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Characterization of interactions of the Type IV secretion system core component VirB8Sivanesan, Durga 09 1900 (has links)
<p> Type IV secretion systems (T4SS) are essential for the virulence of many gram-negative
pathogens. The systems studied here comprise eleven VirB proteins in case of
Agrobacterium tumefaciens and twelve in case of Brucella suis. The VirB proteins
associate in the cell envelope and form a complex that mediates the translocation of
virulence factors into host cells. In this report, VirB8, a core component of T4SS, is
characterized with regards to its interaction with itself and with other VirB proteins. </p> <p> VirB8 was found to exist in monomer-dimer equilibrium and the self-association
was demonstrated by analytical ultracentrifugation, analytical gel filtration, surface
plasmon resonance and bacterial two-hybrid assay. The above experiments demonstrated
that residues M102, Y105 and E214 o fVirB8 from B. suis are involved in self-association
and mutagenesis of these residues led to the impairment of T4SS function in B. suis.
Furthermore, this information was utilized to unravel the contribution of VirB8 self-association
towards T4SS assembly and function. To this end dimerization variants of
VirB8 from Agrobacterium tumefaciens were created and the effects were assessed with
purified proteins in vitro. Following this, the effects of VirB8 dimer site changes were
assessed in vivo. Introduction of a cysteine residue at the predicted interface (V97C)
supported DNA transfer but not T-pilus formation. Variants that reduced the self-association
did not support T4SS functions and T-pilus formation. Moreover, VirB2-
VirB5 co-fractionated with high molecular mass components from membranes of A.
tumefaciens and VirB8 dimerization was shown to be necessary for VirB2 association
with the high molecular mass components. Using purified VirB8 and VirB5 it was shown that VirB5 interacts with VirB8 via its globular domain and this interaction dissociates
VirB8 dimers. Taking these results together, a mechanistic contribution of VirB8
dimerization to T4SS assembly was proposed. </p> <p> Next, the interactions of VirB8 with other core components (VirB9 and VirBlO)
were analyzed by using various in vitro and in vivo experiments. Purified soluble
periplasmic domains of VirB8, VirB9 and VirB10 were used in enzyme-linked
immunosorbent assays, circular dichroism, and surface plasmon resonance experiments.
The pair-wise interactions and self-association of VirB8, VirB9 and VirB 10 were
demonstrated with the in vitro experiments. In addition, a ternary complex formation
between VirB8, VirB9, and VirBlO was identified. Using the bacterial two-hybrid
system, the dynamics of the interactions between VirB8-VirB9-VirB 10 full-length
proteins were analyzed demonstrating that VirB9 stimulates VirB8 self-association, but
that it inhibits the VirB10-VirB10 as well as the VirB8-VirB10 interaction. Based on
these results, a dynamic model for secretion system assembly is proposed where VirB8
plays a role as an assembly factor that is not closely associated with the functional core
complex comprising VirB9 and VirB10. </p> <p> The work reported in this thesis advances the understanding of VirB8 self-association
and its contribution to T4SS assembly and function. Furthermore, the
establishment of the bacterial two-hybrid system to detect VirB interactions has helped
identify inhibitors for the VirB8 dimerization through collaboration with Dr. Athanasios
Paschos. Moreover, techniques such as ELISA, analytical ultracentrifugation, circular dichroism and surface plasmon resonance will be utilized routinely to characterize other
VirB-VirB interactions in future. </p> / Thesis / Doctor of Philosophy (PhD)
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Structural and Biochemical Characterization of VirB8 Protein in Type IV Secretion SystemsSharifahmadian, Mahzad 07 1900 (has links)
