Over 40 gram-positive pathogenic species from genera such as Streptococcus, Listeria, Brevibacterium, Clostridium, Bacillus, Arcanobacterium and Gardnerella express a member of the pore-forming cholesterol-dependent cytolysin (CDC) toxin family. Collectively, these pathogens represent a significant health burden worldwide and cause a wide variety of diseases, which include pneumonia, strep throat, earaches, wound infections and food-borne illnesses. The CDCs contribute to these diseases and are secreted by these pathogens as soluble monomeric proteins that bind mammalian host cells via membrane receptors, primarily cholesterol. These cell-bound monomers oligomerize into an oligomer complex comprised of 35-40 monomers, which then assembles and inserts a large ?-barrel pore into the plasma membrane of eukaryotic cells. Our studies of the assembly of the CDC pore complex have provided insight into the rational design of a new CDC-based vaccine and provided the first insights into the mechanism of mammalian immune defense proteins. Herein we will continue to investigate the molecular basis of cellular recognition and assembly of the large ?-barrel pore by the CDCs.
In Aim 1, we will address the hypothesis that the structural differences in the membrane binding interface of the CDCs directs their binding to cholesterol that resides in specific domains on the cell wherein pore formation induces cellular effects that are unique to different CDCs. Understanding the basis of these differences will change the fundamental paradigms of how CDCs recognize and alter cellular function.
In Aim 2 the fundamental features by which the CDCs control the assembly of the ?-barrel pore and its insertion to the membrane will be investigated. These studies will not only deepen our understanding of the CDC mechanism of pore formation, but will have relevance to a wide variety of bacterial and mammalian immune defense pore-forming proteins.
In Aim 3, we will continue the investigation of the allosteric pathway by which the CDCs activate monomers upon cell binding to assemble into the giant CDC pore. Overall, the results of these studies will reveal new paradigms of CDC pore formation, provide a deeper understanding of how CDCs recognize and alter cellular function and impact the study of other pore-forming toxins and proteins.

Public Health Relevance

This project has the overarching goal of providing a deep understanding of the mechanism by which the cholesterol-dependent cytolysin (CDC) family of toxins form pores in mammalian cells. The CDCs not only provide a tractable system to study the basic mechanism of pore-forming proteins, but our studies have and will continue to facilitate the design of better CDC-based vaccines, enhance our understanding of how other pore-forming proteins function and provide important insights into how they contribute to the myriad diseases caused by CDC-producing bacterial pathogens. The proposed studies herein will show that the CDCs are far more sophisticated toxins than have been appreciated and will change our understanding of how they contribute to disease progression, as well as provide new tools for testing their contribution to pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI037657-21
Application #
9234451
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Ranallo, Ryan
Project Start
1997-04-01
Project End
2021-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
21
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
878648294
City
Oklahoma City
State
OK
Country
United States
Zip Code
73104
Lawrence, Sara L; Gorman, Michael A; Feil, Susanne C et al. (2016) Structural Basis for Receptor Recognition by the Human CD59-Responsive Cholesterol-Dependent Cytolysins. Structure 24:1488-98
Lukoyanova, Natalya; Kondos, Stephanie C; Farabella, Irene et al. (2015) Conformational changes during pore formation by the perforin-related protein pleurotolysin. PLoS Biol 13:e1002049
Lawrence, Sara L; Feil, Susanne C; Morton, Craig J et al. (2015) Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation. Sci Rep 5:14352
Feil, Susanne C; Ascher, David B; Kuiper, Michael J et al. (2014) Structural studies of Streptococcus pyogenes streptolysin O provide insights into the early steps of membrane penetration. J Mol Biol 426:785-92
Sato, Takehiro K; Tweten, Rodney K; Johnson, Arthur E (2013) Disulfide-bond scanning reveals assembly state and ?-strand tilt angle of the PFO ?-barrel. Nat Chem Biol 9:383-9
Dowd, Kelley J; Farrand, Allison J; Tweten, Rodney K (2012) The cholesterol-dependent cytolysin signature motif: a critical element in the allosteric pathway that couples membrane binding to pore assembly. PLoS Pathog 8:e1002787
Lawrence, Sara L; Feil, Susanne C; Holien, Jessica K et al. (2012) Manipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin. Front Immunol 3:330
Feil, Susanne C; Lawrence, Sara; Mulhern, Terrence D et al. (2012) Structure of the lectin regulatory domain of the cholesterol-dependent cytolysin lectinolysin reveals the basis for its lewis antigen specificity. Structure 20:248-58
Kennedy, Catherine L; Lyras, Dena; Cordner, Leanne M et al. (2009) Pore-forming activity of alpha-toxin is essential for clostridium septicum-mediated myonecrosis. Infect Immun 77:943-51
Flanagan, John J; Tweten, Rodney K; Johnson, Arthur E et al. (2009) Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding. Biochemistry 48:3977-87