Bacteriophages are the most abundant biological entities on earth and the selective pressures imposed by these pervasive predators have a profound impact on the composition and the behavior of microbial communities in every ecological setting. The human body is a compilation of complex ecosystems that rely on bacteria. In fact, the number of bacteria living in a healthy human is estimated to be ten times greater than the number of human cells and phage infections that perturb these microbial communities have recently been implicated in a wide range of human disorders. Furthermore, phages are major purveyors of genes that confer virulence and antibiotic resistance and thus phages play a major role in the evolution of bacterial pathogenesis. Clustered regularly interspaced short palindromic repeats (CRISPR) are essential components of a recently discovered, nucleic-acid-based adaptive immune system that is widespread in bacteria and archaea, and these immune systems play a central role in controlling the horizontal dissemination of virulence associated genes. The long-term goal of our research is to understand the impact of CRISPR-mediated immune systems on the evolution and ecology of human associated microbial communities. Specifically, the work outlined in this proposal is aimed at explicating the mechanisms of CRISPR RNA-guided surveillance and targeted elimination of foreign DNA by the adaptive immune system in Escherichia coli. We anticipate that this work will result in high-resolution structures of the CRISPR RNA-guided surveillance complex from Escherichia coli and that these structures will be invaluable to our understanding of how these surveillance machines work. However, structures will only capture snap-shots of the complex in specific poses. To understand the dynamics of these machines we complement our structural studies with real-time kinetic analysis aimed at probing the mechanism and rates of target surveillance. Finally, we implement our structural and biochemical insights to design programmable gene silencing systems. CRISPR-mediated gene silencing offers a novel approach for systematically controlling gene expression in both prokaryotic and eukaryotic systems. Overall, this proposal provides promising new insight to the public and scientific community regarding the mechanisms of adaptive immunity in bacteria that will have significant impacts in biotechnology and medicine.

Public Health Relevance

Bacteriophages play a major role in the behavior and virulence of their bacterial hosts. CRISPR-mediated adaptive immune systems are important regulators of phage-mediated gene transfer and this proposal aims to clarify the molecular mechanisms associated with acquired immunity in bacterial populations. We anticipate that insights from this work will lead to new approaches for treating diseases that arise from dysbiosis in human associated microbial populations, and will contribute to the development of new methodologies for programmable control of gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM108888-01
Application #
8613224
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2014-01-01
Project End
2018-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
1
Fiscal Year
2014
Total Cost
$273,600
Indirect Cost
$83,600
Name
Montana State University - Bozeman
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
625447982
City
Bozeman
State
MT
Country
United States
Zip Code
59717
Jackson, Ryan N; Golden, Sarah M; van Erp, Paul B G et al. (2014) Structural biology. Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli. Science 345:1473-9
Jackson, Ryan N; Lavin, Matthew; Carter, Joshua et al. (2014) Fitting CRISPR-associated Cas3 into the helicase family tree. Curr Opin Struct Biol 24:106-14
van der Oost, John; Westra, Edze R; Jackson, Ryan N et al. (2014) Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nat Rev Microbiol 12:479-92
Wilkinson, Royce; Wiedenheft, Blake (2014) A CRISPR method for genome engineering. F1000Prime Rep 6:3