Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont

Temporal classes of gene expression during the Protochlamydia developmental cycle. A total of 797 genes were detected as differentially expressed; tRNA genes (20 genes), rRNA genes (2 genes), and genes detected only in a single replicate (38 genes) were excluded from further analysis. Clustering identified three main temporal classes of gene sets (colored bars to the left of the heatmap) that could be further divided into five large subclasses (colored bars to the right of the heatmap). The largest group of genes was most highly expressed early (n = 304), whereas the expression of the smallest group of genes peaked at midcycle when only RBs were present (n = 161). The third main gene cluster generally showed highest expression at the end of the cycle and the extracellular stage (n = 273; see Data Set S1 in the supplemental material). Gene products detected in the EB proteome in a previous study (n = 231) (39) are indicated in the bar plot next to the heatmap. To illustrate the course of gene expression for each subcluster, the expression values (log₂ RPKM plotted on the y axis) were averaged per time point (x axis) and visualized as line plots (error bars indicate standard deviations). Selected gene names are shown for each of the temporal clusters. RPKM, reads per kilobase per million; hpi, h postinfection; extracell., extracellular; PG, peptidoglycan synthesis; T3SS, type III secretion system.

Enrichment of functional categories by temporal class. The overrepresentation of functional categories among genes assigned to temporal classes provides evidence for stage-specific activities during the Protochlamydia developmental cycle. Only functional categories that were significantly enriched with a false-discovery rate (FDR) of ≤0.05 are shown here; the color code indicates the degree of significance (dark red indicates highly significant). The significant enrichment of putatively type III secreted gene products was tested using Fisher’s exact test, and only P values of ≤0.05 are shown. cat, category.

Course of Protochlamydia type III secretion system activity during the developmental cycle. This model is based on the observation that structural components of the type III secretion system and its (putative) effectors are expressed at different time points during the developmental cycle (Fig. S4). This suggests a scenario in which novel, fully assembled, and thus functional secretion systems occur only late in the developmental cycle, and type III secretion reaches its full capacity and highest activity during early stages of the infection. The indicated polarity of the active type III secretion system has been shown for C. trachomatis (54), but it is unclear whether this is also true for P. amoebophila, as the symbionts reside within single-cell inclusions. The color code for type III secretion components and effectors (nomenclature according to Hueck [105]) refers to the respective temporal gene expression classes (Fig. 2 and Fig. S4). Circles inside the cells represent chaperones. Differentially expressed components/effectors are labeled with asterisks; PEX1 and PEX2 refer to members of the expanded effector gene families in Protochlamydia (33); pc0309 is an ortholog of the putative chaperone encoded by CT274 (106). inc, inclusion membrane; hcm, host cell membrane; im, inner membrane; om, outer membrane.

Expression maps of selected metabolic pathways of Protochlamydia. A pronounced expression of the ATP/ADP translocase (ntt1) at midcycle and an early-to-mid activity of genes involved in amino acid breakdown to pyruvate is observed, whereas pathways involved in central carbon metabolism and energy generation were generally only upregulated at later stages (with the ATPases indicated by purple boxes being notable exceptions). This suggests that a major metabolic shift occurs during the developmental cycle and provides evidence for a stage-specific metabolism. All genes marked with an asterisk were detected to be significantly differentially expressed. RPKM, reads per kilobase per million; hpi, h postinfection; extracell., extracellular.