Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

Abstract

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans and Holospora obtusa. With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus, of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as 'Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWC9 as 'Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp. strain UWE39 as 'Candidatus Paracaedibacter symbiosus'. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit <92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.

Acanthamoeba T4 and T15 genotypes associated with keratitis infections in Italy

“…The analysis of the 18S rDNA proved to be an effective tool for describing dissimilarities in the genus, thus identifying 12 distinct sequence types (T1-T12) [3,9]. Three more sequence types, T13 [4], T14 [5] and T15 [6], so far not related to a precise pathogenic picture, have been further described.…”

“…The taxonomy of the genus is mainly based on morphological features such as size and cyst morphology [2], and only recently the application of molecular tools, in particular sequencing of 18S rRNA genes, has allowed the classification of Acanthamoeba into 15 genotype groups designated as T1-T15 [3][4][5][6][7]. Amoebic keratitis is caused by a wide variety of Acanthamoeba spp., most of which belonging to the T4 rDNA complex [3].…”

Phylogenetic evidence for a new genotype of Acanthamoeba (Amoebozoa, Acanthamoebida)

“…By analysing more than 50 strains from the three morphogroups, they identified 12 sequence types or genotypes, named T1 to T12, where each genotype should correspond to natural species or species complex (Gast et al 1996;Stothard et al 1998). This approach has been largely adopted, and three new genotypes have been established, called T13 to T15 (Horn et al 1999;Gast 2001;Hewett et al 2003). …”

Morphological, physiological, molecular and phylogenetic characterization of new environmental isolates of Acanthamoeba spp. from the region of Bratislava, Slovakia

“…Sequencing the complete 18S rDNA, ranging between 2,300-2,700 base pairs (bp), provided sufficient data for reliable determination of the relationships among particular genera and toward understanding their phylogenesis (Gast et al 1996;Stothard et al 1998). Research carried out to date has revealed that the new Acanthamoeba classification system based on 18S rDNA consists of 15 different sequence types: T1-T12 (Stothard et al 1998), T13 (Horn et al 1999), T14 (Gast 2001), and T15 (Hewett et al 2003).…”