little is known about the role of the AIDA-I protein in pathogenesis of porcine enteric disease caused by AIDA-I positive E. coli and the properties of AIDA-I protein expressed by porcine AIDA-I positive <i>E. coli</i> isolates and its receptors. <p>In this study, we demonstrated that AIDA-I adhesin isolated from porcine AIDA-I positive <i>E. coli</i> PD20 and PD58 is an acidic protein consisting of five isoforms. It has a molecular weight (100 kDa) similar to the AIDA-I adhesin expressed by human AIDA-I positive <i>E. coli</i> strain 2787 and has a relatively high amino acid homology (78-87%) with it. Immunodetection of AIDA-I positive <i>E. coli</i> strains using polyclonal anti-AIDA-I antibodies had relatively low sensitivity and specificity, accordingly these tests are unlikely to be used for regular diagnostic detection. <p>Using affinity chromatography, we isolated from porcine intestinal mucus proteins that bind to purified AIDA-I adhesin. These proteins were separated by one- and two-dimensional electrophoresis and subjected to overlay Western blot with purified AIDA-I adhesin and AIDA-I positive <i>E. coli</i> to demonstrate 65 and 120 kDa (p65 and p120) proteins as AIDA-I binding proteins. The identity of p65 was not determined based on LCMS/MS data, whereas p120 was matched to two nuclear proteins (namely, DNA damage binding protein and splicing factor 3b) and one cytoplasmic protein, which is an IgG Fc binding protein. Based on similar amino acid homology, molecular weight, structural similarity to mucin and reported evidence of being secreted by goblet cells into the intestinal lumen, we think that the IgG Fc binding protein is the most likely candidate to serve as a potential receptor in intestinal mucus for AIDA-I adhesin.

A relatively high percentage of porcine <i>Escherichia coli</i> isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding the adhesin involved in diffuse adherence I (AIDA-I). This gene and its corresponding protein were first identified and characterized in <i>E. coli</i> strain 2787 isolated from human infantile diarrhea. Little is known about the role of the AIDA-I protein in pathogenesis of porcine enteric disease caused by AIDA-I positive E. coli and the properties of AIDA-I protein expressed by porcine AIDA-I positive <i>E. coli</i> isolates and its receptors. <p>In this study, we demonstrated that AIDA-I adhesin isolated from porcine AIDA-I positive <i>E. coli</i> PD20 and PD58 is an acidic protein consisting of five isoforms. It has a molecular weight (100 kDa) similar to the AIDA-I adhesin expressed by human AIDA-I positive <i>E. coli</i> strain 2787 and has a relatively high amino acid homology (78-87%) with it. Immunodetection of AIDA-I positive <i>E. coli</i> strains using polyclonal anti-AIDA-I antibodies had relatively low sensitivity and specificity, accordingly these tests are unlikely to be used for regular diagnostic detection. <p>Using affinity chromatography, we isolated from porcine intestinal mucus proteins that bind to purified AIDA-I adhesin. These proteins were separated by one- and two-dimensional electrophoresis and subjected to overlay Western blot with purified AIDA-I adhesin and AIDA-I positive <i>E. coli</i> to demonstrate 65 and 120 kDa (p65 and p120) proteins as AIDA-I binding proteins. The identity of p65 was not determined based on LCMS/MS data, whereas p120 was matched to two nuclear proteins (namely, DNA damage binding protein and splicing factor 3b) and one cytoplasmic protein, which is an IgG Fc binding protein. Based on similar amino acid homology, molecular weight, structural similarity to mucin and reported evidence of being secreted by goblet cells into the intestinal lumen, we think that the IgG Fc binding protein is the most likely candidate to serve as a potential receptor in intestinal mucus for AIDA-I adhesin.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les E. coli entérotoxinogènes (ETEC) sont souvent la cause de diarrhée post-sevrage chez le porc. Deux types d’entérotoxines sont retrouvées chez les ETEC, soit les thermolabiles, comme la toxine LT, et les thermostables, comme EAST-1, STa et STb. Cette dernière est composée de 48 acides aminés et est impliquée dans la pathologie causée par les ETEC. Pour la première fois un variant de la toxine STb fut découvert dans une étude. Nous avons alors émis l’hypothèse qu’il y a présence de variants dans la population de souches ETEC du Québec. Dans les 100 souches STb+ analysées, 23 possédaient le gène de la toxine avec une variation dans la séquence génétique : l’asparagine était présente en position 12 remplaçant ainsi l’histidine. Une corrélation entre la présence du variant et la présence de facteurs de virulence retrouvés dans ces 100 souches ETEC étudiées a été effectuée. Ce