Scuticociliatosis, causing severe mortality in South Korea, has been considered one of the most serious fish diseases than any other countries
[6, 7]. Although several reports have revealed that some chemotherapeutics showed effective scuticocidal activities in vitro[25–27], finding effective chemotherapeutics is still remained to be difficult in vivo. Therefore, efficient vaccine development is urgently to prevent diseases. Several studied have attempted to develop vaccines using whole cells and have shown some positive results by obtaining good protection against scuticociliate infection following vaccination
[28–30]. However, as there are some limitations for in vitro mass and economic culture of scuticociliates for commercial use, subunit vaccines should be developed with development of cost-effective methods of antigens. Selecting suitable target antigens that can induce effective protective responses may be the most important factor to develop effective subunit vaccines. Among scuticociliate antigens, the crucial roles of peptidases in infection of host fish have been already reported by previous studies
In the present study, M. avidus were starved for at least 1 month by inoculation in the filtered seawater without any additional nutrients and cells. Similar to what occurs during the free-living stage, these starved ciliates may lose their ability to destroy fish tissue due to a reduction in protease activities they need. Quantitative RT-PCR was performed to investigate mRNA expression of the identified peptidase genes in relation with the cell-feeding parasitic stage of M. avidus. The results showed that two cysteine peptidases, a leishmanolysin-like peptidase and a peptidase family M49 protein genes were up-regulated more than 2-fold in the cell-fed ciliates than in the starved ciliates. Among them, one cysteine peptidase gene (MaPro 4) and one leishmanolysin-like peptidase gene (MaPro 14) showed 100-fold higher expression in the cell-fed ciliates. Expression of two other leishmanolysin-like peptidase genes (MaPro 15 and MaPro 16) was detected only in the cell-fed ciliates.
The differential expression of the cysteine peptidases in the cell-fed ciliates might be an important part of M. avidus pathogenesis as shown in previous studies
[8–12]. Many parasite cysteine peptidases and their important roles in pathogenesis have been well documented in a variety of parasites and inhibitors of cysteine peptidases have been developed for anti-parasitic chemotherapy, as cysteine peptidase inhibitors can selectively inhibit parasite peptidases without untoward toxicity to the host
[32–36]. In this study, we cloned seven different cysteine peptidases genes and two of them (MaPro 4 and MaPro 7) were differentially up-regulated in the cell-fed ciliates. We obtained the mRNA sequences of three leishmanolysin-like family proteins containing the metalloprotease M8 domain and found differential mRNA expression in the cell-fed ciliates. Leishmanolysin, which is also known as the gp63 protein, is a metalloprotease found in protozoan parasites including Leishmania and Trypanosoma. This protein is the most abundant cell surface protein during the promastigote stage of the parasite and is attached to the membrane by a glycosylphosphatidylinositol anchor
[37–40]. The protective effects of gp63 immunization have been recently demonstrated using various vaccine formulations by many researchers
[41, 42]. In fish pathogenic haemoflagellates Cryptobia spp., metallopeptidase activity was only found in the pathogenic strain of C. salmositica, its activity decreased significantly with long-term in vitro culture, and the purified metallopeptidases could lyse fish red blood cells
[43–45]. Moreover, metallopeptidase activities could be neutralized by either a monoclonal antibody or a natural anti-peptidase or the antibody against the DNA vaccines
Recent technological advances in whole genome sequence analyses and comparative genomic analyses have revealed that there are more than 90 peptidase homologs in a single organism such as Plasmodium falciparum, Tetrahymena thermophila and Ichthyophthirius multifiliis[49–51]. In protozoa, 254, 578, 480 and 95 peptidase genes have been identified in Ichthyophthrius multifiliis, Paramecium tetraurelia, Tetrahymena thermophile, and Plasmodium falciparum, respectively
. Like other parasites, M. avidus may express many peptidase proteins to undergo various biological processes including parasite survival and pathogenesis. In this study, we obtained 17 different peptidase genes from a M. avidus cDNA library by ESTs sequence screening, and the results of differential mRNA expression related to pathogenesis were also obtained. Based on the analysis of structurally conserved regions and motifs presented in the deduced amino acid sequences of each peptidase proteins, five cathepsin L-like cysteine peptidases, one cathepsin B-like cysteine peptidase, one cathepsin C-like cysteine peptidase, four serine carboxypeptidase, 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 although there were some structurally differences with previously reported similar proteins. Although the number of identified peptidase genes obtained from this study was relatively lower than expected, this is the first report of cloning and mRNA expression of peptidase gene homologs as important virulence factors in M. avidus. Moreover, the information of exact protein sequence obtained from this study could help to perform futher studies to develop specific inhibitors.
We are currently performing studies on the actual activities using recombinant proteins of cloned peptidase genes to understand whether these proteins are biologically active at the protein level. We will further analyze of M. avidus genome using large scale-genome analysis techniques to identify more peptidase sequences, and will perform combined research of transcriptional analysis and enzymatic activities of each peptidase proteins.