The broad, long-term objectives of this proposal are to understand the structural steps required for bacterial pathogens to invade mammalian host cells. Host cell invasion is a critical step in the life cycle of intracellular pathogens and parasites, which are major causes of human morbidity and mortality. The mechanism of cell invasion is being investigated in the pathogen Listeria monocytogenes, a cause of recent outbreaks of human illness and death. A protein attached to the cell wall of L. monocytogenes, internalin B (67 kD), is solely responsible for triggering the uptake of the bacterium into several nonphagocytic mammalian cell types. Included among these are hepatocytes, which are the major locus of bacterial proliferation in vivo. Internalin B acts by binding to a mammalian receptor, gClq receptor (gClq-R), and activating host phosphoinositide (PI) 3-kinase, leading to induction of phagocytosis. A 25 kD mammalian cell effector domain of internalin B is necessary and sufficient to activate PI-3 kinase, whereas the intact molecule is required for bacterial uptake. How internalin B activates host signaling pathways and causes uptake is not understood structurally.
The specific aims of the proposal are to: (1) Determine the structure of the 25 kD effector domain of internalin B. Crystals of the 25 kD effector domain that diffract x-rays to 1.5 A resolution have been grown and heavy-atom phasing has been achieved, allowing an interpretable electron density map to be calculated. (2) Determine the structure of intact internalin B. Crystals of intact internalin B that diffract x-rays to 3.15 A resolution have been grown and phasing information is being sought. (3) Co-crystallize and determine the structure of internalin B bound to gCIq-R. For these studies, a number of internalin B constructs are available in milligram quantities, as is gCIq-R in a form that crystallizes. The proposed structures are important to revealing the stereochemical basis by which internalin B induces phagocytosis and causes host cell invasion. This knowledge will be generally applicable to devising strategies to combat L. monocytogenes and other intracellular pathogens.
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