variant semble fortement associé à la toxine STa puisque toutes les souches variantes ont hybridé avec le gène codant pour cette dernière. Étant donné sa présence répandue dans la population de souches ETEC du Québec, nous avons de plus émis l’hypothèse que ce variant a des caractéristiques biologiques altérées par rapport à la toxine sauvage. L’analyse par dichroïsme circulaire a montré que le variant et la toxine sauvage ont une structure secondaire ainsi qu’une stabilité similaires. Par la suite, l’attachement au récepteur de la toxine, le sulfatide, a été étudié par résonnance plasmonique de surface (biacore). Le variant a une affinité au sulfatide légèrement réduite comparativement à la toxine sauvage. Puisque l’internalisation de la toxine fut observée dans une étude précédente et qu’elle semble liée à la toxicité, nous avons comparé l’internalisation du variant et de la toxine sauvage à l’intérieur des cellules IPEC-J2. L’internalisation du variant dans les cellules est légèrement supérieure à l’internalisation de la toxine sauvage. Ces résultats suggèrent que le variant est biochimiquement et structurellement comparable à la toxine sauvage. / Enterotoxigenic Escherichia coli (ETEC) are a major cause of post-weaning diarrhea. STb is one of two heat-stable toxins produced by ETEC and is mostly associated with pathogenic porcine isolates. For the first time, a variant of the toxin was observed in a study in 2003. Our hypothesis is that STb variants are present in ETEC strains from Quebec. To screen for alterations at the gene level, a collection of 100 STb+ ETEC strains isolated from diseased pigs was randomly selected and analyzed. A total of 23 strains had a change from His12 to Asn. An association between the presence of the variant and virulence factors present in those strains was done. These strains were also positive for STa. Since this variant seems to be widely distributed in Quebec, we hypothesize that the variant has different biological properties compared to the wild-type STb. First, the secondary structure of the variant and wild-type toxin and their thermal stability was determined by circular dichroism. Both show similar structures and thermal stability. In addition, the binding affinity with the toxin receptor, the sulfatide, was determined by surface plasmon resonance. The affinity of the wild-type for the sulfatide is slightly superior to the variant. Finally, the internalization inside IPEC-J2 cells of the variant was compared to the wild-type. The variant is able to internalize more cells than the wild-type. Altogether, these results suggest that both the variant and the wild-type toxin are biochemically and structurally similar.

Este trabalho realizou importantes avanços na elucidação do potencial das toxinas termo-lábeis do tipo II (LT-IIs) como adjuvantes por via intradérmica e transcutânea. Os dados gerados indicam que LT-IIb e LT-IIc nativas atuam como potentes adjuvantes vacinais por via intradérmica induzindo respostas imunológicas antígeno específicas, como medido pela produção de anticorpos sistêmicos (IgG) e ativação de linfócitos T CD8+ citotóxicos. Soma-se ao potencial adjuvante demonstrado para as LT-IIs por essa via a baixa reatogenicidade das moléculas, com menores níveis de edema e reduzida migração leucocitária para o sítio de inoculação em comparação a LT-I. Os resultados indicam também que o efeito adjuvante das LTs aplicadas por via transcutânea pode estar relacionado à capacidade de ligação ao gangliosídeo GM1 já que a toxina LT-IIb, incapaz de interagir com este receptor, não apresenta efeito adjuvante por essa via. O conjunto de dados apresentados abre perspectivas para o emprego das LT-IIs nativas como adjuvantes parenterais em vacinas para animais e humanos. / This work has made significant advances in the understanding of the potential of type II heat-labile toxins (LT-IIs) as vaccine adjuvants by intradermal and transcutaneous route. The generated data indicate that native forms of LT-IIb and LT-IIc act as potent vaccine adjuvants when intradermally injected inducing antigen-specific immune responses, as measured by the generation of systemic serum antibody (IgG) and activation of cytotoxic CD8+ T lymphocytes. Besides the adjuvant effects, LT-IIs show reduced side effects, measured by the lesser edema formation and reduced leukocytes migration to the site of injection, in comparison to LT-I. The results also indicate that the adjuvant activity of LTs applied transcutaneously may be related to the the ganglioside GM1 binding property since the LT-IIb toxin, unable to interact with this receptor, has no adjuvant effect by this route. The presented data set opens prospects for the employment of native LT-IIs as parenteral adjuvants in vaccines for animals and humans.

