Project description:Community-acquired pneumonia (CAP) is a common disease responsible for significant morbidity and mortality. However, the definite etiology of CAP often remains unresolved, suggesting that unknown agents of pneumonia remain to be identified. The recently discovered members of the order Chlamydiales, Chlamydia-related bacteria (CRB), are considered as possible emerging agents of CAP. Parachlamydia acanthamoebae is the most studied candidate. It survives and replicates inside free-living amoeba, which it might potentially use as a vehicle to infect animals and humans. A Mycoplasma pneumoniae outbreak was observed in Kymenlaakso region in Southeastern Finland during August 2017-January 2018. We determined the occurrence of Chlamydiales bacteria and their natural host, free-living amoeba in respiratory specimens collected during this outbreak with molecular methods. Altogether, 22/278 (7.9%) of the samples contained Chlamydiales DNA. By sequence analysis, majority of the CRBs detected were members of the Parachlamydiaceae family. Amoebal DNA was not detected within the sample material. Our study further proposes that Parachlamydiaceae could be a potential agent causing atypical CAP in children and adolescents.
Project description:Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37 °C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30 °C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30 °C compared to at 37 °C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells.
