Temporal analysis of Coxiella burnetii morphological differentiation - PubMed (original) (raw)
Temporal analysis of Coxiella burnetii morphological differentiation
Sherry A Coleman et al. J Bacteriol. 2004 Nov.
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.
Figures
FIG. 1.
Transmission electron micrograph showing SCV purified from aged Vero cell cultures. SCV were purified from infected cells cultured for 4 weeks as described in Materials and Methods. (A) Immunogold labeling of a mix of C. burnetii morphological forms showing specific labeling of SCV by serum against the SCV-specific protein, ScvA. (B) Immunogold labeling of C. burnetii purified from aged Vero cell cultures showing uniform heavy labeling with anti-ScvA serum. (C) C. burnetii purified from aged Vero cell cultures showing morphological and ultrastructural characteristics typical of SCV, e.g., size of 0.2 to 0.5 μm and electron-dense compacted chromatin. (The fixation of C. burnetii depicted in panels A and B was optimized for retention of ScvA antigenicity, and consequently, the condensed chromatin of the SCV is not obvious in these panels. The fixation of C. burnetii depicted in panel C was optimized for preservation of ultrastructure.) Bar, 0.5 μm.
FIG. 2.
Temporal analysis of C. burnetii morphological development in Vero cells. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Infected cells were fixed and processed for TEM at 0, 8, and 16 h and 1, 2, 3, 4, 6, and 8 days p.i. Prototypic SCV and LCV are designated in selected panels with white and black arrows, respectively. Bar, 0.5 μm.
FIG. 3.
One-step growth curves of C. burnetii. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Replating FFU and genome equivalent assays were conducted to quantify C. burnetii replication as described in Materials and Methods. The approximate times p.i. of C. burnetii morphological changes and LCV replication are indicated above the graph. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.
FIG. 4.
Relative expression levels of selected C. burnetii genes during morphological differentiation as detected by quantitative RT-PCR. Assays were performed using TaqMan primers and probes specific for each gene. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Total RNA was extracted at the indicated times. Transcriptional activity is expressed as relative expression, with transcript copy number normalized to the number of C. burnetii genomes present in each sample. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.
FIG. 5.
Quantification of immunogold labeling of ScvA. Vero cells were infected with SCV and processed for immunogold TEM at 8 h, 3 days, and 6 days p.i. by using rabbit polyclonal anti-ScvA serum. The purified SCV inoculum was also labeled. The relative ScvA content of individual C. burnetii organisms (at least 100 organisms per time point) was determined by counting the number of gold particles per organism on representative micrographs. Organisms were scored as having more or fewer than 10 gold particles. Representative micrographs of immunogold labeling of C. burnetii at 3 and 6 days p.i. are depicted above the graph. Bar, 0.5 μm.
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