Pore-forming cytolysins are produced by virtually all genera of pathogenic Gram-positive bacteria. The most widely distributed group is the CDC family of which Streptolysin O (SLO) is a prominent member. SLO is produced by Streptococcus pyogenes, an important cause of pharyngitis and other serious diseases including scarlet fever, rheumatic fever and necrotizing faciitis. However, how SLO acts to promote the pathogenesis of any of these diseases is not well-understood. In this project, we have found that SLO plays a central role in the Cytolysin-Mediated Translocation (CMT) pathway, a novel pathway that acts to translocate the S. pyogenes NAD-glycohydrolase (SPN) across the host cell membrane and into its cytosol. CMT likely makes an important contribution to pathogenesis as cytosolic SPN is cytotoxic for cultured cells. However, how SLO functions to translocate SPN, and how SPN contributes to cytotoxicity is not clearly understood. CMT is a polarized process in which the majority of the exported SPN is destined for the host cell cytosol, and is not released into the extracellular milieu. Also, CMT requires specific domains in SLO and SPN that are absolutely required for translocation, but not for pore-formation or glycohydrolase activity. We have also recently found that SLO pore-formation itself is completely dispensable for CMT. Thus, how SLO and SPN interact with the membrane and each other and how this results in polarized translocation is not clear. In addition, our recent data suggest that glycohydrolase activity may not serve as the only or most important basis of SPN's cytotoxic effect as variants of SPN that lack glycohydrolase activity are still cytotoxic and that cytotoxicity requires concomitant formation of the SLO pore. The present study will investigate the mechanism of translocation and cytotoxicity and will be furthered by our recent determination of the SPN crystal structure and our detailed kinetic analysis of SPN enzymology. Specific questions to be addressed include an analysis of a predicted glycan-binding domain in SPN, the role of SLO-membrane interactions in SPN uptake, the basis for the glycohydrolase-independent cytotoxicity and how the various activities of both SLO and SPN interact to promote toxicity and to modulate host cell signaling. Further analysis of CMT will reveal details of a novel pathway for effector translocation and how different toxins synergize to modulate host cell behavior to produce specific pathogenic outcomes.

Public Health Relevance

Cytolysin-mediated translocation (CMT) is a novel pathway for toxin delivery in the Gram-positive pathogen Streptococcus pyogenes that uses the pore-forming cytolysin Streptolysin O (SLO) to translocate the streptococcal NAD+ glycohydrolase (SPN) across the host cell membrane and into its cytosol. Both SLO and cytosolic SPN then act to cause host cell death. In this project we will exploit our recent observations that pore-formation and glycohydrolase activity are dispensable for CMT and toxicity, respectively, along with our determination of the crystal structure of SPN to produce mutants of SPN and SLO that will be used to elucidate how SLO acts to translocate SPN. We will also use these mutants to investigate how SLO and SPN interact to both kill and manipulate the cytokine signaling responses of cells. This information will be important for understanding how the multiple toxins of S. pyogenes interact with each other and with host cells to unravel how this pathogen can cause such a diverse range of different diseases.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacterial Pathogenesis Study Section (BACP)
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GU, Xin-Xing
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Washington University
Schools of Medicine
Saint Louis
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Chandrasekaran, Sukantha; Caparon, Michael G (2016) The NADase-Negative Variant of the Streptococcus pyogenes Toxin NAD? Glycohydrolase Induces JNK1-Mediated Programmed Cellular Necrosis. MBio 7:e02215-15
Port, Gary C; Paluscio, Elyse; Caparon, Michael G (2015) Complete Genome Sequences of emm6 Streptococcus pyogenes JRS4 and Parental Strain D471. Genome Announc 3:
Cusumano, Zachary T; Caparon, Michael G (2015) Citrulline protects Streptococcus pyogenes from acid stress using the arginine deiminase pathway and the F1Fo-ATPase. J Bacteriol 197:1288-96
Mozola, Cara C; Caparon, Michael G (2015) Dual modes of membrane binding direct pore formation by Streptolysin O. Mol Microbiol 97:1036-50
Chandrasekaran, Sukantha; Caparon, Michael G (2015) The Streptococcus pyogenes?NAD(+) glycohydrolase modulates epithelial cell PARylation and HMGB1 release. Cell Microbiol 17:1376-90
Mozola, Cara C; Magassa, N'Goundo; Caparon, Michael G (2014) A novel cholesterol-insensitive mode of membrane binding promotes cytolysin-mediated translocation by Streptolysin O. Mol Microbiol 94:675-87
Cusumano, Zachary T; Watson Jr, Michael E; Caparon, Michael G (2014) Streptococcus pyogenes arginine and citrulline catabolism promotes infection and modulates innate immunity. Infect Immun 82:233-42
Port, Gary C; Paluscio, Elyse; Caparon, Michael G (2013) Complete Genome Sequence of emm Type 14 Streptococcus pyogenes Strain HSC5. Genome Announc 1:
Chandrasekaran, Sukantha; Ghosh, Joydeep; Port, Gary C et al. (2013) Analysis of polymorphic residues reveals distinct enzymatic and cytotoxic activities of the Streptococcus pyogenes NAD+ glycohydrolase. J Biol Chem 288:20064-75
Smith, Craig L; Ghosh, Joydeep; Elam, Jennifer Stine et al. (2011) Structural basis of Streptococcus pyogenes immunity to its NAD+ glycohydrolase toxin. Structure 19:192-202

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