Probing the Phylogenomics and Putative Pathogenicity Genes of Pythium insidiosum by Oomycete Genome Analyses.

Pythium insidiosum is a human-pathogenic oomycete. Many patients infected with it lose organs or die. Toward the goal of developing improved treatment options, we want to understand how Py. insidiosum has evolved to become a successful human pathogen. Our approach here involved the use of comparative genomic and other analyses to identify genes with possible functions in the pathogenicity of Py. insidiosum. We generated an Oomycete Gene Table and used it to explore the genome contents and phylogenomic relationships of Py. insidiosum and 19 other oomycetes. Initial sequence analyses showed that Py. insidiosum is closely related to Pythium species that are not pathogenic to humans. Our analyses also indicated that the organism harbours secreted and adhesin-like proteins, which are absent from related species. Putative virulence proteins were identified by comparison to a set of known virulence genes. Among them is the urease Ure1, which is absent from humans and thus a potential diagnostic and therapeutic target. We used mass spectrometric data to successfully validate the expression of 30% of 14,962 predicted proteins and identify 15 body temperature (37 °C)-dependent proteins of Py. insidiosum. This work begins to unravel the determinants of pathogenicity of Py. insidiosum.

Chemically induced disseminated pythiosis in BALB/c mice: A new experimental model for Pythium insidiosum infection.

Pythiosis is a severe and life-threatening disease that affects humans and various animal species. We report a model of vascular/disseminated pythiosis occurring after subcutaneous inoculation of 2 x 104 Pythium insidiosum zoospores/mL in immunocompromised BALB/c mice. For this model, we carried out two rounds of experiments. First, we evaluated two protocols of immunosuppression before inoculation: cyclophosphamide at 150 mg/kg (CYP group) and cyclophosphamide 200 mg/kg plus hydrocortisone acetate at 250 mg/kg (CYP+HCA group). It was not possible to obtain mortality in the CYP group; however, the combination of CYP+HCA altered disease outcomes, with mortality rates reaching 60%. Second, we used the CYP+HCA immunosuppression protocol to analyze the histological and immunological statuses triggered by disease. When we inoculated immunocompetent mice with P. insidiosum zoospores, self-healing occurred via increased levels of IL-2, IFN-γ and IL-17A, which are characteristic of the Th1/Th17 cytokine response. For infected and immunosuppressed mice, the cytokine profiles showed high levels of IL-10, IL-6 and TNF-α. Increased IL-10 values are related to fungal infection susceptibility and led us to speculate that infection may be established through suppression of the host immune response. In addition, histopathological evaluation of the kidneys and liver demonstrated the presence of hyphae and the cellular findings suggested an acute vascular inflammation that mimics vascular/disseminated pythiosis in humans. This is the first murine model for pythiosis that is useful both for understanding the pathogenesis of this disease and for evaluating new treatment approaches.

Geographic variation in the elicitin-like glycoprotein, ELI025, of Pythium insidiosum isolated from human and animal subjects.

Oomycetes are fungus-like in appearance, but form a distinct clade within the eukaryotes. While most pathogenic oomycetes infect plants, the understudied oomycete Pythium insidiosum infects humans and animals, and causes a life-threatening infectious disease, called pythiosis. Phylogenetic analyses divide P. insidiosum into 3 groups, according to geographic origins: Clade-I (Americas), Clade-II (Asia and Australia), and Clade-III (Thailand). Surgical removal of the infected organ is the inevitable treatment for patients with pythiosis, but it is often too late or unsuccessful, and many patients die from advanced infection. Understanding P. insidiosum's basic biology could lead to improved infection control. Elicitins, a unique group of proteins found only in oomycetes, are involved in sterol acquisition and stimulation of host responses. Recently, we identified glycosylated and non-glycosylated forms of the elicitin-like protein, ELI025, which is secreted by P. insidiosum, and detected during P. insidiosum infection. In this study, we investigated geographic variation of ELI025 in 24 P. insidiosum strains isolated from humans, animals, and the environment. Genotypes of ELI025, based on 2 sets of PCR primers, correlated well with rDNA-based phylogenetic grouping. Unlike strains in Clade-I and -II, Clade-III strains secreted no glycosylated ELI025. Sera from 17 pythiosis patients yielded a broad range of antibody responses against ELI025, and ∼30% lacked reactivity against the protein. Selective production or secretion of glycosylated ELI025 by different P. insidiosum strains might contribute to the variable host antibody responses. In conclusion, ELI025 was secreted by all P. insidiosum strains isolated from different hosts and geographic origins, but the protein had different biochemical, and immunological characteristics. These finding contribute to the better understanding of the biology and evolution of P. insidiosum, and could lead to appropriate clinical application of the ELI025 protein for diagnosis or treatment of pythiosis.

Insights into the pathophysiology of iron metabolism in Pythium insidiosum infections.

Pythium insidiosum causes life-threatening disease in mammals. Animals with pythiosis usually develop anemia, and most human patients are reported to have thalassemia and the major consequence of thalassemia, iron overload. Therefore, this study evaluated the iron metabolism in rabbits experimentally infected with P. insidiosum. Ten infected rabbits were divided into two groups: one groups received a placebo, and the other was treated with immunotherapy. Five rabbits were used as negative controls. The hematological and biochemical parameters, including the iron profile, were evaluated. Microcytic hypochromic anemia was observed in the infected animals, and this condition was more accentuated in the untreated group. The serum iron level was decreased, whereas the transferrin level was increased, resulting in low saturation. The level of stainable iron in hepatocytes was markedly decreased in the untreated group. A high correlation was observed between the total iron binding capacity and the lesion size, and this correlation likely confirms the affinity of P. insidiosum for iron. The data from this study corroborate the previous implications of iron in the pathogenesis of pythiosis in humans and animals.

A comparative study of the histopathology and immunohistochemistry of pythiosis in horses, dogs and cattle.

Twenty-one cases of pythiosis in horses (n = 10), dogs (n = 9) and cattle (n = 2) were investigated. The aetiology in all cases was confirmed by immunohistochemistry. Data related to the clinical course and outcome and localization of the lesions were obtained from pathology reports. The equine lesions consisted of fibrotic tissue with multiple, often coalescing, areas of immature granulation tissue encircling eosinophilic cores. Affected dogs had gastrointestinal and/or cutaneous lesions with either or both of a granulomatous/pyogranulomatous or necrotizing eosinophilic inflammatory reaction. In cattle, cutaneous lesions were characterized by multifocal to coalescing granulomas with surrounding fibrosis. The number of intralesional hyphae, the distribution of hyphae, the presence of angioinvasion and the nature of the local inflammatory reactions were associated with the different types of lesions observed.