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.

Satoshi Oguri,Junji Matsuo,Tomohiro Yamazaki,Sumire Yamazaki,Kasumi Ishida,Mitsutaka Yoshida,Yasuhiro Hayashi,Yimin Yimin,Shinji Nakamura,Natsumi Shouji,Chikayo Yamane,Hiroyuki Yamaguchi

doi:10.1371/journal.pone.0116486

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.

Highlights

The obligate intracellular bacteria, chlamydiae, have successfully adapted to several distinct hosts
To confirm that Parachlamydia Bn9 did not adapt at 37°C, we used a previously established amoeba-infectious unit (AIU) assay [13] with 4’,6-diamidino-2-phenylindole (DAPI) staining to determine whether the bacteria could grow in C3 amoebae at 37°C
The findings confirmed that the bacteria adapted and grew well at a low temperature in the host amoebae

Summary

Introduction

The obligate intracellular bacteria, chlamydiae, have successfully adapted to several distinct hosts. While the evolution of pathogenic chlamydiae has involved a decrease in genome size to approximately 1.0–1.2 Mb, which may be a strategy to evade the host immune network, resulting in a shift to parasitic energy and metabolic requirements [17], the genomes of representative environmental chlamydiae are not decreasing and have stabilized at 2.4–3.0 Mb [12, 18, 19]. These findings indicate that ancient pathogenic features are strongly selected for in environmental chlamydiae. Ancient pathogenic chlamydiae could not readily grow in mammalian cells within a stable niche of approximately 37°C and there might be a significant temperature gap that ancient pathogenic chlamydiae had to overcome to successfully adapt to mammals, including humans

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Results

Discussion

Conclusion