Secretome Profiling by Proteogenomic Analysis Shows Species-Specific, Temperature-Dependent, and Putative Virulence Proteins of Pythium insidiosum

Abstract

In contrast to most pathogenic oomycetes, which infect plants, Pythium insidiosum infects both humans and animals, causing a difficult-to-treat condition called pythiosis. Most patients undergo surgical removal of an affected organ, and advanced cases could be fetal. As a successful human/animal pathogen, P. insidiosum must tolerate body temperature and develop some strategies to survive and cause pathology within hosts. One of the general pathogen strategies is virulence factor secretion. Here, we used proteogenomic analysis to profile and validate the secretome of P. insidiosum, in which its genome contains 14,962 predicted proteins. Shotgun LC–MS/MS analysis of P. insidiosum proteins prepared from liquid cultures incubated at 25 and 37 °C mapped 2980 genome-predicted proteins, 9.4% of which had a predicted signal peptide. P. insidiosum might employ an alternative secretory pathway, as 90.6% of the validated secretory/extracellular proteins lacked the signal peptide. A comparison of 20 oomycete genomes showed 69 P. insidiosum–specific secretory/extracellular proteins, and these may be responsible for the host-specific infection. The differential expression analysis revealed 14 markedly upregulated proteins (particularly cyclophilin and elicitin) at body temperature which could contribute to pathogen fitness and thermotolerance. Our search through a microbial virulence database matched 518 secretory/extracellular proteins, such as urease and chaperones (including heat shock proteins), that might play roles in P. insidiosum virulence. In conclusion, the identification of the secretome promoted a better understanding of P. insidiosum biology and pathogenesis. Cyclophilin, elicitin, chaperone, and urease are top-listed secreted/extracellular proteins with putative pathogenicity properties. Such advances could lead to developing measures for the efficient detection and treatment of pythiosis.

PacBio long read-assembled draft genome of Pythium insidiosum strain Pi-S isolated from a Thai patient with pythiosis

“…This study aims to obtain a better-quality genome of P. insidiosum using the PacBio long-read. The P. insidiosum strain Pi-S was selected because it has been widely referred to in recent immunological, genomic, transcriptomic, and proteomic studies [8, [14][15][16][17][18][19][20]. The short-read version of the strain Pi-S genome data shows 53.2 Mb in size and comprises 1,192 contigs (average length: 44,664 bases; N 50 : 146,252 bases), 52% G-C content, 10% N composition, and 14,962 open reading frames (ORFs) [8].…”