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.

Zinc binding proteome of a phytopathogen Xanthomonas translucens pv. undulosa

“…Prior studies provide evidence that Clp ATPase causes cell proteolysis and plays central roles in virulence, gene expression, stress response and antimicrobial resistance [123]. Also, the role of Zn metallopeptidase M41 was previously listed in proteolysis and virulence [124].…”

Biochemical changes and drug residues in ascidian Halocynthia roretzi after formalin–hydrogen peroxide treatment regimen designed against soft tunic syndrome

“…Highly active protease enzymes are produced and excreted from A. hoyamushi cells (Jang et al 2012). Although the disease spreads very rapidly, safe and effective measures have not been established to control the spread of STS in farms.…”

Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome¿affected ascidian Halocynthia roretzi using real-time polymerase chain reaction

“…Due to low fiber density, the tunics become thin and soft and finally rupture leading to leakage of internal tissues and death [6][7][8][9]. Breakdown of the tunics is mediated by a metalloprotease enzyme secreted by A. hoyamushi [10].…”