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Diversity of bacterial endosymbionts of environmental acanthamoeba isolates - PubMed

Diversity of bacterial endosymbionts of environmental acanthamoeba isolates

Stephan Schmitz-Esser et al. Appl Environ Microbiol. 2008 Sep.

Abstract

Free-living amoebae are frequent hosts for bacterial endosymbionts. In this study, the symbionts of eight novel environmental Acanthamoeba strains isolated from different locations worldwide were characterized. Phylogenetic analysis revealed that they were related to one of four evolutionary lineages of amoeba symbionts recognized previously. This study provides evidence for the existence of only a small number of phylogenetically well-separated groups of obligate intracellular endosymbionts of acanthamoebae with global distribution.

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Figures

FIG. 1.
FIG. 1.

Phylogenetic relationships of Acanthamoeba host cells. An 18S rRNA-based TREE-PUZZLE tree (HKY nucleotide substitution model) (52) is shown. A filter considering only positions which are conserved in at least 50% of all amoebal 18S rRNA sequences was used for tree calculations. Selected Acanthamoeba 18S rRNA sequence types (51) are indicated. Black dots represent nodes with TREE-PUZZLE support and PHYLIP maximum parsimony bootstrap values (1.000 resampling) (18) greater than 80%. GenBank accession numbers are given in parentheses. The arrow indicates toward the out-group. The bar at the bottom represents 10% of the estimated evolutionary distance.

FIG. 2.
FIG. 2.

Phylogenetic relationships of Acanthamoeba symbionts. 16S rRNA-based trees calculated using the TREE-PUZZLE algorithm (HKY nucleotide substitution model) (52) are shown for the proteobacterial symbionts (A), the Bacteroidetes symbionts (B) and the chlamydial symbionts (C). A filter considering only positions which are conserved in at least 50% of all Bacteria strains was used for tree calculations. Black dots represent nodes with TREE-PUZZLE support and PHYLIP maximum parsimony bootstrap values (1.000 resampling) (18) greater than 80%. GenBank accession numbers are given in parentheses. Arrows indicate toward the out-groups. The bar at the bottom of the figure represents 10% of the estimated evolutionary distance.

FIG. 3.
FIG. 3.

Identification and intracellular localization of Acanthamoeba symbionts by FISH. Probes EUK516 labeled with Cy5 (and shown in blue), targeting most Eukarya, and EUB-Mix labeled with Fluos dye (green), targeting most Bacteria strains, were used in all experiments in combination with Cy3-labeled symbiont-specific probes (red) (Table 2); the combined signal from bacterial and symbiont-specific probes appears yellow. At least three independent experiments were performed and ≥100 individual Acanthamoeba host cells were examined, all of which were infected; representative confocal laser scanning micrographs are shown. (A) Parachlamydia sp. isolate EI1 in Acanthamoeba sp. isolate EI1 (probe Bn9-658). (B) Protochlamydia sp. isolate EI2 in Acanthamoeba sp. isolate EI2 (probe Bn9-658). (C) “Candidatus Paracaedibacter” EI3 in Acanthamoeba sp. isolate EI3 (probe Cc23a). (D) “Candidatus Amoebophilus” EI4 in Acanthamoeba sp. isolate EI4 (probe Aph1180). (E) “Candidatus Procabacter” EI5 in Acanthamoeba sp. isolate EI5 (probe Proca438). (F) Parachlamydia EI6 in Acanthamoeba sp. isolate EI6 (probe Bn9-658). (G) “Candidatus Amoebophilus” EIDS3 in Acanthamoeba sp. isolate EIDS3 (probe Aph1180). (H) “Candidatus Amoebophilus” 5a2 in Acanthamoeba sp. isolate 5a2 (probe Aph1180). The white bars in the bottom right corner of each panel represent 10 μm.

FIG. 4.
FIG. 4.

Ultrastructure of symbionts within Acanthamoeba host cells. Representatives from each phylogenetic group of symbionts are shown. (A) Parachlamydia sp. isolate EI1. Elementary (black arrowhead) and reticulate (white arrowhead) bodies within the chlamydial inclusion can be seen. (B) “Candidatus Amoebophilus” EI4. (C) “Candidatus Paracaedibacter” EI3. An electron-translucent space, indicative of a capsule or slime layer, surrounding “Candidatus Paracaedibacter” EI3 is clearly visible. (D) “Candidatus Procabacter” EI5 is surrounded by a membrane (black arrow). (E) Protochlamydia sp. isolate EI2. Each Protochlamydia sp. isolate EI2 cell is surrounded by an inclusion membrane. Mitochondria are labeled “m.” The lengths of bars in the bottom right corner of each panel represent 1 μm.

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