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

Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as ‘Candidatus Cochliophilus cryoturris’. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.

Here we report on the first characterization of a bacterial symbiont found in the testate amoeba Cochliopodium. The rod-shaped, Gram-negative bacterial symbiont replicates in host-derived vacuoles within its amoeba host cell and represents a distinct, yet uncharacterized lineage within the family Coxiellaceae (Gammaproteobacteria, Legionellales).

Results and Discussion
Cochliopodium minus from a cooling tower water sample. During a cooling tower water screening study 37 , an amoeba was isolated that could be readily propagated on non-nutrient agar plates coated with E. coli. 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][32][33][34][35] . Highest 18S rRNA sequence similarity (>99%) was observed with Cochliopodium sp. F-117 (ATCC ® 30936 ™ ) and various Cochliopodium minus strains. Characteristic for members of the genus, the trophozoites of this new isolate are covered with a dorsal monolayer of scale-like structures, the so called tectum (Fig. 1A,E,I,J). Lightmicrographs show the thin, scaled-covered hyaloplasmic sheet surrounding the granuloplasm, as well as the presence of subpseudopodiae (Fig. 1A,B). Contractile vacuoles of various stages, able to undergo fusion, are present in the cytoplasm (Fig. 1B,C). Occasionally, we could observe various encystation stages, including rounded trophozoites, the beginning of the encystation process (Fig. 1D). Transmission electron microscopy demonstrated that arrangement and fine structure of the scales are consistent with those described for other Cochliopodium minus isolates 39 (Fig. 1I,J). We thus refer to the novel isolate as C. minus strain 9B. It is interesting to note that another C. minus isolate was previously reported to contain bacterial symbionts, which could not be further characterized at the time 40 .
Intracellular bacteria in Cochliopodium minus 9B Staining of C. minus 9B trophozoites with the DNA dye DAPI readily revealed small, rod-shaped bacteria within the amoeba cytoplasm that differed in fluorescence intensity, quantity and size from E. coli cells, which were primarily observed outside of the trophozoites (data not shown). The presence of bacteria other than E. coli was further confirmed by fluorescence in situ hybridization (FISH), and 16S rRNA gene sequencing recovered a sequence with highest similarity to members of the bacterial order Legionellales (Gammaproteobacteria). We designed an oligonucleotide probe for the specific detection of this sequence; its application in FISH together with general bacterial and eukaryotic probes demonstrated unambiguously the presence of bacterial endosymbionts in C. minus (Fig. 2). All analyzed trophozoites were infected, and the bacteria were always located in the amoeba cytoplasm, being notably absent in the nucleus 19,41 . The number of symbionts per amoeba cell varied and ranged from a few to over 100. The infection did not compromise the host's capability to encyst as described for some other symbionts 16,42 , nor did we observe pronounced lysis of the amoeba at room temperature or at 28 °C. Over a period of two years, the amoebae remained infected, demonstrating that this symbiont-amoeba relationship is a stable long-term association. Failed attempts at host-free cultivation in diverse nutrient-rich and complex media under oxic and micro-oxic conditions indicate that the symbiont is dependent on its amoeba host and should therefore be considered obligate intracellular.
A novel clade of endosymbionts in the Coxiellaceae. The near full length 16S rRNA sequence (1,506 bp) of the C. minus 9B symbiont showed highest sequence similarity to a clone sequence from a soil sample (91%; accession number GQ263960.1). The most similar cultivated representative was Coxiella burnetii RSA 331 (CP000890.1), with only moderate sequence similarity (86%). Phylogenetic analysis confirmed that the symbiont is affiliated with the order Legionellales, in which it forms a well-supported monophyletic group together with a number of uncultured microbes predominantly from diverse marine environments (Fig. 3). This yet uncharacterized group represents a sister clade of the Rickettsiella/Diplorickettsiella/Aquicella group, three genera in the family Coxiellaceae. Notably, the bacterial symbiont of C. minus 9B is not closely affiliated with any known amoeba endosymbiont. However, its moderate relationship with members of the Legionellales is intriguing, as this order comprises a number of bacterial taxa associated with eukaryotes, including human and animal pathogens as well as parasites of amoebae (Fig. 3).
Members of the genera Rickettsiella and Diplorickettsia are parasites and symbionts of arthropods, including insects, crustaceans, and arachnids 43 . Aquicella species were first isolated from borehole and spa water samples and later shown to be able to thrive in co-culture with Vermamoeba vermiformis 44 . The genus Coxiella currently includes a single recognized species, C. burnetii, with numerous pathovars; these obligate intracellular bacteria are associated with insects and can cause severe infections in humans (aka Q fever) 45 ; they might also be able to thrive in Acanthamoeba castellanii 10 . Berkiella species have recently been identified as intranuclear symbionts of Acanthamoeba polyphaga 41 . Furthermore, the Legionellaceae comprise a large number of facultative intracellular bacteria able to infect protists and animals including humans 46,47 .
Taking into account current thresholds for the delineation of bacterial genera and families 48 , the C. minus symbiont identified in this study represents a novel genus. We thus propose to tentatively classify this microbe as Cochliophilus cryoturris PDD8 (Cochliophilus, pertaining to the obligate intracellular lifestyle in its native host Cochliopodium minus; cryoturris, pertaining to the origin of the water sample, a cooling tower, from which the amoeba host was isolated). Currently, the novel genus is placed within the Coxiellaceae, although we noted that the sequence similarity of C. cryoturris and its relatives to other members of the Coxiellaceae is below the commonly used family level threshold of 86.5% (Fig. 3) 48 . C. cryoturris is currently the sole isolated representative of this novel genus; whether the other uncultured members of this clade are also naturally associated with protists is still unclear.   Vacuolar location of C. cryoturris. The appearance and host-associated intracellular lifestyle of Cochliophilus cryoturris PDD8 is reminiscent of that of many of its relatives in the Legionellales. C. cryoturris cells are small and show a short rod-shaped morphology, measuring 0.5 ± 0.1 µm in width and around 1 ± 0.2 µm in length; they show a Gram-negative type cell envelope and frequently a condensed, electron-dense central region in the cytoplasm (Fig. 1F,G). The symbionts are not located directly within the amoeba cytoplasm but in membrane-bound compartments (Fig. 1E,F). These clearly differ from the food vacuoles containing (degraded) E. coli cells, which can also be seen in the amoeba cytoplasm (Fig. 1E,F). Mitochondria are frequently located in the vicinity of the symbiont-containing vacuoles (Fig. 1E-H).
Bacterial strategies for escaping phagolysosomal degradation differ. Coxiella burnetii, the closest cultured relative of C. cryoturris, is able to resist the harsh conditions after fusion of the phagosome with lysosomes and modifies the phagolysosome to interact with the autophagic pathway, promoting metabolic activity and replication 49,50 . Legionella pneumophila takes an alternative route and prevents lysosomal fusion to establish a heavily modified vacuolar compartment resembling endoplasmic reticulum (ER) membranes 51,52 . How C. cryoturris establishes its intracellular niche is currently unknown. We were, however, unable to infect different Acanthamoeba species, which are otherwise permissive to an array of phylogenetically diverse intracellular bacteria. This suggests that C. cryoturris is well adapted to infection of its natural Cochliopodium host but might have a limited host range.
In conclusion, we discovered and identified the first naturally occurring Cochliopodium endosymbiont together with its amoeba host. The symbiont is a representative of a hitherto uncharacterized clade of microbes found in diverse aquatic environments and related to other intracellular bacteria in the family Coxiellaceae. Together this indicates that relationships between free-living amoebae and bacterial symbionts are more widespread than currently recognized.

