Analysis of Chlamydia pneumoniae growth in cells by reverse transcription-PCR targeted to bacterial gene transcripts.
Chlamydia pneumoniae is an obligate intracellular bacterium and has a unique development cycle consisting of an elementary body (EB) and reticular body (RB). EBs survive in extracellular environments as well as infect susceptible host cells. However, EBs display no measurable metabolic activity. In contrast, RBs are metabolically active and can replicate in a host cell but are noninfectious. Therefore, analysis of C. pneumoniae growth in infected cells by conventional bacterial culture may not permit sufficient information about growth of the bacteria in cells. In this study, therefore, we examined the usefulness of the reverse transcription (RT)-PCR method for analysis of bacterial transcripts to evaluate C. pneumoniae growth in HEp-2 cells because the levels of bacterial gene transcripts are known to show the metabolic activity of bacteria. The transcripts for the C. pneumoniae hsp60 gene and 16S rRNA in the cells were easily detected just after infection, followed by a marked increase. In contrast, pyk and omcB transcripts slowly increased after a latent period. The hydrocortisone treatment of C. pneumoniae-infected cells induced an increase of all bacterial transcripts tested compared with the control group. The treatment of the infected cells with the antibiotic minocycline showed a selective inhibition of bacterial gene transcripts, even though the complete inhibition of EB production determined by the bacterial culture assay was evident. These results indicate that the determination of bacterial gene transcripts by RT-PCR might be a powerful method to analyze in detail growth of C. pneumoniae in host cells, particularly altered bacterial growth caused by agents such as antimicrobials.
Haranaga
Shusaku
S
Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
Ikejima
Hideaki
H
Yamaguchi
Hiroyuki
H
Friedman
Herman
H
Yamamoto
Yoshimasa
Y
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002690:Chlamydia Infections / Q000175:diagnosis*; D016993:Chlamydophila pneumoniae / Q000235:genetics* / Q000254:growth & development*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D008911:Minocycline / Q000494:pharmacology; D012329:RNA, Bacterial / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D000900:Anti-Bacterial Agents; D000893:Anti-Inflammatory Agents; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D008911:Minocycline; D006854:Hydrocortisone