Zugl.: Berlin, Humboldt-Univ., Diss., 2009

Two posttranslational modifications, ISGylation and phosphorylation, impact the replication of influenza A virus, a human pathogen responsible for high mortality pandemics. The ubiquitin-like ISG15 protein is induced by type 1 interferon (IFN) and is conjugated to many cellular proteins by three enzymes that are also induced by IFN. Experiments using ISG15-knockout (ISG15-/-) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus, but inhibition was not detected in mouse embryo fibroblasts in tissue culture. The present study is focused on the effect of ISG15 and/or its conjugation on the replication of influenza A virus in human cells in tissue culture. IFN-induced antiviral activity against influenza A virus in human cells was significantly alleviated by blocking ISG15 conjugation using small interfering RNAs (siRNAs) against ISG15 conjugating enzymes. IFN-induced antiviral activity against influenza A virus gene expression and replication was reduced 10-20-fold by suppressing ISG15 conjugation. Unconjugated ISG15 does not contribute to this antiviral activity. Consequently human tissue culture cells can be used to delineate how ISG15 conjugation inhibits influenza A virus replication. SiRNA knockdowns were also used to demonstrate that other IFN-induced proteins, specifically p56, MxA and phospholipid scramblase 1, also inhibit influenza A virus gene expression in human cells. The research on phosphorylation focused on the viral NS1A protein, a multifunctional virulence factor. Although threonine phosphorylation of the NS1A protein was discovered 30 years ago, the sites of phosphorylation and its function had not been identified. A recombinant influenza A virus encoding an epitope-tagged NS1A protein was generated, enabling the purification of NS1A protein from infected cell extracts. Mass spectrometry identified phosphorylation at T49 and T215. A recombinant virus in which phosphorylation at 215 was abolished by replacing T with A is attenuated, and an apparently aberrant NS1A protein is produced. Attenuation did not occur when T was changed to E to mimic a constitutively phosphorylated state, or surprisingly when T was changed to P to mimic avian NS1A proteins. These results suggest that T215 phosphorylation in human viruses and P215 in avian viruses can support analogous functions. / text

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a foodborne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the interferon gamma (IFNγ) pro-inflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this thesis was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants, stimulated with IFNγ, and then protein extracts were analyzed by immunoblotting. The data showed that IFNγ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using 2D-Difference Gel Electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated, complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFNγ-mediated cell activation. I conclude that E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a foodborne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the interferon gamma (IFNγ) pro-inflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this thesis was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants, stimulated with IFNγ, and then protein extracts were analyzed by immunoblotting. The data showed that IFNγ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using 2D-Difference Gel Electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated, complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFNγ-mediated cell activation. I conclude that E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

No description available.

Les protéoglycannes exercent de nombreuses fonctions par le biais de leur partie protéique ou de leurs glycosaminoglycannes. La caractérisation des interactions entre les glycosaminoglycannes et des protéines a ouvert de larges champs d'applications. Dans ce cadre, deux thèmes de recherche ont été développés. Premièrement, nous nous sommes intéressés à un nouveau protéoglycanne appelé endocan. La développement des capacités de production et de purification de cette macromolécule, nous a permis par différentes approches de déterminer le profil structural de sa chaîne glycannique et de sa partie protéique, mais aussi d'étudier les interactions avec plusieurs de ses partenaires protéiques dont l'interféron γ et l'hépatocyte growth factor, impliqués respectivement dans l'inflammation et le développement tumoral. Parallèlement, une étude structurale et fonctionnelle de l'interaction entre l'interféron γ et des glycosaminoglycannes de type héparanes sulfates a conduit au développement de mimes oligosaccharidiques obtenus par synthèse chimique. Parmi ces molécules, certaines permettent de moduler in vitro l'activité de la cytokine, et constituent une base possible pour le développement de nouveaux médicaments.

Mild hypothermic conditions (30ºC to 33ºC) have previously been shown to increase cell specific productivity (Qp) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titres. This thesis describes the isolation of a novel population of Chinese Hamster Ovary (CHO) cells that have been adapted to low temperature growth by continuous subculture at low temperature for a duration of 400 days. This adapted cell population achieved a growth rate 2-fold greater than non-adapted cells under low temperature conditions (32ºC) while maintaining an elevated level of cell specific expression of recombinant beta-interferon. The volumetric titre of beta-interferon was enhanced by 70% in stationary cultures and by more than 2-fold by application of a temperature-shift strategy involving a growth to production phase. However, the low temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem, caused by a weakened vimentin intermediate filament network, was resolved by the use of macroporous microcarriers which were demonstrated to entrap and protect the cold-adapted cells. Cold-adapted microcarrier cultures were able to achieve high cell densities (greater than 5x10^6 nuclei/mL) cultures under hypothermic conditions. This resulted in a 3-fold enhancement of volumetric titre of monomeric beta-interferon compared to the original control culture at 37ºC.

Mild hypothermic conditions (30ºC to 33ºC) have previously been shown to increase cell specific productivity (Qp) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titres. This thesis describes the isolation of a novel population of Chinese Hamster Ovary (CHO) cells that have been adapted to low temperature growth by continuous subculture at low temperature for a duration of 400 days. This adapted cell population achieved a growth rate 2-fold greater than non-adapted cells under low temperature conditions (32ºC) while maintaining an elevated level of cell specific expression of recombinant beta-interferon. The volumetric titre of beta-interferon was enhanced by 70% in stationary cultures and by more than 2-fold by application of a temperature-shift strategy involving a growth to production phase. However, the low temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem, caused by a weakened vimentin intermediate filament network, was resolved by the use of macroporous microcarriers which were demonstrated to entrap and protect the cold-adapted cells. Cold-adapted microcarrier cultures were able to achieve high cell densities (greater than 5x10^6 nuclei/mL) cultures under hypothermic conditions. This resulted in a 3-fold enhancement of volumetric titre of monomeric beta-interferon compared to the original control culture at 37ºC.