No description available.

No description available.

No description available.

The Type Three Secretion Systems (T3SSs) of Gram-negative bacteria, including Shigella, Salmonella, and Enteropathogenic/Enterohaemorrhagic Escherichia coli (EPEC/EHEC), pass virulence factors directly into the host to mediate invasion. Prior to secretion down the narrow T3SS channel, effector proteins associate with chaperone proteins. The binding enables the T3SS to keep effectors soluble and partially unfolded for secretion. In the first part of this thesis, the association of one promiscuous chaperone, Spa15 of Shigella flexneri, with three of its cognate effectors has been studied. In addition to the role this plays in secretion, the binding of one particular substrate leads to Spa15 being involved in the regulation of the T3SS. The oligomerisation and impact of substrate binding upon Spa15 has been determined by crystallography and EPR. Once secreted, T3SS effectors subvert the host cytoskeleton for the benefit of the bacteria. Soluble homologues of Spa15 effectors from EHEC and Salmonella have been purified, and their interactions with host GTPases which lead to stress fibre phenotypes observed. The Twin Arginine Translocation (Tat) pathway provides a contrasting view of bacterial secretion. Instead of preventing folding in the cytoplasm, it is a criterion of transport that the protein be folded. One of the reasons for internal folding is the necessity to insert cofactors which could not be incorporated externally. In the second part of this thesis, a protein which exemplifies this necessity is studied. This is PhoD, the model protein for Tat export from Bacillus subtilis. PhoD is an alkaline phosphodiesterase expressed to scavenge phosphate in times of phosphate deficiency. The structure of PhoD has been solved, and the protein is shown to be able to cleave a component of its own cell wall. It uses an unusual catalytic site more reminiscent of the eukaryotic purple acid phosphatases than of other currently known alkaline phosphatases. Furthermore this site appears to require metal binding before export from the bacterial cytoplasm.

<p>The pathogenic bacteria Yersinia pestis and Y. pseudotuberculosis are related to the degree where the former is considered a subspecies of the latter, and still they cause disease of little resemblance in humans. Y. pestis is the causative agent of lethal bubonic and pneumonic plague, while Y. pseudotuberculosis manifests itself as mild gastroenteritis. An important virulence determinant for these species is their ability to secrete and inject toxins (Yop effectors) into immune cells of the infected host, in a bacterium-cell contact dependent manner. This ability depends on the extensively studied type III secretion system, a highly complex multicomponent structure resembling a needle. The induction of Yop secretion is a strictly controlled event. The two structural type III secretion components YscU and YscP are here shown to play a crucial role in this process, which is suggested to require an YscP mediated conformational change of the C-terminus of YscU. Proteolytic cleavage of YscU within this domain is further revealed to be a prerequisite for functional Yop secretion. The needle subcomponent itself, YscF, is recognised as a regulatory element that controls the induction of Yop effectors and their polarised delivery into target cells. Potentially, the needle might act as a sensor that transmits the inducing signal (i.e. target cell contact) to activate the type III secretion system. Secondly a, for Yersinia, previously unexplored system, the Twin arginine translocation (Tat) pathway, is shown to be functional and absolutely required for virulence of Y. pseudotuberculosis. A range of putative Yersinia Tat substrates were predicted in silico, which together with the Tat system itself may be interesting targets for future development of antimicrobial treatments.</p>

The pathogenic bacteria Yersinia pestis and Y. pseudotuberculosis are related to the degree where the former is considered a subspecies of the latter, and still they cause disease of little resemblance in humans. Y. pestis is the causative agent of lethal bubonic and pneumonic plague, while Y. pseudotuberculosis manifests itself as mild gastroenteritis. An important virulence determinant for these species is their ability to secrete and inject toxins (Yop effectors) into immune cells of the infected host, in a bacterium-cell contact dependent manner. This ability depends on the extensively studied type III secretion system, a highly complex multicomponent structure resembling a needle. The induction of Yop secretion is a strictly controlled event. The two structural type III secretion components YscU and YscP are here shown to play a crucial role in this process, which is suggested to require an YscP mediated conformational change of the C-terminus of YscU. Proteolytic cleavage of YscU within this domain is further revealed to be a prerequisite for functional Yop secretion. The needle subcomponent itself, YscF, is recognised as a regulatory element that controls the induction of Yop effectors and their polarised delivery into target cells. Potentially, the needle might act as a sensor that transmits the inducing signal (i.e. target cell contact) to activate the type III secretion system. Secondly a, for Yersinia, previously unexplored system, the Twin arginine translocation (Tat) pathway, is shown to be functional and absolutely required for virulence of Y. pseudotuberculosis. A range of putative Yersinia Tat substrates were predicted in silico, which together with the Tat system itself may be interesting targets for future development of antimicrobial treatments.

No description available.