The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents

Abstract

A 7?5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1-L3), ten trachoma isolates (serovars A-C) and nine urogenital isolates (serovars D-J). The results indicated an average plasmid copy number of 4?0±0?8 (mean±95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7?6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7?4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1?3±0?2 plasmids per chromosome (mean±95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number. INTRODUCTIONMembers of the Chlamydiaceae are obligately intracellular, Gram-negative bacteria of significant importance in both human and animal pathogenesis. These organisms exhibit a unique developmental cycle, in which a metabolically active reticulate body (RB) gives rise to a dense, infectious elementary body (EB) (Rockey & Matsumoto, 2000). The family comprises two genera, Chlamydophila and Chlamydia (Everett et al., 1999), although this classification remains controversial (Schachter et al., 2001).Chlamydophila pneumoniae is an important human pathogen implicated in arterial disease (Saikku et al., 1992); infection of the respiratory tract causes an atypical pneumonia (Hahn et al., 2002). Although predominantly a human pathogen, C. pneumoniae strains have been isolated from animal species, e.g. equines (Everett et al., 1999;Storey et al., 1993; Wills et al., 1990) and koala (Wardrop et al., 1999).Chlamydia trachomatis is not only the major infectious agent of preventable blindness in the developing world (Thylefors et al., 1995), but also the commonest cause of non-specific urethritis in developed countries (Burstein & Zenilman, 1999). Sexually transmitted disease caused by C. trachomatis is frequently undiagnosed in women, whereupon an ascending infection may lead to pelvic inflammatory disease, salpingitis and consequent infe...

Chlamydia trachomatis Load at Matched Anatomic Sites: Implications for Screening Strategies

“…Three dilutions of the EB standard were quantified relative to the plasmid standard in duplicate runs. The results showed that there were 7.72 Ϯ 0.68 (mean Ϯ SD) plasmid copies per EB, consistent with previously obtained values (21,32). This value was used to convert plasmid copies into equivalent EBs per 100 l. Analysis of the 15 matched sets each of male and female specimens revealed a pronounced correlation (r 2 ϭ 0.929) between EB counts determined by QLCR and those determined by QPCR (Fig.…”

“…A partially purified preparation of EBs from C. trachomatis (serovar L1), which is readily propagated in buffalo green monkey kidney cells, was provided by I. Clarke (University of Southampton, United Kingdom) and used as the standard for EB quantitation. Although most of the infected patients were likely to harbor C. trachomatis serovars D through K, the primer sets for both the LCR and QPCR assays correspond to conserved regions of the C. trachomatis cryptic plasmid and are therefore able to detect all C. trachomatis serovars (16,21,31). The EB concentration of the standard was determined by electron microscopy.…”

A plasmid-cured Chlamydia muridarum strain displays altered plaque morphology and reduced infectivity in cell culture

“…The ability to cure C. muridarum strain Nigg would suggest that, at appropriate concentrations of novobiocin, plasmid replication is sufficiently inhibited that plasmid-deficient isolates can be recovered; this is in contrast to previous reports that at low concentrations of the drug (20 mg ml 21 in conjunction with 20 mg imipramine ml 21 ), plasmid copy number increases (Pickett et al, 2005). The apparently elevated concentrations of novobiocin required to suppress chlamydial replication were striking.…”

“…A highly conserved plasmid of approximately 7?5 kb is present in almost all strains of Chlamydia trachomatis, with copy numbers estimated to range from 4 (Pickett et al, 2005) to 10 (Tam et al, 1992) copies per cell. The role of this plasmid is unknown.…”

Optimal Method of Collection of First-Void Urine for Diagnosis of Chlamydia trachomatis Infection in Men

“…The FVU specimens for qPCR analysis were extracted as described by Magbanua et al (11). qPCR was performed using the method of Pickett et al (14) using amplification conditions described previously (12). All qPCR analysis was performed within 30 days of specimen collection.…”