Members of the spotted fever group (SFG) of the genus Rickettsia are obligate intracellular bacteria that cause serious human illnesses such as Rocky Mountain Spotted Fever. They require the host actin cytoskeleton to facilitate internalization into host cells, and to power intracytoplasmic movement that enables spread between cells during infection. Therefore, elucidating how SFG Rickettsia manipulate the actin cytoskeleton is critical for understanding the process of pathogenesis. In addition, determining the mechanism of actin-based motility is of great interest from a cell biological perspective because actin filaments in Rickettsia comet tails differ considerably in their organization compared to filaments assembled by well-studied pathogens such as Listeria monocytogenes, suggesting that Rickettsia use different molecules and mechanisms to promote motility. Despite the importance of actin in entry and motility, very little is known about the molecular mechanisms used by the bacteria to manipulate the cytoskeleton. We recently identified a Rickettsia protein called RickA that is conserved among SFG species and stimulates actin polymerization by activating the host Arp2/3 complex, providing the first molecular insight into how Rickettsia harness actin. We now need to answer important questions about the role of RickA and Arp2/3 in infection. For example, do RickA and Arp2/3 complex play a functionally important role, and how to they act? When and how is RickA introduced into host cells to stimulate actin polymerization? What other cytoskeletal proteins are required to polymerize and organize actin filaments? Based our preliminary results, we propose a unifying hypothesis that RickA is translocated by bacteria into host cells using a type IV secretion system, where it activates Arp2/3 complex to initiate actin assembly during entry and/or motility. We further hypothesize that, during actin-based motility, RickA and Arp2/3 act transiently, and other cyoskeletal proteins are subsequently needed to generate the unique organization of actin filaments in comet tails. To test this hypothesis, we propose the following aims: (1) Determine the timing and localization of the secreted RickA protein and the mechanism of secretion, (2) Examine the function of Arp2/3 complex during entry and actin- based motility, and (3) test the role of the full spectrum of actin cytoskeletal proteins in entry and motility. Determining the mechanisms used by SFG Rickettsiae to manipulate the host actin cytoskeleton will shed light on an essential and poorly understood aspect of Rickettsia pathogenesis, and will also illuminate the mechanisms used by host cells to regulate the functions of the cytoskeleton. In the long-term these studies may result in the discovery of new paradigms for understanding host-pathogen interactions, and new approaches to diagnose and treat infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI074760-05
Application #
8122252
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Perdue, Samuel S
Project Start
2007-08-15
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
5
Fiscal Year
2011
Total Cost
$361,792
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Lamason, Rebecca L; Welch, Matthew D (2017) Actin-based motility and cell-to-cell spread of bacterial pathogens. Curr Opin Microbiol 35:48-57
Welch, Matthew D (2015) Why should cell biologists study microbial pathogens? Mol Biol Cell 26:4295-301
Reed, Shawna C O; Lamason, Rebecca L; Risca, Viviana I et al. (2014) Rickettsia actin-based motility occurs in distinct phases mediated by different actin nucleators. Curr Biol 24:98-103
Welch, Matthew D; Way, Michael (2013) Arp2/3-mediated actin-based motility: a tail of pathogen abuse. Cell Host Microbe 14:242-55
Reed, Shawna C O; Serio, Alisa W; Welch, Matthew D (2012) Rickettsia parkeri invasion of diverse host cells involves an Arp2/3 complex, WAVE complex and Rho-family GTPase-dependent pathway. Cell Microbiol 14:529-45
Welch, Matthew D; Reed, Shawna C O; Lamason, Rebecca L et al. (2012) Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion. PLoS One 7:e37310
Huang, Ju; Birmingham, Cheryl L; Shahnazari, Shahab et al. (2011) Antibacterial autophagy occurs at PI(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase. Autophagy 7:17-26
Haglund, Cat M; Welch, Matthew D (2011) Pathogens and polymers: microbe-host interactions illuminate the cytoskeleton. J Cell Biol 195:7-17
Campellone, Kenneth G; Welch, Matthew D (2010) A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol 11:237-51
Haglund, Cat M; Choe, Julie E; Skau, Colleen T et al. (2010) Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility. Nat Cell Biol 12:1057-63

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