Survival and transfer ability of phylogenetically diverse bacterial endosymbionts in environmental Acanthamoeba isolates

Abstract

SummaryObligate intracellular bacteria are commonly found as endosymbionts of acanthamoebae, however, their survival in and ability to transfer to amoebae are currently uncharacterized.In this study, six bacterial endosymbionts, found in five environmental Acanthamoeba isolates (S13, R18, S23, S31, S40) from different locations of Sapporo city, Japan, were characterized. Phylogenetic analysis revealed that three-bacterial endosymbionts (eS31, eS40a, eS23) belonged to α-and β-Proteobacterium phyla and the remaining endosymbionts (eR18, eS13, eS40b) belonged to the Chlamydiales phylum. The Acanthamoeba isolate (S40) contained two phylogenetically different bacterial endosymbionts (eS40a, eS40b). Fluorescent in situ hybridization analysis showed that all bacterial endosymbionts were diffusely localized within amoebae. Transmission electron microscopy also showed that the endosymbionts were rod-shaped (eS31, eS40a, eS23) or sphere-or crescent-shaped (eR18, eS13, eS40b). No successful culture of these bacteria was achieved using conventional culture methods, but the viability of endosymbionts was confirmed by live/dead staining and RT-PCR methods. However, endosymbionts (except eR18) derived from original host cells lost the ability to be transferred to another amoeba strain (Acanthamoebae ATCC C3). Taken together, our data demonstrate that phylogenetically diverse bacterial endosymbionts found in amoebae are viable and maintain a stable interaction with amoebae.

Environmental Chlamydiae Alter the Growth Speed and Motility of Host Acanthamoebae

“…Figure 1A is a representative FISH image showing the amoebae before treatment with antibiotics, indicating that the bacterial endosymbionts localized to the cytoplasm of the amoebae, as described previously (23, 25). After antibiotic treatment, successful elimination of the endosymbionts was confirmed by DAPI staining (Fig.…”

“…As described previously (23, 25), two different amoeba strains, R18WT and S13WT, which were persistently infected with Protochlamydia endosymbiont (eR18) or Neochlamydia (eS13), were isolated from a river water sample and a soil sample, respectively. We have previously confirmed that the prevalence of amoebae with endosymbionts, as defined by 4′,6-diamidino-2-phenylindole (DAPI) staining, was always approximately 100% (for both R18WT [26] and S13WT [data not shown]).…”

“…FISH was performed as previously reported (23). In brief, amoebae were fixed and hybridized with Alexa Fluor 488-labeled EUK 516 (green), which targets eukaryotic 18S rRNA (5′-ACCAGACTTGCCCTCC-3′) (1), or Alexa Fluor 532-labeled C 22 -658 (red), which is specific to the Parachlamydiaceae family (5′-TCCATTTTCGTCTAC-3′) (10).…”

“…Endosymbiotic bacteria have been detected in Acanthamoeba , with a possibly stable interaction, although amoebae possess a powerful bactericidal mechanism (4, 9, 11, 15–17, 23, 25, 30). Investigations of Acanthamoeba are facilitated by the fact that these organisms grow easily and rapidly in simple media regardless of the presence of bacterial endosymbionts.…”

“…Previously, we isolated environmental Acanthamoeba infected with endosymbiotic bacteria, and successfully established models of environmental Acanthamoeba persistently infected with Protochlamydia (R18 symbiotic amoebae: R18WT [wild type]) or Neochlamydia (S13 symbiotic amoebae: S13WT) (23, 25). These endosymbionts have been assigned to the order Chlamydiales (14), which includes Chlamydia trachomatis and Chlamydophila pneumoniae , so-called pathogenic chlamydiae that are linked to sexually transmitted disease (19) and respiratory infection (16, 19), respectively.…”