Bacterial invasion reconstructed molecule by molecule

We propose to visualize the initial stages of bacterial infection of a human host cell with unmatched spatial and temporal resolution. This work will develop a new capability for the laboratory (super-resolution optical imaging), will test unresolved scientific hypotheses regarding host-pathogen interaction dynamics, and leverages state of the art 3D molecular tracking instrumentation developed recently by our group. There is much to be gained by applying new single molecule tools to the important and familiar problem of pathogen entry into a host cell. For example, conventional fluorescence microscopy has identified key host receptors, such as CD44 and {alpha}5{beta}1 integrin, that aggregate near the site of Salmonella typhimurium infection of human cells. However, due to the small size of the bacteria ({approx} 2 {micro}m) and the diffraction of the emitted light, one just sees a fluorescent 'blob' of host receptors that aggregate at the site of attachment, making it difficult to determine the exact number of receptors present or whether there is any particular spatial arrangement of the receptors that facilitates bacterial adhesion/entry. Using newly developed single molecule based super-resolution imaging methods, we will visualize how host receptors are directed to the site of pathogen adhesion and whether host receptors adoptmore » a specific spatial arrangement for successful infection. Furthermore, we will employ our 3D molecular tracking methods to follow the injection of virulence proteins, or effectors, into the host cell by the pathogen Type III secretion system (TTSS). We expect these studies to provide mechanistic insights into the early events of pathogen infection that have here-to-fore been technically beyond our reach. Our Research Goals are: Goal 1--Construct a super-resolution fluorescence microscope and use this new capability to image the spatial distribution of different host receptors (e.g. CD44, as {alpha}5{beta}1 integrin) at the point of bacterial attachment to a human cell. Goal 2--Determine if the transport method of host cell receptors to the site of Salmonella infection depends upon their initial spatial location. We hypothesize that host receptors originally near the infection site will diffuse in the host membrane till 'caught' by the bacteria, while receptors further away from the bacteria will be driven by actin to the infection site in a highly directed fashion. Goal 3--Measure the 3D path taken by a bacterial effector to find its host counterpart. We hypothesize that bacterial effectors, after passing into the host via the TTSS, fmd their host target proteins through an un-biased diffusion process (e.g. a random walk, rather than active transport). This hypothesis will be tested on the bacterial effector SopE as it searches for its host partner, Racl.« less