Cloning and characterization of the major outer membrane protein of Coxiella burnetii /
Coxiella burnetii, the etiological agent of Q fever, is a gram negative, obligate intracellular organism that resides in an acidified phagolysosome. The ability of the bacteria to persist within the environment has been attributed to the existence of different life cycle variants: the large cell va...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2002.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=764786521&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Coxiella burnetii, the etiological agent of Q fever, is a gram negative, obligate intracellular organism that resides in an acidified phagolysosome. The ability of the bacteria to persist within the environment has been attributed to the existence of different life cycle variants: the large cell variant (LCV), the small cell variant (SCV), and the small dense cell (SDC). Aside from varying extracellular stabilities, these morphological variants differ from one another with respect to metabolic activity and gene expression. The major outer membrane protein (MOMP) of C. burnetii, P1, is one example of this differential expression; abundantly expressed in LCV, downregulated in SCV, and not apparent in SDC. MOMP is a 29 kDa surface component that is resistant to detergent solubilization and found to elicit an immune response in C. burnetii challenged mice. The latter points led me to hypothesize that P1 functioned as a porin in the bacteria. In order to address this, I developed methodology that allowed me to purify the protein to levels suitable for peptide analysis. Amino acid sequences were used to PCR amplify a large internal region of the gene. Using inverse PCR, I was able to clone the remaining 5 and 3 regions. Sequence analysis of the full clone revealed P1 secondary structure to be primarily β sheet, similar to other porins. Attempts to express the recombinant protein were unsuccessful, so native P1 was purified and used in planar lipid bilayer studies in order to address the question of possible porin function. Assays indicated that P1 did function as a porin and that channel function was modulated by external pH. In addition, it was shown that the porin is an anion selective channel with a possible substrate preference of glutamic acid. Characterization of P1 function has led to increased understanding of life cycle variants. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". |
| Physical Description: | ix, 77 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 47-58). |