Amoebal Endosymbiont Parachlamydia acanthamoebae Bn9 Can Grow in Immortal Human Epithelial HEp-2 Cells at Low Temperature; An In Vitro Model System to Study Chlamydial Evolution

Abstract

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.

Amoebal endosymbiont Neochlamydia protects host amoebae against Legionella pneumophila infection by preventing Legionella entry

“…Rabbit serum containing anti-Parachlamydia Bn 9 antibodies against formalin fixed the bacteria was produced by Iwaki (Tokyo, Japan) [19]. In addition, each of the amoebae (S13WT ameobae, S13RFP amoebae, C3 amoebae) (1×10 cells/well) were cultured in PYG with 50μl of virus solution for up to h. Amoebal lysis was observed under a light microscope.…”

Environmental Metagenomic Assemblies Reveal Seven New Highly Divergent Chlamydial Lineages and Hallmarks of a Conserved Intracellular Lifestyle

“…On the other hand, the F-like conjugative DNA transfer system identified by Greub et al ( 2004 ) in Protochlamydia amobophila was restricted to Simkaniaceae and Parachlamydiaceae (Bertelli et al, 2016 ). Since then, multiple genomes belonging to the family Parachlamydiaceae were sequenced from strains in pure culture, including one representative of a new genus named Rubidus (Domman et al, 2014 ; Ishida et al, 2014 ; Yamaguchi et al, 2015 ; Yamane et al, 2015 ; Bertelli et al, 2016 ; Bou Khalil et al, 2016 , 2017 ; Fukumoto et al, 2016 ). In addition, genome sequences representing two distinct genera of the family Criblamydiaceae were recently released (Bertelli et al, 2014 , 2015 ).…”

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation

“…Meanwhile, the bacteria failed to grow in a representative immortal human epithelial cell line, HEp-2. Surprisingly, the amount of bacteria significantly decreased during the culture period compared to a reference strain, Parachlamydia Bn 9 [25]. While these findings indicate a rapid elimination of bacteria from the cells, it raises a possible scenario that the host cells can rapidly sense the bacteria, which stimulates pro-inflammatory cytokines.…”

“…Meanwhile, when compared with the other bacteria (UWE25 and R18), the type IV secretion system, which delivers effector proteins associated with host cell modulation [32], was not seen, indicating a divergent evolution of environmental chlamydiae. Meanwhile, because environmental chlamydiae ( Parachlamydia ) lacking the type IV secretion system replicated in immortal epithelial cells, albeit not at body temperature [25], Protochlamydia W-9 has the potential to adapt to human cells.…”