It is now widely appreciated that bacterial cells have a dramatically complex structural organization, with many individual proteins distributed in the cells in a highly nonuniform pattern that may change rapidly as the cell grows. In the context of bacterial infection and pathogenesis, the dynamic behavior of proteins in the cell envelope is likely to be particularly important, since it is the outside surface of the bacterial cell that directly contacts the host. Technical barriers have made it difficult to directly examine the distribution and mobility of bacterial surface proteins, particularly integral outer membrane proteins in Gram-negative bacterial pathogens.
The aim of the project described in this proposal is to exploit several recently developed techniques that allow observation of surface protein dynamics in living bacterial cells to study the role of membrane protein mobility in the persistence and pathogenesis of disease caused by several enteric bacteria, including the category B pathogens Shigella flexneri, Salmonella enterics, Yersinia spp., and enteropathogenic Escherichia coli (EPEC). These new techniques, based on quantitative analysis of videomicroscopy images, are capable of tracing both large-scale protein distributions as they change over time and small-scale movements of individual protein molecules on the bacterial surface. They will be used to answer three specific questions about surface protein mobility in the context of bacterial infection: 1) How does mobility of IcsAA/irG in the outer membrane contribute to protein polarization on the surface of Shigella flexneri? 2) How are the mobility and activity of virulence factors in the outer membrane of Salmonella, Yersinia, and EPEC affected by the lipopolysaccharide remodeling associated with infection? and, 3) What is the organization and dynamic behavior of multidrug resistance (MDR) efflux pumps in Gram-negative bacteria, prior to and during drug exposure? .A fourth goal of this project is to develop a suite of high- throughput, automated computational image/analysis techniques that can facilitate analysis of protein dynamics and cell-to-cell variation in experiments on live bacteria, which we will make freely available to the research community.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI067712-05
Application #
7766299
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Alexander, William A
Project Start
2006-02-01
Project End
2013-01-31
Budget Start
2010-02-01
Budget End
2013-01-31
Support Year
5
Fiscal Year
2010
Total Cost
$238,469
Indirect Cost
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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Zhou, Xiaoxue; Halladin, David K; Rojas, Enrique R et al. (2015) Bacterial division. Mechanical crack propagation drives millisecond daughter cell separation in Staphylococcus aureus. Science 348:574-8
Harris, Leigh K; Dye, Natalie A; Theriot, Julie A (2014) A Caulobacter?MreB mutant with irregular cell shape exhibits compensatory widening to maintain a preferred surface area to volume ratio. Mol Microbiol :
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Weber, Stephanie C; Spakowitz, Andrew J; Theriot, Julie A (2012) Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci. Proc Natl Acad Sci U S A 109:7338-43
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Dye, Natalie A; Pincus, Zachary; Fisher, Isabelle C et al. (2011) Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter. Mol Microbiol 81:368-94
Weber, Stephanie C; Theriot, Julie A; Spakowitz, Andrew J (2010) Subdiffusive motion of a polymer composed of subdiffusive monomers. Phys Rev E Stat Nonlin Soft Matter Phys 82:011913
Wilson, Cyrus A; Tsuchida, Mark A; Allen, Greg M et al. (2010) Myosin II contributes to cell-scale actin network treadmilling through network disassembly. Nature 465:373-7
Weber, Stephanie C; Spakowitz, Andrew J; Theriot, Julie A (2010) Bacterial chromosomal loci move subdiffusively through a viscoelastic cytoplasm. Phys Rev Lett 104:238102

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