Telomere-to-Telomere genome assemblies of human-infecting Encephalitozoon species

Abstract

Background Microsporidia are diverse spore forming, fungal-related obligate intracellular pathogens infecting a wide range of hosts. This diversity is reflected at the genome level with sizes varying by an order of magnitude, ranging from less than 3 Mb in Encephalitozoon species (the smallest known in eukaryotes) to more than 50 Mb in Edhazardia spp. As a paradigm of genome reduction in eukaryotes, the small Encephalitozoon genomes have attracted much attention with investigations revealing gene dense, repeat- and intron-poor genomes characterized by a thorough pruning of molecular functions no longer relevant to their obligate intracellular lifestyle. However, because no Encephalitozoon genome has been sequenced from telomere-to-telomere and since no methylation data is available for these species, our understanding of their overall genetic and epigenetic architectures is incomplete. Methods In this study, we sequenced the complete genomes from telomere-to-telomere of three human-infecting Encephalitozoon spp. —E. intestinalis ATCC 50506, E. hellem ATCC 50604 and E. cuniculi ATCC 50602— using short and long read platforms and leveraged the data generated as part of the sequencing process to investigate the presence of epigenetic markers in these genomes. We also used a mixture of sequence- and structure-based computational approaches, including protein structure prediction, to help identify which Encephalitozoon proteins are involved in telomere maintenance, epigenetic regulation, and heterochromatin formation. Results The Encephalitozoon chromosomes were found capped by TTAGG 5-mer telomeric repeats followed by telomere associated repeat elements (TAREs) flanking hypermethylated ribosomal RNA (rRNA) gene loci featuring 5-methylcytosines (5mC) and 5-hemimethylcytosines (5hmC), themselves followed by lesser methylated subtelomeres and hypomethylated chromosome cores. Strong nucleotide biases were identified between the telomeres/subtelomeres and chromosome cores with significant changes in GC/AT, GT/AC and GA/CT contents. The presence of several genes coding for proteins essential to telomere maintenance, epigenetic regulation, and heterochromatin formation was further confirmed in the Encephalitozoon genomes. Conclusion Altogether, our results strongly support the subtelomeres as sites of heterochromatin formation in Encephalitozoon genomes and further suggest that these species might shutdown their energy-consuming ribosomal machinery while dormant as spores by silencing of the rRNA genes using both 5mC/5hmC methylation and facultative heterochromatin formation at these loci.

Near chromosome–level genome assembly of the microsporidium Hamiltosporidium tvaerminnensis

“…2021 ). The structural completeness of our assembly was assessed using scripts from Mascarenhas dos Santos et al . (2023) , which allow for the identification of telomeres.…”

“…The highest quality genomes within the microsporidia are from the genus Encephalitozoon , which have an order of magnitude smaller genomes than H . tvaerminnensis and telomeric repeats of (TTAGG) n on both ends of all eleven chromosomes ( Mascarenhas dos Santos et al . 2023 ).…”

“…2018 ) and manual curation. The absence of contaminants in this final assembly was ascertained by BLAST homology searches against the NCBI database, and its completeness was assessed with check_for_telomeres.pl v.0.3 ( Mascarenhas dos Santos et al . 2023 ).…”

“…2022 ). Following recent successful efforts to obtain telomere-to-telomere assemblies in Encephalitozoon ( Mascarenhas dos Santos et al . 2023 ), a genus of microsporidia with small genomes, we focus here on a large-genome species, Hamiltosporidium tvaerminnensis .…”

SYNY: a pipeline to investigate and visualize collinearity between genomes

“…Microsporidia from the genus Encephalitozoon are human-infecting pathogens causing chronic diarrhea, bronchitis, conjunctivitis and/or encephalitis in afflicted patients [9]. With tiny yet complete nuclear genomes totaling less than 3 Mbp, these obligate intracellular pathogens constitute paradigms of genome streamlining in eukaryotes [10]. Their closest known relatives from the genus Ordospora , whose members infect crustaceans but not humans, also arbor diminutive genomes [11].…”