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Temporal Analysis of Coxiella burnetii Morphological Differentiation

Temporal Analysis of Coxiella burnetii Morphological Differentiation Temporal Analysis of Coxiella burnetii Morphological Differentiation Sherry A. Coleman , Elizabeth R. Fischer , Dale Howe , David J. Mead , and Robert A. Heinzen * Coxiella Pathogenesis Unit, Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana ABSTRACT Coxiella burnetii undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study C. burnetii morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of scvA , which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of C. burnetii is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of C. burnetii developmental forms. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Bacteriology American Society For Microbiology

Temporal Analysis of Coxiella burnetii Morphological Differentiation

Temporal Analysis of Coxiella burnetii Morphological Differentiation

Journal of Bacteriology , Volume 186 (21): 7344 – Nov 1, 2004

Abstract

Temporal Analysis of Coxiella burnetii Morphological Differentiation Sherry A. Coleman , Elizabeth R. Fischer , Dale Howe , David J. Mead , and Robert A. Heinzen * Coxiella Pathogenesis Unit, Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana ABSTRACT Coxiella burnetii undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study C. burnetii morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of scvA , which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of C. burnetii is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of C. burnetii developmental forms.

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References (62)

Publisher
American Society For Microbiology
Copyright
Copyright © 2004 by the American society for Microbiology.
ISSN
0021-9193
eISSN
1098-5530
DOI
10.1128/JB.186.21.7344-7352.2004
pmid
15489446
Publisher site
See Article on Publisher Site

Abstract

Temporal Analysis of Coxiella burnetii Morphological Differentiation Sherry A. Coleman , Elizabeth R. Fischer , Dale Howe , David J. Mead , and Robert A. Heinzen * Coxiella Pathogenesis Unit, Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana ABSTRACT Coxiella burnetii undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study C. burnetii morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of scvA , which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of C. burnetii is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of C. burnetii developmental forms.

Journal

Journal of BacteriologyAmerican Society For Microbiology

Published: Nov 1, 2004

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