Les E. coli entérotoxinogènes (ETEC) sont souvent la cause de diarrhée post-sevrage chez le porc. Deux types d’entérotoxines sont retrouvées chez les ETEC, soit les thermolabiles, comme la toxine LT, et les thermostables, comme EAST-1, STa et STb. Cette dernière est composée de 48 acides aminés et est impliquée dans la pathologie causée par les ETEC. Pour la première fois un variant de la toxine STb fut découvert dans une étude. Nous avons alors émis l’hypothèse qu’il y a présence de variants dans la population de souches ETEC du Québec. Dans les 100 souches STb+ analysées, 23 possédaient le gène de la toxine avec une variation dans la séquence génétique : l’asparagine était présente en position 12 remplaçant ainsi l’histidine. Une corrélation entre la présence du variant et la présence de facteurs de virulence retrouvés dans ces 100 souches ETEC étudiées a été effectuée. Ce variant semble fortement associé à la toxine STa puisque toutes les souches variantes ont hybridé avec le gène codant pour cette dernière. Étant donné sa présence répandue dans la population de souches ETEC du Québec, nous avons de plus émis l’hypothèse que ce variant a des caractéristiques biologiques altérées par rapport à la toxine sauvage. L’analyse par dichroïsme circulaire a montré que le variant et la toxine sauvage ont une structure secondaire ainsi qu’une stabilité similaires. Par la suite, l’attachement au récepteur de la toxine, le sulfatide, a été étudié par résonnance plasmonique de surface (biacore). Le variant a une affinité au sulfatide légèrement réduite comparativement à la toxine sauvage. Puisque l’internalisation de la toxine fut observée dans une étude précédente et qu’elle semble liée à la toxicité, nous avons comparé l’internalisation du variant et de la toxine sauvage à l’intérieur des cellules IPEC-J2. L’internalisation du variant dans les cellules est légèrement supérieure à l’internalisation de la toxine sauvage. Ces résultats suggèrent que le variant est biochimiquement et structurellement comparable à la toxine sauvage. / Enterotoxigenic Escherichia coli (ETEC) are a major cause of post-weaning diarrhea. STb is one of two heat-stable toxins produced by ETEC and is mostly associated with pathogenic porcine isolates. For the first time, a variant of the toxin was observed in a study in 2003. Our hypothesis is that STb variants are present in ETEC strains from Quebec. To screen for alterations at the gene level, a collection of 100 STb+ ETEC strains isolated from diseased pigs was randomly selected and analyzed. A total of 23 strains had a change from His12 to Asn. An association between the presence of the variant and virulence factors present in those strains was done. These strains were also positive for STa. Since this variant seems to be widely distributed in Quebec, we hypothesize that the variant has different biological properties compared to the wild-type STb. First, the secondary structure of the variant and wild-type toxin and their thermal stability was determined by circular dichroism. Both show similar structures and thermal stability. In addition, the binding affinity with the toxin receptor, the sulfatide, was determined by surface plasmon resonance. The affinity of the wild-type for the sulfatide is slightly superior to the variant. Finally, the internalization inside IPEC-J2 cells of the variant was compared to the wild-type. The variant is able to internalize more cells than the wild-type. Altogether, these results suggest that both the variant and the wild-type toxin are biochemically and structurally similar.