Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium

Abstract

Chlamydia trachomatis is among the most clinically significant human pathogens, yet their obligate intracellular nature places severe restrictions upon research. Chlamydiae undergo a biphasic developmental cycle characterized by an infectious cell type known as an elementary body (EB) and an intracellular replicative form called a reticulate body (RB). EBs have historically been described as metabolically dormant. A cell-free (axenic) culture system was developed, which showed high levels of metabolic and biosynthetic activity from both EBs and RBs, although the requirements differed for each. EBs preferentially used glucose-6-phosphate as an energy source, whereas RBs required ATP. Both developmental forms showed increased activity when incubated under microaerobic conditions. Incorporation of isotopically labeled amino acids into proteins from both developmental forms indicated unique expression profiles, which were confirmed by genome-wide transcriptional analysis. The described axenic culture system will greatly enhance biochemical and physiological analyses of chlamydiae.

Fig. 1.

Differential energy source utilization by EBs and RBs. Purified EBs and RBs were incubated in medium containing G6P, ATP, or both. (A) Scintillation counting and (B) autoradiography of bacterial lysates show differential energy source utilization by the two cell forms. (C) Silver staining shows equal loading of samples with distinct protein profiles. Data are expressed as the mean (n = 4) ± SEM. Background signal from heat-inactivated organisms was subtracted from all samples. An autoradiograph from a representative experiment is shown. Molecular weight markers are in kilodaltons.

Fig. 2.

Comparison of EB and RB protein synthesis in axenic media. Protein synthesis by EBs and RBs normalized to total protein content was assessed in CIP-1 and two previously described media by measuring incorporation of [³⁵S]Cys-Met into bacterial proteins after 6 h of incubation. (A) Scintillation count analysis showed strong incorporation of radiolabel in both EBs and RBs when incubated in CIP-1 medium, and moderate radiolabel incorporation by RBs, but not EBs when incubated in Hatch medium. Medium DGM-21A supported negligible levels of radiolabel incorporation by either EBs and RBs. (Inset) Counts per minute detected in EB and RB lysates after incubation in DGM-21A on adjusted scale. Data are expressed as the mean (n = 4) ± SEM. The asterisk indicates statistical significance (P < 0.05). Background signal measured from incubation with heat-inactivated organisms was subtracted from all samples. (B) SDS/PAGE and autoradiography of bacterial lysates confirmed incorporation into bacterial proteins. An autoradiograph from a representative experiment is shown. Molecular weight markers are in kilodaltons.

Fig. 3.

Pharmacologic inhibition of chlamydial axenic protein synthesis. C. trachomatis de novo translation and transcription was assessed by analyzing incorporation of [³⁵S]Cys-Met after incubation of EBs in medium containing ethanol solvent, or medium supplemented with 10 µg/mL chloramphenicol or rifampicin. (A and B) Data are expressed as the mean (n = 6) ± SEM. The asterisk indicates statistical significance (P < 0.05). Background signal measured from heat-inactivated organisms was subtracted from all samples. A representative autoradiograph is shown. Molecular weight markers are in kilodaltons.

Fig. 4.

Developmental stage-specific transcription of purified EBs and RBs. EBs and RBs were incubated in CIP-1 for 3 h under 2.5% oxygen and RNA extracted for microarray analysis. (A) Heat map showing axenic EB gene expression relative to axenic RBs. (B) Number of genes up-regulated more than threefold in EBs, up-regulated more than threefold in RBs, or expressed at relatively equivalent levels in both. Seventeen transcripts were not detected.