Material and Methods
Amoeba isolation and cultivation. A water sample was collected from the tank-bottom of a cooling tower and stored at 4 °C. 250 µl were filtered onto a cellulose-nitrate filter (Sartorius Lab Instruments GmbH & Co. KG, Göttingen, Germany; pore-size 0.2 µm). The filter was cut into two pieces, which were transferred onto non-nutrient agar plates (PAS; 0.12 g l −1 NaCl, 0.004 g l −1 MgSO 4 *7 H 2 O, 0.004 g l −1 CaCl 2 *2H 2 O, 0.142 g l −1 Na 2 HPO 4 , 0.136 g l −1 KH 2 PO 4 , 1.5 g l −1 agar) coated with Escherichia coli and stored at room temperature. Detected amoebae were cloned by daily serial sub-culturing of single cells onto fresh E. coli-coated agar plates using a sterile inoculation loop. 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. Fresh bacteria suspended in PAS were added to the agar plate once per week, and once per month an agar piece was transferred to a new plate.
To facilitate microscopic and molecular analysis, an agar piece containing amoebae was transferred to a culture flask (Nunclon delta-surface, Thermo Scientific, St. Leon-Rot, Germany) containing 6 ml PAS. Amoebae were allowed to attach for 12 h and washed twice with PAS to remove E. coli. Amoeba cells were collected for further analysis by detaching through vigorous shaking.

