title: RNA-seq-based metatranscriptomic and microscopic investigation reveals novel metalloproteases of Neobodo sp. as potential virulence factors for soft tunic syndrome in Halocynthia roretzi.
abstract: Bodonids and trypanosomatids are derived from a common ancestor with the bodonids being a more primitive lineage. The Neobodonida, one of the three clades of bodonids, can be free-living, commensal or parasitic. Despite the ecological and evolutionary significance of these organisms, however, many of their biological and pathological features are currently unknown. Here, we employed metatranscriptomics using RNA-seq technology combined with field-emission microscopy to reveal the virulence factors of a recently described genus of Neobodonida that is considered to be responsible for ascidian soft tunic syndrome (AsSTS), but whose pathogenesis is unclear. Our microscopic observation of infected tunic tissues suggested putative virulence factors, enabling us to extract novel candidate transcripts; these included cysteine proteases of the families C1 and C2, serine proteases of S51 and S9 families, and metalloproteases grouped into families M1, M3, M8, M14, M16, M17, M24, M41, and M49. Protease activity/inhibition assays and the estimation of expression levels within gene clusters allowed us to identify metalloprotease-like enzymes as potential virulence attributes for AsSTS. Furthermore, a multimarker-based phylogenetic analysis using 1,184 concatenated amino acid sequences clarified the order Neobodo sp. In sum, we herein used metatranscriptomics to elucidate the in situ expression profiles of uncharacterized putative transcripts of Neobodo sp., combined these results with microscopic observation to select candidate genes relevant to pathogenesis, and used empirical screening to define important virulence factors.
authors.affiliation: Aquatic Biotechnology Center of WCU Project, College of Veterinary Medicine, Gyeongsang National University, Jinju, South Korea.
authors.forename: Ho Bin
authors.initials: HB
authors.lastname: Jang
authors.forename: Young Kyu
authors.initials: YK
authors.lastname: Kim
authors.forename: Carmelo S
authors.initials: CS
authors.lastname: Del Castillo
authors.forename: Seong Won
authors.initials: SW
authors.lastname: Nho
authors.forename: In Seok
authors.initials: IS
authors.lastname: Cha
authors.forename: Seong Bin
authors.initials: SB
authors.lastname: Park
authors.forename: Mi Ae
authors.initials: MA
authors.lastname: Ha
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Hikima
authors.forename: Sung Jong
authors.initials: SJ
authors.lastname: Hong
authors.forename: Takashi
authors.initials: T
authors.lastname: Aoki
authors.forename: Tae Sung
authors.initials: TS
authors.lastname: Jung
mesh_terms: D000818:Animals; D056986:Euglenozoa Infections / Q000469:parasitology*; D005407:Flagella / Q000201:enzymology / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D020869:Gene Expression Profiling; D016830:Kinetoplastida / Q000201:enzymology / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure*; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D058977:Molecular Sequence Annotation; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D016053:RNA, Protozoan / Q000235:genetics; D017423:Sequence Analysis, RNA; D013045:Species Specificity; D014561:Urochordata / Q000469:parasitology*; D037521:Virulence Factors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D011480:Protease Inhibitors; D016053:RNA, Protozoan; D037521:Virulence Factors; D045726:Metalloproteases