Functional and immunological characterization of an elicitin-like protein of the pathogenic oomycete Pythium insidiosum

Abstract

Pythiosis is a life-threatening infectious disease of humans and animal living in tropical and subtropical countries, and caused by the pathogenic oomycete Pythium insidiosum. Pythiosis in humans has been reported mostly from Thailand. Most patients usually come with clinical symptoms resulting from arterial (vascular pythiosis) or corneal (ocular pythiosis) infection. Morbidity and mortality of pythiosis are markedly high. Unfortunately, there is no effective medical treatment for pythiosis, leaving extensive surgical removal the only options. Most patients with vascular pythiosis had their infected leg amputated, and those with ocular infection had their eye removed. Many patients died from the advanced stage of the disease. Basic information on pathogenesis of this disease, which could lead to a new method of infection control, is very limited. Recently, we have generated an expressed sequence tag database (ESTdb) of P. insidiosum. Bioinformatics analysis of the ESTdb revealed a number putative virulence factors. Among them, elicitins are an interesting group of protein, due to they are unique (not present in other human pathogens), and sterol binding molecules. A recombinant elicitin-like protein#025 (rELI025) was successfully expressed in Escherichia coli, and purified using an affinity column. Biological and immunological properties of ELIs have been explored. Molecular techniques using rabbit anti-ELI antibody showed that ELI was a secreted protein. Different strains of P. insidiosum produced different amount of ELI025. Serum samples from different patients with pythiosis showed various immunoreactivity against rELI025. Based on the rDNA sequence, P. insidiosum can be grouped into three phylogenetic clades: Clade I-III. The ELI025-encoding gene from the strains in Clade III cannot be detected by PCR. In conclusion, we report here rELI025 as the first recombinant protein of P. insidiosum to be molecularly investigated, which could be a virulence factor necessary for pathogenesis, a phylogenetic marker, and a potential therapeutic target.