Several intracellular bacterial pathogens, such as Listeria ssp, Rickettsia spp. and Shigella spp., rely on actin-based motility to spread from infected cells to neighboring cells and disseminate within their host. Genetic and biochemical studies have revealed that actin-based motility relies on the production of virulence factors at the bacterial surface that interact with host cell cytoskeleton factors, including the ARP2/3 complex. In spite of these seminal contributions, the host cell factors supporting actin-based motility are still poorly understood. To fill this gap in knowledge, we have developed innovative approaches combining automated fluorescence microscopy and computer-assisted image analysis in order to visualize and quantify bacterial pathogen spread. We have used this newly developed approach to screen the human kinome and identified CK1 and CK2 as host cell kinases required for Listeria spread. We recently demonstrated that, similar to WASP and WAVE2, the affinity of ActA for the ARP2/3 complex is modulated by CK2-mediated phosphorylation, a notion that we refer to as regulatory mimicry. In this proposal we propose genetic and biochemical approaches designed to explore the role(s) of CK1 in the regulation of Listeria actin-based motility (Aim1). In addition, we propose to extend the approach used for investigating Listeria spread to several bacterial pathogens displaying actin-based motility, including Shigella and Rickettsia (Aim2). These exploratory studies may provide important and novel insights both into the mechanisms supporting Listeria, Shigella and Rickettsia pathogenesis and into the fundamental processes involved in the regulation of the actin cytoskeleton.

Public Health Relevance

Various intracellular pathogens have evolved the ability to manipulate host cell processes in order to spread from infected cells into the neighboring cells. Most of he host factors contributing to the cell-to-cell spread of pathogens are unknown. In this proposal, we present our plans to determine and investigate the host cell kinases involved in Listeria spp., Shigella spp. and Rickettia spp. actin-based motility. 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI094228-02
Application #
8223156
Study Section
Special Emphasis Panel (ZRG1-IDM-A (80))
Program Officer
Mills, Melody
Project Start
2011-03-01
Project End
2013-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
2
Fiscal Year
2012
Total Cost
$207,239
Indirect Cost
$82,239
Name
Yale University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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; Talman, Arthur M; Agaisse, Herve (2013) Bruton's tyrosine kinase regulates Shigella flexneri dissemination in HT-29 intestinal cells. Infect Immun 81:598-607