High‐temperature adapted primitive Protochlamydia found in Acanthamoeba isolated from a hot spring can grow in immortalized human epithelial HEp‐2 cells

Abstract

To elucidate how ancient pathogenic chlamydiae could overcome temperature barriers to adapt to human cells, we characterized a primitive chlamydia found in HS-T3 amoebae (Acanthamoeba) isolated from a hot spring. Phylogenetic analysis revealed the primitive species to be Protochlamydia. In situ hybridization staining showed broad distribution into the amoebal cytoplasm, which was supported by transmission electron microscopic analysis showing typical chlamydial features, with inclusion bodies including both elementary and reticular bodies. Interestingly, although most amoebae isolated from natural environments show reduced growth at 37°C, the HS-T3 amoebae harbouring the Protochlamydia grew well at body temperature. Although infection with Protochlamydia did not confer temperature tolerance to the C3 amoebae, the number of infectious progenies rapidly increased at 37°C with amoebal lysis. In immortalized human epithelial HEp-2 cells, fluorescence microscopic study revealed atypical inclusion of the Protochlamydia, and quantitative real-time polymerase chain reaction analyses also showed an increase in 16S ribosomal RNA DNA amounts. Together, these results showed that the Protochlamydia found in HS-T3 amoebae isolated from a hot spring successfully adapted to immortalized human HEp-2 cells at 37°C, providing further information on the evolution of ancient Protochlamydia to the present pathogenic chlamydiae.

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

“…In fact, we have previously shown that environmental chlamydiae ( Protochlamydia R18, Parachlamydia Bn 9 ) failed to grow in the immortalized epithelial cell line, HEp-2, at 37°C [ 13 – 15 ]. However, in contrast to these findings, we have also found that an amoebal endosymbiont, Protochlamydia , found in HS-T3 Acanthamoeba isolated from a hot spring, successfully adapted to HEp-2 cells at 37°C with active replication [ 16 ]. More importantly, 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 ].…”

Acanthamoeba S13WT relies on its bacterial endosymbiont to backpack human pathogenic bacteria and resist Legionella infection on solid media

“…An aposymbiotic strain was established from S13WT by treatment with rifampicin (RFP; 64 mg ml 21 ), and designated S13RFP (Okude et al, 2012). Acanthamoeba strain HS-T3, persistently infected with endosymbiont Protochlamydia HS-T3, was originally isolated from a hot spring located in Kanagawa Prefecture, Japan (Sampo et al, 2014). We generated a reconstructed S13RFP symbiont strain containing Protochlamydia HS-T3, designated S13HS-T3, for use in this study.…”

“…With this in mind, we previously isolated several environmental Acanthamoeba strains infected with endosymbiotic bacteria from natural and built environments, including soil, hot spring water and hospital floors. These strains have proven useful as models for understanding host-parasite relationships Nakamura et al, 2010;Sampo et al, 2014;Fukumoto et al, 2016). Among them, soil-borne Acanthamoeba strain S13WT, harbouring environmental nonpathogenic chlamydial strain Neochlamydia S13, was of particular interest for several reasons .…”

“…Third, amoebae harbouring the Neochlamydia endosymbiont can resist Legionella, a natural pathogen of amoebae . Thus, the presence of the bacterial endosymbiont obviously hinders in the growth of the host amoebae, and despite extensive experiments in liquid media Nakamura et al, 2010;Okude et al, 2012;Sampo et al, 2014;Fukumoto et al, 2016), the biological advantage of maintaining the endosymbiont remains to be fully understood.…”

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

“…During this period, the amoebal cells were regularly collected to assess bacterial numbers using quantitative real-time PCR (qPCR), the AIU assay and DAPI staining, according to methods described previously [10, 15]. Meanwhile, the immortal human cell line, HEp-2, was also infected with the bacteria at a MOI of 1–5.…”