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Protochlamydia, an environmental chlamydia and obligate amoebal endosymbiotic bacterium, evolved to survive within protist hosts, such as Acanthamobae, 700 million years ago. However, these bacteria do not live in vertebrates, including humans. This raises the possibility that interactions between Protochlamydia and human cells could induce a novel cytopathic effect, leading to new insights into host-parasite relationships. Therefore, we studied the effect of Protochlamydia on the survival of human immortal cell line, HEp-2 cells and primary peripheral blood mononuclear cells (PBMC). Using mainly 4′,6-diamidino-2-phenylindole staining, fluorescent in situ hybridization, transmission electron microscopy, and also TUNEL and Transwell assays, we demonstrated that the Protochlamydia induced apoptosis in HEp-2 cells. The attachment of viable bacterial cells, but not an increase of bacterial infectious progenies within the cells, was required for the apoptosis. Other chlamydiae [Parachlamydia acanthamoebae and Chlamydia trachomatis (serovars D and L2)] did not induce the same phenomena, indicating that the observed apoptosis may be specific to the Protochlamydia. Furthermore, the bacteria had no effect on the survival of primary PBMCs collected from five volunteers, regardless of activation. We concluded that Protochlamydia induces apoptosis in human-immortal HEp-2 cells and that this endosymbiont could potentially be used as a biological tool for the elucidation of novel host-parasite relationships.

Symbiosis between living beings is an important driver of evolutionary novelty and ecological diversity; however, understanding the mechanisms underlying obligate mutualism remains a significant challenge. Regarding this, we have previously isolated two different Acanthamoeba strains harboring endosymbiotic bacteria, Protochlamydia (R18 symbiotic amoebae: R18WT) or Neochlamydia (S13 symbiotic amoebae; S13WT). In this study, we treated the symbiotic amoebae R18WT and S13WT with doxycycline (DOX) and rifampicin (RFP), respectively, to establish the aposymbiotic amoebae R18DOX and S13RFP, respectively. Subsequently, we compared the growth speed, motility, phagocytosis, pinocytosis, and morphology of the symbiotic and aposymbiotic amoebae. The growth speed of R18DOX was decreased, although that of S13RFP was increased. A marked change in motility was observed only for R18DOX amoebae. There was no difference in phagocytic and pinocytic activities between the symbiotic and aposymbiotic amoebae. Meanwhile, we observed a significant change in the phalloidin staining pattern and morphological changes in R18DOX (but not S13RFP) aposymbiotic amoebae, indicating a change in actin accumulation upon removal of the Protochlamydia. Infection of C3 (a reference strain) or S13RFP amoebae with Protochlamydia had a harmful effect on the host amoebae, but R18DOX amoebae re-infected with Protochlamydia showed recovery in both growth speed and motility. Taken together, we conclude that endosymbiont environmental chlamydiae alter the growth speed and/or motility of their host Acanthamoeba, possibly implying an close mutual relationship between amoebae and environmental chlamydiae.