My research is focused on understanding how intracellular bacterial pathogens hijack host pathways of intercellular communication to promote cell-to-cell spread. My interest in the cell biology of host pathogen interactions evolved from my graduate training at Johns Hopkins University School of Medicine. There I studied how T cells use cell signaling pathways to respond to antigen, and how these responses go awry in cancer. I discovered how changes in the subcellular architecture and molecular complexes of positive regulators altered the activation intensity of signals to NF-?B. This spurred an interest in understanding additional modes of signaling regulation and led me to investigate how intracellular bacterial pathogens hijack host cell signaling pathways. In particular, I have focuse on the process of cell-to-cell spread, which relies on vesicular-mediated traffic of bacteria between cells and likely involves pathways of intercellular communication. In the long-term, my goal is to discover how bacterial proteins target host cell signaling molecules to enable cell-to-cell spread, and to build a more complete picture of the molecular mechanisms that govern the spreading process. To achieve my long-term goals, I have developed a 2-year training plan that will allow me to learn cutting-edge methods such as genome editing and super resolution microscopy, and to expand my expertise in research fields such as endocytosis and microbiology. These will be essential for the future success of my independent research program. This additional time will allow me to participate in necessary career development activities, including mentoring students, writing papers, and presenting talks. To complete the work during the mentored phase, I have assembled a panel of experts that includes my mentor Dr. Matthew Welch, an expert in cell biology and the actin cytoskeleton, and my co-mentors Dr. Daniel Portnoy, a leader in the field of bacterial pathogenesis, Dr. David Drubin, an expert in membrane trafficking mechanisms, and finally Dr. Xavier Darzacq, a pioneer in super resolution microscopy. The combination of this excellent advisory team as well as the phenomenal facilities provided by UC Berkeley will ensure that I make significant progress towards my research and career goals. This training plan will prepare me for a successful transition into an independent investigator position and lay the groundwork for research to be done during the independent phase. The goal of the research plan outlined in this proposal is to understand how two bacterial pathogens, Rickettsia parkeri and Listeria monocytogenes, hijack host pathways of intercellular communication to promote spread, a process that is crucial for their virulence. These pathogens live in the host cell cytosol and spread from cell to cell by mobilizing the host's actin cytoskeleton for intracellular motility and transport to the plasma membrane. Once at the cell periphery, they induce the formation of membrane protrusions that are engulfed by neighboring cells. Despite recent advances, we lack critical information about how these stages of spread occur. Based on my preliminary work, this proposal will investigate the specific hypothesis that bacterial pathogens promote spread by hijacking proteins normally involved in cell adhesion, membrane remodeling, and endocytosis; processes critically involved in intercellular communication. Furthermore, I hypothesize that distinct host pathways are hijacked by each pathogen. I propose the following experiments to test these hypotheses.
In Aim 1, I will identify which endocytic, cell adhesion, and membrane remodeling proteins promote spread, with a focus on the role of caveolin endocytosis in the K99 phase, and a focus on cell adhesion and membrane remodeling factors in the R00 phase. This will define the key host molecules involved in spread. To provide further mechanistic insight, in Aim 2 I will utilize live cell imagig to determine the dynamics of host protein recruitment, and super-resolution imaging to define the subcellular architecture of those host proteins studied in Aim 1. This will reveal the order in which host proteins are recruited, and their spatial organization during spread, providing key insights into their mechanisms of action. Finally, to examine how bacterial effector proteins hijack host factors to promote spread, in Aim 3 I will examine how the R. parkeri secreted factor Sca4 targets the cell adhesion protein vinculin to promote spread. In the K99 phase I will focus on the localization and interaction of these factors. During the R00 phase I will examine the regulation of Sca4 secretion and the role of vinculin in spread. As a whole, this work will delineate how bacterial effectors and host pathways cooperate to enable spread, and reveal similarities and differences in the molecular strategies of spread for diverse pathogens. Discerning the targets of spread should also improve our understanding of basic cellular mechanisms, like vesicular traffic and intercellular communication, and how these go awry in disease.
Intracellular bacterial pathogens spread between host cells by targeting molecules critical to cellular communication. This project will characterize how pathogens spread by manipulating host proteins normally involved in cell adhesion, membrane remodeling, and endocytosis; processes critically involved in intercellular communication. This work will improve our understanding of bacterial virulence mechanisms, provide insights into host cell biology, and may lead to improved diagnostics or treatments for infection.
|Lamason, Rebecca L; Kafai, Natasha M; Welch, Matthew D (2018) A streamlined method for transposon mutagenesis of Rickettsia parkeri yields numerous mutations that impact infection. PLoS One 13:e0197012|
|Lamason, Rebecca L; Welch, Matthew D (2017) Actin-based motility and cell-to-cell spread of bacterial pathogens. Curr Opin Microbiol 35:48-57|