Bacillus anthracis interaction with non-phagocytic cells : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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College Station, Tex. :
Texas A&M University System Health Science Center,
2008.
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| Subjects: |
| Abstract: | ABSTRACT: Dissemination of Bacillus anthracis spores from the lung is a critical early event in the establishment of inhalation anthrax. The current model for B. anthracis dissemination in vivo focuses on macrophages as carriers. However, recent evidence suggest that other host cells may also play a role in the process. We tested the possibility of B. anthracis being internalized by a human fibroblast cell line, HT1080, and an epithelial cell line, Caco-2. A combinaiton of gentamicin protection assays, scanning and transmission electron microscopy (EM) and fluorescence microscopy (FM) was used to demonstrate for the first time that both spores and vegetative bacilli of B. anthracis Sterne strain 7702 were able to adhere to and be internalized by HT1080 and Caco-2 cells. Spore adherence to and internalization by HT1080 cells were not affected by a germination inhibitor suggesting that factors on dormant spores were sufficient for these processes. Vegetative bacterial adherence to and internalization by both cell lines were growth-phase dependent. We also showed that internalization of both spores and vegetative rods required active functions of the host cell cytoskeleton. Using gentamicin protection assays and/or EM, we found that 7702 spores were able to adhere to and be internalized by polarized A549 (a Type II alveolar cell line) and primary human small airway epithelial cells. Fully virulent Ames and UT500 strain spores adhered to A549 cells at a frequency similar to 7702, whereas the capsule in germinated Ames and UT500 spores prevented adherence. FM revealed that dormant Ames and 7702 spores were internalized at a similar frequency. We showed that internalized spores were able to survive and that spores could translocate across an A549 cell barrier from the apical side to the basolateral side without disrupting the barrier integrity, suggesting a transcellular route. An intratracheal mouse model of infection was used to demonstrate intracellular 7702 spores inside epithelial cells isolated from the lung in vivo. FM analysis of lung sections from 2 and 4 hours infected mice revealed spores adhered to cells of the alveoli. Confocal scanning FM revealed intracellular spores surrounded by actin in cells of the alveoli. We developed a novel method of identifying and quantifying the presence of intracellular bacteria in epithelial cells. FM analysis of crude lung cell suspensions isolated from 4 hour infected mice revealed extracellularly adhered and intracellular spores inside epithelial cells. We reported [is equilavent to] 20,000 epithelial cells in the lung contain an intrcellular spore. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies of The Texas A&M University System Health Science Center in partial fulfillment for the requirements for the degree of Doctor of Philosophy May 2008." Approved as to style and content by: Xi Yu, Magnus A Hook, Theresa M. Koehler, Eric L. Brown, Magnus A. Hook. |
| Physical Description: | xiv, 152 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 141-151). |