The Characterization of Circular Extrachromosomal rDNA Elements in Naegleria SPP
ABSTRACT Naegleria are unicellular, eukaryotic amoebas characterized by their ability to shift between multiple life stages. These organisms are ubiquitous in nature and found in bodies of freshwater and soil. The Naegleria life stages include: the trophozoite stage, a growth and replication stage characterized by pseudopodic movement, the cyst stage, a hibernation stage characterized a double-walled membrane, and the flagellate stage, a mobility stage that is characterized by the presence of flagella. Naegleria are predominantly non-pathogenic except for Naegleria fowleri, which is the only species of Naegleria to cause disease in humans. Organisms of the Naegleria genus are further characterized by a circular extrachromosomal rDNA element (CERE) that is localized to the nucleolus. The CERE is unique because it contains the genes for rDNA production, which are not found in the chromosome. Naegleria species contain thousands of copies of the CERE which can replicate autonomously and independently of the chromosome. To date, the full-length CERE sequences were only known for different strains of N. gruberi, N. fowleri, and N. lovaniensis. We investigated the CERE further by studying N. gruberi and other Naegleria species that represent varying levels of phylogenetic relatedness to N. gruberi: N. australiensis, N. jadini, and N. pringsheimi. Long-read sequencing of these CERE demonstrated a unique sequence composition for each species, predominantly within the non-ribosomal sequence (NRS). Sequencing revealed sizes ranging from 11.8 kbp in N. jadini up to 16.1 kbp in N. pringsheimi. Analysis of the full-length CERE indicate potential shifts in phylogenetic relatedness between species as well as the presence of predicted structures such as G-quadruplexes that are both unique and conserved across the genus. We further explored the molecular dynamics of the N. gruberi CERE by transfecting cells with the molecular clone, pGRUB. Transfection with pGRUB demonstrated transient propagation across several culture passages, encystment and excystment. N. gruberi trophozoites transfected with constructs that either have or do not have the putative ori demonstrated diminished propagation, indicating other components of the NRS may be necessary for replication. The objectives of this research were to provide further understanding of the Naegleria CERE and to significantly contribute to the Naegleria sequence database. The data from these results will function as a basis study the characteristics of N. fowleri and non-pathogenic Naegleria.
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