Facultative Associations between Protists and Rickettsial Symbionts: Morphological and Molecular Characterization Functional Implications, and Insights on Emerging Intracellular Parasites

Rickettsiae and Rickettsia-like organisms (RLOs) are Gram-negative prokaryotes known as obligate intracellular parasites of a variety of eukaryotic hosts, including humans. Recent studies reported the presence of these organisms also in protists, although no specific researches were accomplished. This work was aimed to investigate the diffusion of RLOs among protists, and to get insights about their host range and host shift capabilities. The first goal was achieved by systematical screenings of natural populations of ciliated protists from different habitats trough the “full-cycle rRNA approach” (individuation of symbionts through FISH, 16S rDNA characterization and use of specifically designed oligonucleotide probes for in situ detection). Descriptions of symbionts were refined through electron microscopy techniques and phylogenetic analysis. Six novel species of RLOs were identified in five different ciliates, namely Pseudomicrothorax dubius (Nassophorea), Spirostomum minus (Heterotrichea), Euplotes octocarinatus (Spirotrichea), Paramecium multimicronucleatum (Oligohymenophorea) and Diophrys oligothrix (Spirotrichea), which harbours two different symbionts. Phylogenetic inferences supported their belonging to the family Rickettsiaceae as candidate novel genera, except for symbionts of S. minus and P. multimicronucleatum that branches within the genus Rickettsia. These latter also share motile flagella, which were never reported among Rickettsiaceae. Analysis revealed the belonging of symbionts of E. octocarinatus and of one of that of D. oligothrix to the same novel species, which was further retrieved in three unrelated ciliate hosts. The finding accounted for possible host shifts through horizontal transfer. Both inter- and intraspecific transmissibility were tested using the other symbiont of D. oligotrix as infector and uninfected conspecific and allospecific (E. harpa) strains as hosts. Experiments evidenced its capability to undergo horizontal host shifts at both levels. Preliminary results indicate that some RLOs (symbionts of P. multimicronucleatum and one of that of D. oligothrix) can survive in association with lab-cultured metazoan cell lines. The present work contributed to reveal an unexpected, intriguing phylogenetic and morphological diversity among ciliate-borne Rickettsiaceae. The frequency of occurrence of symbioses between these latter and protists, together with the retrieval of the same RLO species from different hosts and the capability of horizontal shift (in case of symbionts of D. oligothrix) account for their polyxenic and likely opportunistic nature. Given the parasitic/pathogenic nature of the majority of known Rickettsiaceae, ciliated protists could effectively play the role of both natural reservoir and vectors for potentially hazardous rickettsial pathogens.