Spatiotemporal Regulation of Protrusion Dynamics During Intracellular Bacterial Dissemination

点击翻译按钮获取中文摘要

PROJECT SUMMARY/ABSTRACT Infections by intracellular bacterial pathogens are a leading cause of illness and mortality worldwide. Listeria monocytogenes (Lm) is a gram-positive food-borne pathogen that replicates within the cytosol of host cells and causes listeriosis, a life-threatening disease that affects newborns, immunocompromised individuals, pregnant people, and the elderly. A key aspect of Lm pathogenesis is the ability of bacteria to use actin-based motility to spread into neighboring cells via Lm-induced protrusions. The formation and uptake of Lm protrusions is a multi- stage process that includes protrusion initiation, elongation, stabilization, and engulfment by neighboring cells. The host and bacterial factors required for each step of protrusion dynamics have remained unclear. We have discovered that a host protein, Abi1, localizes to Lm-induced protrusions and is required for efficient Lm cell-to- cell spread in human epithelial cells. Moreover, prior host factor screens have indicated that Abi1 is critical for infection by additional intracellular bacterial pathogens. The focus of this proposal is to determine the requirement of Abi1 and potential host accessory proteins for Lm protrusion dynamics and cell-to-cell spread. In Aim I, the requirement of Abi1 for cell-to-cell spread will be determined by infecting host cells of different lineages in which Abi1 expression has been knocked down or knocked out with Lm strains expressing GFP. Assessment of foci of infections by fluorescence microscopy and plaquing assays will be used to evaluate cell-to-cell spread. Furthermore, immunofluorescence (IF) microscopy will be used to determine the localization of Abi1 during infection and to characterize protrusions within wild-type and Abi1-negative host cells. Transmission electron microscopy will be used to determine differences in the architecture of Lm-induced protrusions in Abi1-negative cells. In addition, spinning disk confocal live-cell imaging with GFP-Abi1 expressing host cells and Lm expressing TagRFP will be used to determine the spatiotemporal localization of Abi1 during Lm infection. In Aim II, we will use host cells expressing full-length and truncated GFP-Abi1 variants with IF microscopy to identify the domain(s) of Abi1 responsible for Abi1 localization to Lm protrusions. Plaquing assays will be performed to determine if Abi1 truncation variants can complement cell-to-cell spread in ABI1 knockout cells. We will determine the requirement of known Abi1 interacting proteins for localization of Abi1 and Lm protrusion formation using RNAi in conjunction with IF and spinning disk confocal microscopy. Plaquing assays in cells depleted for known Abi1 interacting proteins or the Abi1 interacting proteins and Abi1 will be performed to determine the contribution of the accessory proteins in Lm cell-to-cell spread. To identify additional accessory proteins that may play a role in Lm protrusion dynamics and bacterial dissemination, we will determine the complete proteomic profile of isolated Lm-induced protrusions by mass spectrometry. The proposed studies will provide significant insights into the role of Abi1 in regulating Lm protrusion dynamics and cell-to-cell spread and provide fundamental knowledge into a process shared by multiple intracellular bacterial pathogens for dissemination within the host.