S-layer function and assembly in Bacillus anthracis.
|Author / Creator:||Kern, Justin W.|
|Local Note:||School code: 0330.|
|URL for this record:||http://pi.lib.uchicago.edu/1001/cat/bib/9369969|
|Other authors / contributors:||University of Chicago.|
|Notes:||Advisor: Olaf Schneewind.|
Thesis (Ph.D.)--The University of Chicago, Division of the Biological Sciences, and The Pritzker School of Medicine, 2010.
Dissertation Abstracts International, Volume: 71-04, Section: B, page: 2162.
|Summary:||Anthrax disease is a fulminate and lethal infection of mammalian hosts by the Gram-positive pathogen Bacillus anthracis. Although traditionally a disease of herbivorous mammals, anthrax has gained increased notoriety of late due to its use as the biowarfare and bioterrorism agent of choice, highlighting the need for new vaccines and countermeasures against strains engineered to be multi-drug and vaccine resistant. B. anthracis causes disease when introduced into the host as a metabolically inactive spore, eventually replicating to high titer throughout all tissues and organ systems and causing death by bacteremia and toxemia. During all stages of infection, B. anthracis must use its surface organelles and secreted effectors to interact with and subvert its host environment. The envelope of B. anthracis is similar to that of the well-studied Staphylococcus aureus and Bacillus subtilis, with three notable exceptions. One, B. anthracis does not produce teichoic acids and the cell-wall peptidoglycan is instead decorated with a secondary cell wall polysaccharide, two, fully virulent strains of B. anthracis are encapsulated by a thick layer of poly-gamma-D-glutamic acid (PDGA) peptide capsule, and, three, the envelope of B. anthracis includes a para-crystalline sheet of protein called a Surface-Layer (S-layer). S-layers are found in microorganisms from all clades of the Eubacteria and Archaebacteria and are thought to serve myriad functions, ranging from exoskeleton to virulence factor. The assembly of S-layer proteins into arrays on the Gram-positive envelope occurs via a poorly understood, non-covalent interaction between S-layer proteins and a cell wall linked polysaccharide. The genome of B. anthracis encodes 24 genes predicted to be a component of the S-layer, only a handful of which have known or predicted functions. This thesis focuses on the function of one such S-layer protein as an adhesin required for anthrax pathogenesis and on the mechanism of S-layer assembly into the Gram-positive envelope.|
In Chapter II, the discovery of BslA, an S-layer protein, as the molecule responsible for B. anthracis adherence to host tissues is described. The necessity and sufficiency of this gene as an adhesin, its requirements for synthesis, and its tissue tropism were studied. This study identified the molecule responsible for the adherence of vegetative forms to host tissues and assigned a novel function to the B. anthracis S-layer. Anthrax disease is characterized in humans by a virulent meningitis and chapter III describes the role of BslA in adhering to and penetrating the blood brain barrier to cause that meningitis. This study provided a role that BslA-mediated adherence plays in the dissemination and pathogenesis of anthrax disease, as well as potential mechanism that BslA may exploit to cross the blood brain barrier. Chapter IV investigates the role that BslA plays in encapsulated, fully virulent anthrax infections. This study demonstrated that BslA is required for the dissemination of B. anthracis throughout the host during infection, that it can interact with the host despite its localization underneath the PDGA capsule, and that mutations in BslA abrogate the virulence properties of this organism, validating efforts to develop therapeutics targeting BslA. The final chapter of this thesis investigates the structural requirements for S-layer assembly in Gram-positive bacteria. This study describes the purification of polysaccharides that bind the S-layer and the structural characterization of these polymers. This work linked for the first time a pyruvyl modification necessary for S-layer binding to the structure of the major secondary cell wall polysaccharide, a well-studied polysaccharide, providing a model for how S-layers bind to the cell wall via this molecule. Moreover, the study uncovers an essential role for the enzymes of the teichoic acid ( tag) pathway in S-layer assembly, demonstrating that the genetic machinery for secondary cell wall polymer synthesis is highly conserved between S. aureus, B. subtilis, and B. anthracis, even if the structure of those polymers is not.
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