Infection of Acanthamoeba.
Amoebae were harvested from cell culture flasks, and the cell suspension was transferred to a Dounce tissue grinder (Sigma-Aldrich Handels GmbH, Vienna, Austria) using the tight pestle for 15 times to break up the cells and release the endosymbionts. The suspension was filtered (Macherey-Nagel, Düren, Germany; pore size 5 µm) twice to remove remaining intact amoebae. The symbiont suspension was added to cultures of Acanthamoeba castellanii Neff (ATCC 30010), Acanthamoeba sp. 5a2 18 , and Acanthamoeba sp. UWC12 56 . The outcome of the infection progress was monitored by fluorescence in situ hybridization.
Transmission electron microscopy. Amoebae were fixed (2.5% glutaraldehyde in 3 mM cacodylate buffer containing 0.1 M sucrose, pH 6.5) in culture flasks for one hour, detached using a cell-scraper, and concentrated by centrifugation (2900 rcf, 6 min). The pelleted cells were washed with 0.1 M cacodylate-sucrose buffer (pH 7.2-7.4) for three times and then resuspended in 40 µl 1% agarose (Low melting point agarose; Promega, Mannheim, Deutschland). The agar pellet was solidified on ice for 45 min and then cut into smaller pieces with 1 mm thickness, which were fixed in 1% OsO 4 for 1 h and dehydrated in an increasing ethanol series. Agar blocks were embedded in Low Viscosity resin (Agar Scientific ® ) and polymerized for 48 h at 60 °C. Ultrathin sections placed on Formvar ® coated slot grids were stained with 0.5% uranyl acetate and 3% lead citrate prior to imaging with a Zeiss ® Libra 120 transmission electron microscope.
In order to sequence the bacterial 16S rRNA gene, amoebae were collected from an E. coli-depleted culture flask, and 2 ml of the suspension were transferred to a 2 ml microcentrifuge tube. DNA extraction of the amoeba culture was carried out using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Amplification of bacterial 16S rRNA genes was performed using primers 616F (5′-AGAGTTTGATYMTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′) at 52 °C annealing temperature and 35 cycles 66,67 . PCR reactions contained 100 ng template DNA, 50 pmol/µl of each primer, 1 unit of Taq DNA polymerase (Fermentas, St. Leon-Rot, Germany), 10x Taq buffer with KCl and 2 mM MgCl 2 , and 0.2 mM of each deoxynucleotide in a total volume of 50 µl. PCR products were purified using the PCR Purification Kit (Qiagen) and subsequently cloned using the TOPO XL Cloning Kit (Invitrogen, Darmstadt, Germany) per manufacturer's recommendations. Sanger sequencing of four clones was performed by Microsynth Austria.
Phylogenetic analysis. The obtained 16S rRNA gene sequence was subjected to sequence homology search against the nr/nt database using the BLASTn service available at the NCBI website 68 . The top ten high scoring sequences with a minimum length of 1,400 nt were downloaded, and phylogenetic analysis was performed together with a selection of related taxa retrieved from the SILVA ribosomal RNA gene database 69 . The SINA aligner 70 with standard settings and variability set to "Bacteria" was used for sequence alignment. The alignment was trimmed at both ends to only include positions covered in all sequences. Pairwise sequence similarity was calculated using ARB 57 . Phylogenetic trees were calculated using PhyloBayes 71 with the CAT model 72 and GTR exchange rates. Ten independent chains were calculated with 210 generations each. For the final converged tree, all 10 chains were taken into account whereas the first 20 generations trees were removed. iTOL v3 73 was used to edit and label the tree. Data availability. DNA sequences determined in this study were deposited at Genbank/ENA/DDBJ under accession numbers KU215597 (18S rRNA gene sequence of C. minus 9B) and LT716083 (16S rRNA gene sequence of 'Candidatus Cochliophilus cryoturris' PDD8).