Neochlamydia hartmannellae gen. nov., sp nov (Parachlamydiaceae), an endoparasite of the amoeba Hartmannella vermiformis

Free-living amoebae are increasingly being recognized to serve as vehicles of dispersal for various bacterial human pathogens and as hosts for a varie ty of obligate bacterial endocytobionts. Several Chlamydia-like Acanthamoeb a endocytobionts constituting the recently proposed family Parachlamydiacea e are of special interest as potential human pathogens. In this study cocco id bacterial endocytobionts of a Hartmannella vermiformis isolate were anal ysed. Infection of ii. vermiformis with these bacteria resulted in preventi on of cyst formation and subsequent host-cell lysis. Transfection experimen ts demonstrated that the parasites were not capable of propagating within o ther closely related free-living amoebae but were able to infect the distan tly related species Dictyostelium discoideum. Electron microscopy of the pa rasites revealed typical morphological characteristics of the Chlamydiales, including the existence of a Chlamydia-like life-cycle, but indicated that these endocytobionts, in contrast to Chlamydia species, do not reside with in a vacuole. Comparative 16S rRNA sequence analysis showed that the endocy tobiont of ii. vermiformis, classified as Neochlamydia hartmannellae gen. n ov., sp. nov., is affiliated to the family Parachlamydiaceae. Confocal lase r scanning microscopy in combination with fluorescence in situ hybridizatio n using rRNA-targeted oligonucleotide probes confirmed the intracellular lo calization of the parasites and demonstrated the absence of other bacterial species within the Hartmannella host. These findings extend our knowledge of the phylogenetic diversity of the Parachlamydiaceae and demonstrate for the first time that these endocytobionts can naturally develop within amoeb ae of the genus Hartmannella.