In vitro investigations on extracellular proteins secreted by Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome

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.

Editing the genome of Aphanomyces invadans using CRISPR/Cas9

“…Targeting a serine type protease of A. invadans [ 20 ], gRNA candidates were suggested by the gRNA design tool and further filtered for secondary structure and off-targets. Based on their high total and efficiency scores and zero off-target hits, three gRNA candidates were selected (Table 2 ), synthesized and used for the formation of 3 separate RNP complexes.…”

“…Production of extracellular proteins from A. invadans has been reported by Majeed et al [ 20 ]. These proteins mainly consist of proteases that are believed to be the major virulence factors that contribute to EUS infection.…”

“…Similar to many pathogenic microorganisms, proteases secreted by A. invadans are key in the pathogenesis of EUS [ 19 ]. Different extracellular proteins, most of which are proteases, secreted by A. invadans , have recently been identified and at least one of these proteases (serine protease belonging to peptidase_S8 domain) is believed to be involved in the degradation of the fish immunoglobulin M [ 20 ]. Serine proteases have long been thought of as the virulence factors in many of the true bacteria and fungi [ 21 ].…”

Transcriptome Analysis Based on RNA-Seq in Understanding Pathogenic Mechanisms of Diseases and the Immune System of Fish: A Comprehensive Review

“…These two oomycetes are responsible for significant economic losses in the aquaculture industry [ 121 ]. A. invadans is known to cause the epizootic ulcerative syndrome across fish species and could possibly invade the host by means of some extracellular proteases [ 122 ]. Similarly, S. parasitica causes the saprolegniasis disease, a major threat to the salmonid farming industry.…”

Infectious diseases in aquarium ornamental pet fish

“…Infection with Aphanomyces invadans, also known as epizootic ulcerative syndrome (EUS), is an OIE-listed disease. It has historically caused mass mortality in cultured and wild fish in Asia and Oceania and recently in North America and Africa (Afzali et al, 2015;Majeed et al, 2017;Iberahim et al, 2018; The World Organisation for Animal Health, 2017).…”