Genes required for assembly and function of the protein synthetic system in Chlamydia trachomatis are expressed early in elementary to reticulate body transformation

Abstract

Following binding and internalization into the host cell cytoplasm, elementary bodies (EB) of the obligate intracellular bacterium Chlamydia trachomatis undergo a developmental process resulting in production of reticulate bodies (RB), the vegetative growth form of the organism. EB are metabolically inactive, but EB to RB transformation requires bacterial protein synthesis. Using HeLa cells infected with EB of C. trachomatis serovar C, we examined the time of first appearance of transcripts from several genes whose products are required for assembly and function of the chlamydial protein synthetic system. We monitored appearance of chlamydial RNAs using reverse transcription-polymerase chain reaction assays targeting primary transcripts from the bacterial rRNA operons, and mRNAs encoding the glycyl tRNA synthetase and the ribosomal proteins S5 and L5. Transcripts from the proximal rRNA promoters, and those from the r-protein and tRNA synthetase genes, are detectable as early as 4 h after EB-host binding; transcripts from distal rRNA promoters do not appear until 6 h post-infection. Thus, expression of bacterial genes whose products are required for protein synthesis begins earlier in chlamydial EB to RB development than previously thought.

Chlamydia pneumoniae Expresses Genes Required for DNA Replication but Not Cytokinesis during Persistent Infection of HEp-2 Cells

“…RT was done as previously described (6) with 5 g of total RNA from each preparation, random hexamers as primers, and murine leukemia virus reverse transcriptase (Life Technologies). cDNA from each reaction was treated with RNase A, RNase T1, and RNase H, extracted several times with phenol-chloroform (24:1), and then collected as an ethanol precipitation (6,11). The genes targeted in RT-PCR are given in Table 1, along with the primer sequences employed for amplification.…”

“…RNA preparations were confirmed to be DNA free by PCR targeting the host ␤-actin gene in the absence of RT. The integrity of RNA preparations was assessed by visual analysis of ethidium bromide-stained formaldehyde-agarose electrophoresis gels and by RT-PCR targeting of the host actin gene (6,11).…”

“…Total nucleic acids were prepared from snap-frozen pellets of infected and uninfected HEp-2 cells via homogenization in 65°C buffered phenol and extensive extraction in phenol-chloroform (24:1), as described elsewhere (6). DNA was prepared for quantitative PCR analysis by treatment of total nucleic acid preparations with RNase A plus RNase T1 (Life Technologies).…”

CADD, a Chlamydia Protein That Interacts with Death Receptors

“…The location of Fas ␣-helical segments (underlined) was based on a combination of published structural data derived for the DD of Fas (18) and secondary structure predictions. C, the location of the CADD-encoding gene CT610 within the C. trachomatis genome is depicted showing a portion of the late region of the genome and indicating the relative locations of nearby genes, including the factor-encoding gene rpoD ( 66 ), which produces a transcription factor implicated in late gene expression (13). The arrow gives the orientation of the complementary strand of C. trachomatis genome.…”

“…To determine whether CADD is actually expressed in infected cells, RT-PCR analysis was performed, monitoring CADD gene expression as a function of time during the course of Chlamydia infection (13). For these experiments, HeLa cell cultures were inoculated with C. trachomatis, and RNA was recovered at various times thereafter for RT-PCR analysis.…”