Abstract
BackgroundProteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated.ResultsBLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively.ConclusionsThe identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.
Highlights
Proteases produced by many microorganisms, including oomycetes, are crucial for their growth and development
The results of this study suggest that the secreted proteases may be considered as potential virulence factors of A. invadans in epizootic ulcerative syndrome (EUS)
We investigated A. invadans’ secreted proteases using SDS-PAGE and mass spectrometery followed by BLASTp analysis. 30 peptides belonging to eight major protein domains have been identified (Table 1)
Summary
Proteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. Epizootic ulcerative syndrome is one of the most destructive fish diseases known It is caused by the oomycete Aphanomyces invadans and leads to mass mortali‐ ties of cultured and wild fish in many countries. Extracellular proteases produced by this oomycete are believed to trigger EUS patho‐ genesis in fish To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. Infection with Aphanomyces invadans, known as epizootic ulcerative syndrome (EUS), is an OIE-listed disease [1]. It has historically caused mass mortalities in cultured and wild fish in Asia and Oceania and recently in North America and Africa. The mitochondrial genomes of A. invadans and Aphanomyces astaci were assembled and annotated, which provides good basis for further functional studies and development of diagnostic methods [14]
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