Chlamydiae, such as Chlamydia trachomatis and Chlamydia pneumoniae, can cause chronic infections. It is believed that persistent forms called aberrant bodies (ABs) might be involved in this process. AB formation seems to be a common trait of all members of the Chlamydiales order and is caused by distinct stress stimuli, such as β-lactam antibiotics or nutrient starvation. While the diverse stimuli inducing ABs are well described, no comprehensive morphological characterization has been performed in Chlamydiales up to now. We thus infected mammalian cells with the Chlamydia-related bacterium Waddlia chondrophila and induced AB formation using different stimuli. Their morphology, differences in DNA content and in gene expression were assessed by immunofluorescence, quantitative PCR, and reverse transcription PCR, respectively. All stimuli induced AB formation. Interestingly, we show here for the first time that the DNA gyrase inhibitor novobiocin also caused appearance of ABs. Two distinct patterns of ABs could be defined, according to their morphology and number: (i) small and multiple ABs versus (ii) large and rare ABs. DNA replication of W. chondrophila was generally not affected by the different treatments. Finally, no correlation could be observed between specific types of ABs and expression patterns of mreB and rodZ genes.

Chlamydia trachomatis is an important cause of sexually transmitted infections (STI) in Western countries. It is often asymptomatic, and thus, left untreated, and can have severe negative consequences, such as tubal infertility or adverse pregnancy outcomes. Other sexually transmitted microorganisms, such as Neisseria gonorrhoeae and Trichomonas vaginalis, as well as normal residents of the vaginal flora, such as genital mycoplasmas, also negatively impact human sexual and reproductive health. We evaluated the reliability of the Seegene Allplex STI Essential Assay for C. trachomatis detection using the real-time qPCR routinely used in our diagnostic laboratories as the gold standard. The Seegene assay displayed a sensitivity of 97.8% and a specificity of 98.9%. As this assay can also detect six other urogenital pathogens, we applied it to 404 samples from women who attended Lausanne University Maternity Hospital and obtained the following prevalence rates: 2.5% for C. trachomatis, 3.5% for Mycoplasma hominis, 6.3% for Ureaplasma urealyticum, and 27.7% for Ureaplasma parvum. Two samples were positive for Trichomonas vaginalis, and one sample was positive for Mycoplasma genitalium. Bacterial vaginosis was present in 4.5% of the cases and was strongly associated with M. hominis. Finally, we confirmed the association between C. trachomatis infection and pre-term birth (p = 0.03) but could not detect any association of this condition with other urogenital pathogens (Mycoplasma/Ureaplasma). In conclusion, given its high sensitivity and specificity for C. trachomatis DNA detection as well as its multiplex format, which simultaneously provides results for six other urogenital pathogens, the Seegene Allplex™ STI Essential Assay represents an appealing diagnostic tool in modern microbiology laboratories.

Chronic infections caused by obligate intracellular bacteria belonging to the Chlamydiales order are related to the formation of persistent developmental forms called aberrant bodies (ABs), which undergo DNA replication without cell division. These enlarged bacteria develop and persist upon exposure to different stressful conditions such as β-lactam antibiotics, iron deprivation and interferon-γ. However, the mechanisms behind ABs biogenesis remain uncharted. Using an RNA-sequencing approach, we compared the transcriptional profile of ABs induced by iron starvation to untreated bacteria in the Chlamydia-related species Waddliachondrophila, a potential agent of abortion in ruminants and miscarriage in humans. Consistent with the growth arrest observed following iron depletion, our results indicate a significant reduction in the expression of genes related to energy production, carbohydrate and amino acid metabolism and cell wall/envelope biogenesis, compared to untreated, actively replicating bacteria. Conversely, three putative toxin-antitoxin modules were among the most up-regulated genes upon iron starvation, suggesting that their activation might be involved in growth arrest in adverse conditions, an uncommon feature in obligate intracellular bacteria. Our work represents the first complete transcriptomic profile of a Chlamydia-related species in stressful conditions and sets the grounds for further investigations on the mechanisms underlying chlamydial persistence.

Waddlia chondrophila is an emerging intracellular pathogen belonging to the order of Chlamydiales, and was previously associated with adverse pregnancy outcomes, as well as tubal factor infertility (TFI). In this study, we investigate the link between both W. chondrophila and Chlamydia trachomatis IgG seropositivity and TFI. Antibodies against both bacteria were measured in 890 serum samples of women visiting a fertility clinic. After a hysterosalpingography and/or laparoscopy, they were classified as either TFI-negative (TFI−) or TFI-positive (TFI+). The total seroprevalence was 13.4% for C. trachomatis and 38.8% for W. chondrophila. C. trachomatis antibodies were present significantly more often in the TFI+ group than in the TFI− group, while for W. chondrophila no difference could be observed. In conclusion, our study confirms the association between C. trachomatis seropositivity and TFI, but no association was found between W. chondrophila seropositivity and TFI. The high percentage of W. chondrophila seropositivity in all women attending a fertility clinic does, however, demonstrate the need for further research on this Chlamydia-like bacterium and its possible role in infertility.

Despite broad genetic variability, members of the Chlamydiota phylum share a crucial stress response phenotype, the formation of aberrant bodies. However, how this response operates upon exposure to different kinds of stressors is still largely unknown. In Waddlia chondrophila, wcw_0502 RNA levels are upregulated in aberrant bodies induced by iron starvation. Wcw_0502 is a putative type III secretion system (T3SS) effector and has a homologue in every known chlamydial species, regardless of their host. However, the upregulation of the wcw_0502 gene expression upon iron starvation is not conserved in other chlamydial species such as Chlamydia trachomatis, Chlamydia pneumoniae, Simkania negevensis or Estrella lausannensis. Moreover, among all the stressors examined, only heat shock induced a strong upregulation of wcw_0502 and its C. trachomatis homologue, ctl0271. A Controlling Inverted Repeat of Chaperone Expression sequence is present in the promoter region of wcw_0502 and its homologues. We hypothesized that in the absence of stress, the conserved repressor HrcA, in association with the Hsp60 chaperone, binds this sequence and represses transcription. A decreased occupancy of HrcA and Hsp60 at the wcw_0502 promoter region was observed in aberrant bodies induced by iron starvation when compared to reticulate bodies, which may lead to wcw_0502 upregulation. The precise function of this newly described T3SS effector is still unclear. A cystine knot-like domain, a structural feature never described before in bacterial proteins, was found in the C-terminal region of Wcw_0502. This structure is described as highly resistant to proteolytic, chemical and thermic stressors, an advantageous property for a secreted protein with an increased production during stresses that impact protein integrity.

Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells.