Improved axenization method reveals complexity of symbiotic associations between bacteria and acanthamoebae

Abstract

Bacteria associated with free-living amoebae have attracted considerable attention because of their role in human disease and as models for studying endosymbiosis. However, the identification and analysis of such novel associations are hindered by the limitations of methods for isolation and axenization of amoebae. Here, we replaced the heat-inactivated Escherichia coli, which is typically used as food source during axenization, with a live E. coli tolC knockout mutant strain hypersensitive to antibiotics. Together with the addition of otherwise sublethal amounts of ampicillin, this approach tripled the success rate and reduced the time required for axenization by at least 3 days. Using this method for two environmental samples, 10 Acanthamoeba strains were isolated, seven of which contained bacterial symbionts. In three cases, amoebae harbouring two phylogenetically distinct symbionts were recovered, supporting a more widespread occurrence of multi-partner symbiotic associations among free-living amoebae.

Chlamydial seasonal dynamics and isolation of ‘Candidatus Neptunochlamydia vexilliferae’ from a Tyrrhenian coastal lake

“…Protist-sized particles present in coastal lake water (Lago di Paola, Italy) were counted with a Neubauer counting chamber. Between 1 and 10, protist-sized particles were placed in wells on a 96-well plate (Corning Costar, Sigma-Aldrich, Germany) containing 200 μl artificial sea water (ASW, DSMZ 607) with Escherichia coli tolC serving as food (Lagkouvardos et al, 2014a). Amoebae propagating on these plates were screened for the presence of bacterial endosymbionts with FISH, upscaled and maintained in cell culture flasks (Nunclon delta-surface, Thermoscientifc, Germany) containing ASW and E. coli tolC.…”

Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont

“…Protochlamydia amoebophila (hereafter referred to as Protochlamydia ; a member of the family Parachlamydiaceae ) is a well-studied symbiont (29–31). Of key importance, Protochlamydia in its natural Acanthamoeba host represents an in vivo infection model to study the dynamics of host-microbe interactions, and the model does not rely on the use of immortalized cell lines.…”

‘Candidatus Cochliophilus cryoturris’ (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus

“…Our attempts to establish an axenic culture using various media and a hypersensitive E. coli mutant 38 failed, and the amoeba was thus maintained routinely on agar plates. Morphological analyses, along with molecular identification based on 18S rRNA gene sequencing, confirmed the classification of this isolate as Cochliopodium minus , a testate amoeba found in diverse marine and fresh-water environments 31–35 .…”

“…Clonal cultures were maintained by weekly sub-culturing and morphological identification of the amoeba was accomplished by inverted phase contrast and bright field microscopy (Nikon Eclipse E800) using the identification keys of Page and Smirnov 53, 54 . Amoebae thriving on the agar surface were repeatedly transferred to fresh agar plates containing E. coli JW5503-1 ΔtolC732::kan as food source to facilitate axenization as described 38, 55 . Amoeba were routinely maintained on agar plates covered with E. coli .…”