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Structural characterization of the type III secretion translocon of Pseudomonas aeruginosa


Bacterial pathogens have developed highly specialized processes to target hosts and establish infection. A recently described weapon, the type III secretion system, transfers toxins from the bacterial cytoplasm into the host cell through a pore formed on the host cell membrane upon cell-to-cell contact. In this project, we will use X-ray crystallography to study PopB and PopD, the two membrane proteins required for pore formation by the nosocomial pathogen Pseudomonas aeruginosa, both in their intrabacterial form and as the finalized ring-like pore.

The structural study of membrane proteins has proven to be a challenging endeavor, mainly due to the difficulties presented by the production and purification of membrane proteins in high amounts. PopB and PopD, however, are expressed in the bacterial cytosol in soluble form, complexed to a chaperone, PcrH; in the host laboratory, such complexes can be expressed and purified in milligram quantities, an absolute requirement for successful crystallization experiments. In addition, the host laboratory has already produced preliminary crystals of the PopB:PcrH complex and has expertise at reconstitution of the PopB/PopD pore. Crystallization trials will be performed with TECAN robotics, available at the IBS, and all data will be collected at the ESRF synchrotron in Grenoble.Highly related type III secretion systems are employed by a variety of other pathogens, such as Yersinia pestis, the causative agent of bubonic plague, and Salmonella typhi, the agent of typhoid fever.

Thus, the structural study of the type III secretion pore from P. aeruginosa will provide crucial information regarding the infectivity processes of other pathogens. Targeting a specific host:pathogen interaction which is crucial for bacterial infectivity, such as type III secretion pore formation, will be of central importance for the potential development of novel vaccines, antibiotics, and treatments against pathogenic infections.

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