Date of Award

Summer 8-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Effector translocation is a common strategy used by bacteria to promote pathogenesis via disruption of the immune response, prevention of phagocytosis, or induction of toxicity in the infected cell. The Gram-positive bacterium Streptococcus pyogenes utilizes a unique effector translocation system termed cytolysin-mediated translocation (CMT) to introduce the NAD+ glycohydrolase SPN into host cells during infection, resulting in cytotoxicity. Host cell membrane recognition by the cholesterol-dependent cytolysin (CDC) Streptolysin O (SLO) is a requisite step in this process, but the canonical cholesterol-dependent pore-forming activity of SLO is unnecessary, indicating that SLO is a bifunctional toxin. SLO exhibits extensive homology to other CDCs, yet CMT is highly specific for SLO. SLO lacking its conserved N-terminal extension or SLO chimeras encoding the membrane-binding domains of homologous CDCs are functional cytolysins but do not translocate SPN, demonstrating that these domains have evolved to allow SLO to maintain both lytic and CMT activities. While cholesterol was considered the sole host cell receptor for SLO, we have demonstrated that cholesterol is not required for SLO’s ability to bind host membranes or translocate SPN during infection. Instead, SLO requires the co-expression and membrane localization of SPN to achieve cholesterol-independent membrane binding and CMT. SPN’s membrane localization also requires SLO, and mutants of SLO that cannot perform CMT do not permit SPN-membrane binding, suggesting that a co-dependent mode of binding to an alternative receptor results in SPN translocation. SPN’s conserved non-enzymatic domain, predicted to adopt the structure of a carbohydrate-binding module, plays an active role in this process and may directly bind the CMT receptor. Consistent with structural prediction, mutation of predicted carbohydrate-binding or cation-binding residues abolishes SPN’s recognition of the host cell membrane and translocation. We also investigated the cholesterol-dependent mechanism of binding of SLO and demonstrated that while cholesterol is necessary for the completion of pore formation and sustained membrane binding of SLO, it does not serve as the initial host cell receptor. SLO encodes a carbohydrate-binding site within its membrane-binding domain, and studies of SLO carbohydrate-binding site mutants and carbohydrate-defective host cell lines revealed that recognition of a galactose-containing glycoconjugate is a prerequisite to pore formation in the absence of SPN. However, the SPN-mediated mode of binding also promotes pore formation by SLO, demonstrating that pore formation can occur by distinct pathways during infection. The SPN-dependent mode of binding can be distinguished from the canonical galactose- and cholesterol-dependent mode by differential extraction with the detergent saponin, indicating that SPN mediates SLO’s localization to a more soluble fraction of the plasma membrane or modulates its level of oligomerization or membrane insertion. Binding and translocation of SPN is an important virulence mechanism, particularly in host cell types that are resistant to SLO-induced membrane damage in the absence of SPN.

Language

English (en)

Chair and Committee

Michael G Caparon

Committee Members

Jeffrey Henderson, David Hunstad, Christina Stallings, Niraj Tolia, Joseph Vogel,

Comments

Permanent URL: https://doi.org/10.7936/K7MS3QZ4

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