Differential expression of transcriptional and translational elements by lifecycle variants of Coxiella burnetii /
Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides in an acidified phagolysosome and has remarkable ability to persist in the extracellular environment. C. burnetii has evolved a developmental cycle that includes at least two morphological forms...
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
2000.
|
| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=728408711&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides in an acidified phagolysosome and has remarkable ability to persist in the extracellular environment. C. burnetii has evolved a developmental cycle that includes at least two morphological forms designated large cell variants (LCVs) and small cell variants (SCVs), which have distinct ultrastructural and antigenic characteristics. I hypothesized that LCVs and SCVs differentially express proteins that are specific for the role of each form in a developmental life cycle. I speculated that a description of differentially expressed proteins would allow a more accurate prediction of the functional roles of the life cycle variants. To test this, I proposed to characterize several differentially expressed proteins with the following two-fold strategy. A panel of monoclonal antibodies (Mab) was compared by Western blot for reactivity with LCV and SCV antigens. A 35 kDa (LCV up-regulated/Mab NM7.3) and a 45 kDa reactive (LCV-specific/Mab NM183) antigen were identified as differentially expressed. Genes expressing these immunoreactive antigens were cloned, sequenced and identified as encoding two translation factors, Elongation Factor-Ts and Elongation Factor-Tu respectively. I hypothesized that the alternate sigma factor RpoS, a global regulator of gene expression under starvation and stress conditions in many bacteria, may regulate differential gene expression in lifecycle variants of C. burnetii. To test this hypothesis, I cloned and characterized the major sigma factor, rpoD homologue, and the stress response sigma factor, rpoS homologue. Rickettsia prowazekii and Chlamydia spp., obligate intracellular organisms with recently deduced genome sequences, encode no rpoS homologue, making identification of a C. burnetii rpoS gene evolutionarily intriguing and novel. rpoS was cloned by complementation of an E. coli rpoS null mutant containing a RpoS-dependent lacZ fusion (osmY::lacZ). A GST-RpoS fusion protein was used to develop polyclonal antiserum against C. burnetii RpoS that detected a 40 kDa antigen in LCV, but not in SCV by Western blot. Identification of these key elements of the transcription and translation machinery, and determining their differential expression has lent support to the idea that LCV and SCV play distinct and specialized roles during the pathogenesis of this organism. |
|---|---|
| Item Description: | Vita. "Major Subject: Microbiology". |
| Physical Description: | ix, 102 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 51-66). |