Listeria monocytogenes and Shigella flexneri are human intestinal pathogens that replicate in the cytosol of infected cells and spread directly from primarily infected cells to neighboring cells. The dissemination process is a fundamental aspect of pathogenesis and spreading-defective bacterial mutants are essentially avirulent. The ability of L. monocytogenes and S. flexneri to spread from cell to cell is related to their ability to dispay actin-based motility in the cytosol of infected cells. These bacteria produce virulence factors tha lead to the recruitment at their surface of an essential host cell actin nucleator, the ARP2/3 complex. This results in actin polymerization at one pole of the bacteria, which propels the rods throughout the cytosol. Seminal electron microscopy studies revealed that, when bacteria reach the cell cortex, they form plasma membrane extensions that protrude into the cytosol of neighboring cells. These protrusions are resolved in the receiving cells into double membrane vacuoles, from which the pathogens escape by producing virulence factors that disrupt the integrity of eukaryotic membranes. In contrast to our advanced understanding of the molecular mechanisms supporting cytosolic actin-based motility, the mechanisms supporting pathogen dissemination through membrane protrusion formation are unresolved. To address this gap in knowledge, we have developed innovative procedures for imaging intracellular pathogen dissemination and identified bacterial and cellular factors specifically required for the formation/resolution of membrane protrusions and double membrane vacuoles upon L. monocytogenes infection. Here, we propose to gain the first mechanistic insight into (Aim1) the cellular factors involved in actin network formation in L. monocytogenes protrusions, (Aim2) the bacterial factors involved in L. monocytogenes protrusion formation and resolution, (Aim3) the cellular factors involved in L. monocytogenes double membrane vacuole maturation and (Aim4) the bacterial and cellular factors supporting S. flexneri dissemination.

Public Health Relevance

Various intracellular pathogens have evolved the ability to manipulate host cell processes in order to disseminate from infected cells to neighboring cells. In this application, we present our plans to gain the first mechanistic insight into the bacterial and cellular factors supporting pathogen dissemination through formation of plasma membrane protrusions. The proposed approach will contribute to our general understanding of the mechanisms underlying microbial pathogenesis and may therefore constitute the foundation for the rational design of preventive and therapeutic interventions.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Host Interactions with Bacterial Pathogens Study Section (HIBP)
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Mills, Melody
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Yale University
Schools of Medicine
New Haven
United States
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Kuehl, Carole J; Dragoi, Ana-Maria; Agaisse, Hervé (2014) The Shigella flexneri type 3 secretion system is required for tyrosine kinase-dependent protrusion resolution, and vacuole escape during bacterial dissemination. PLoS One 9:e112738
Dragoi, Ana-Maria; Swiss, Rachel; Gao, Beile et al. (2014) Novel strategies to enforce an epithelial phenotype in mesenchymal cells. Cancer Res 74:3659-72
Dragoi, Ana-Maria; Agaisse, Hervé (2014) Tyrosine kinases, drugs, and Shigella flexneri dissemination. Gut Microbes 5:44-7
Talman, Arthur M; Chong, Ryan; Chia, Jonathan et al. (2014) Actin network disassembly powers dissemination of Listeria monocytogenes. J Cell Sci 127:240-9
Dragoi, Ana-Maria; Agaisse, Hervé (2014) The serine/threonine kinase STK11 promotes Shigella flexneri dissemination through establishment of cell-cell contacts competent for tyrosine kinase signaling. Infect Immun 82:4447-57
Dragoi, Ana-Maria; Talman, Arthur M; Agaisse, Herve (2013) Bruton's tyrosine kinase regulates Shigella flexneri dissemination in HT-29 intestinal cells. Infect Immun 81:598-607
Chong, Ryan; Squires, Raynal; Swiss, Rachel et al. (2011) RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell. PLoS One 6:e23399
Chong, Ryan; Swiss, Rachel; Briones, Gabriel et al. (2009) Regulatory mimicry in Listeria monocytogenes actin-based motility. Cell Host Microbe 6:268-78