Molecular cloning and expression analysis of peptidase genes in the fish-pathogenic scuticociliate Miamiensis avidus

Abstract

Background: Parasite peptidases have been actively studied as vaccine candidates or drug targets for prevention or treatment of parasitic diseases because of their important roles for survival and/or invasion in the host. Like other parasites, the facultative histophagous ciliate Miamiensis avidus would possess peptidases that are closely associated with the invasion into the host tissue and survival in the host.

Results: The 17 genes encoding peptidases, including seven cathepsin-like cysteine peptidases, four serine carboxypeptidases, a eukaryotic aspartyl protease family protein, an ATP-dependent metalloprotease FtsH family protein, three leishmanolysin family proteins and a peptidase family M49 protein were identified from a Miamiensis avidus cDNA library by BLAST X search. Expression of genes encoding two cysteine peptidases, three leishmanolysin-like peptidases and a peptidase family M49 protein was up-regulated in the cell-fed ciliates compared to the starved ciliates. Especially, one cysteine peptidase (MaPro 4) and one leishmanolysin-like peptidase (MaPro 14) were transcribed more than 100-folds in the cell-fed ciliates.

Conclusions: The genetic information and transcriptional characteristics of the peptidases in the present results would be helpful to elucidate the role of peptidases in the invasion of scuticociliates into their hosts.

Figure 1

Multiple alignment of the deduced amino acid sequences of Miamiensis avidus cysteine peptidases. The deduced amino acid sequences of Miamiensis avidus cysteine peptidases MaPro 1 to - MaPro 7 were aligned with Papain (the type peptidase of C1A superfamily, GenBank: AAB02650), Uronema marinum cathepsin L-like protein (UmCatL, GenBank: AAX51228), Homo sapiens cathepsin B protein (HsCatB, GenBank: AAH10240) and Uronema marinum cathepsin B protein (UmCatB, GenBank: AAR19103). The signal peptide, I29 (Inhibitor 29)/ Propeptide, Peptidase C1A domains are boxed. Conserved signatures of cathepsin L family proteins (ERFNIN and GNFD) with slight modifications in I-29 peptide of the cathepsin L-like proteins (MaPro 1- MaPro 5) are highlighted in bold, italic, grey shaded and indicated by ERFNIN and GNFD above the alignment. The catalytic triad residues (C, H and N) are marked in bold, grey shaded and indicated by sharp (#). Conserved proline residues at position 2 of the mature proteins are indicated by asterisk (*) and cysteine residues forming disulphide bonds are in bold, grey shaded and indicated by C above the alignment. S₂ subsite determining enzyme substrate specificity is indicated by a black vertical arrow above the alignment. The predicted ‘occluding loop’, which is the specific feature of cathepsin B-like peptidases, is presented in only MaPro 6 and is indicated with a black thick underline.

Figure 2

Multiple sequence alignment of peptidase M8 domains of the Miamiensis avidus leishmanolysin family proteins. The peptidase M8 domains of M. avidus leishmanolysin family proteins MaPro 14 – MaPro 16 were aligned with the previously deposited protozoan sequences in protein databases. The zinc-binding signature, HEXXH motif conserved in many kinds of metallopeptidases is shown in bold and in square box, and cysteine residues are presented in bold. The GenBank accession numbers of aligned leishmanolysin family proteins of Ichthyophtirius multifiliis, Leishmania donovani and Tetrahymena thermophila are EGR30431, XP_001024633 and AAA29244, respectively.