Secretion is the passage of macromolecules across cellular membranes. In bacteria, secretion is essential for virulence and survival. Gram-negative bacteria use specialized envelope-spanning multiprotein complexes to secrete macromolecules called type IV secretion system (T4SS). T4SSs mediate the secretion of monomeric proteins, multisubunit protein toxins and nucleoprotein complexes. Also, they contribute to the horizontal spread of plasmid-encoded antibiotic resistance genes. Consequently, they are potential targets for antivirulence drugs. Gram- negative bacteria have two membranes that the secretion complex spans. As a result, the T4SS consists of proteins inserted in the membranes and of soluble proteins that face into or out of the bacterial cell. The details of channel assembly and structure are not known, although recent advances have revealed the structure of the core secretion channel. VirB8 is an inner membrane protein of the complex that interacts with many other T4SS subunits and works as nucleation factor for T4SS channel assembly. Biophysical studies and NMR experiments in particular were conducted to characterize the structural aspects of VirB8 interactions. Dynamic regions of VirB8 during monomer-to-dimer transition were identified by NMR spectroscopy. X-ray crystal and NMR analyses revealed structural differences at the helical regions (α-1 and α-4) of wild-type VirB8 and its monomeric variant VirB8M102R. Fragment screening identified small molecules binding to the wild-type and monomeric variant. In silico docking analyses suggested that the surface groove in the VirB8 structure is important for effective binding of the small molecules. NMR experiments and biochemical assays demonstrated that the β-sheet domain (β1 in particular) is the binding interface of VirB8 for the interaction with VirB10. The identified interface has functional importance for T4SS-mediated conjugation. In addition, I used NMR spectroscopy to identify changes in the structure of VirB8 upon interaction with VirB5. Altogether, structural and biochemical studies on periplasmic and full length VirB8 enabled us to characterize the sequence of interactions between VirB8 and other VirB proteins during T4SS complex assembly and function. The results of this research may lead to an innovative strategy for the development of novel antimicrobial drugs. / La sécrétion est le passage de macromolécules à travers les membranes cellulaires. Chez les
bactéries, la sécrétion est essentielle pour la virulence et la survie. Les bactéries à Gramnégatif
utilisent le système de sécrétion de type IV (SST4) pour la sécrétion de toxines et de
nucléoprotéines. Les SST4 contribuent notamment à la propagation des gènes de résistance aux
antibiotiques. Pour cette raison, les composants du SST4 sont des cibles potentielles pour le
développement de médicaments antivirulence. Le SST4 est un complexe protéique qui s’étend
entre la double membrane de la bactérie à Gram-négatif. Les protéines qui le composent sont
insérées dans les membranes cellulaires ou solubles. Bien que la structure du pore central du
SST4 ait été résolue récemment, les détails de l'assemblage et la structure de ce complexe ne
sont pas connus. VirB8 est une protéine de la membrane interne qui interagit avec de
nombreuses autres sous-unités du SST4. Il s’agit d’un acteur central de l'assemblage du SST4.
Des études biophysiques, et notamment des expériences de RMN ont ainsi été réalisées pour
caractériser les aspects structuraux des interactions avec VirB8. Des regions dynamiques dans
la structure de VirB8 ont été identifiées par spectroscopie RMN lors de la transition entre la
forme monomérique et dimérique. Les analyses de cristallographie et de RMN ont révélé des
différences structurales dans les régions hélicoïdales (α1 et α4) de VirB8 wild-type et du variant
monomérique VirB8M102R. Le criblage de fragments a permis d’identifier de petites molécules
capables de se lier à VirB8 ainsi qu’au variant monomérique. Les analyses d’arrimage
moléculaire in silico suggèrent que la rainure de surface dans la structure VirB8 est importante
pour laliaison de ces petites molécules. Les expériences de RMN et les essais biochimiques
révèlent que le feuillet β (β1 en particulier) constitue l'interface d’interaction entre VirB8 et VirB10. Cette interface d’interaction est d’ailleurs importante pour la conjugaison du SST4. De
plus, j'ai identifié des changements dans la structure de VirB8 lors de l'interaction avec VirB5.
Les études sur la protéine VirB8 nous ont permis de caractériser la séquence d'événements
entre VirB8 et d'autres protéines VirB, régulant l'assemblage et la fonction du SST4.
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Caractérisation biochimique, structurale et inhibition du système de sécrétion de type IV par l’étude des protéines VirB8Casu, Bastien 03 1900 (has links)
No description available.
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