The Immunoreactive Exo-1,3-ß-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity.

The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called "pythiosis". Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.

Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-ß-glucanase.

Pythiosis is a life-threatening infectious disease caused by Pythium insidiosum. Early and accurate diagnosis is the key to prompt treatment and an improved prognosis for patients with pythiosis. An alternative to microbiological and immunological approaches for facilitating diagnosis of pythiosis is the PCR-based assay. Until recently, the ribosomal DNA (rDNA) region was the only target available for PCR-based detection of P. insidiosum. Failure to detect P. insidiosum by PCR amplification using the rDNA-specific primers has been reported. PinsEXO1, encoding an exo-1,3-ß-glucanase, is an alternative, novel and efficient target for identification of P. insidiosum by conventional PCR. In this study, we aimed to develop a real-time (RT)-PCR approach targeting PinsEXO1 and compare its performance with conventional PCR for the detection of P. insidiosum. Both conventional and RT-PCR assays were positive for all 35 P. insidiosum strains tested, whilst all 58 control fungi were negative. The turnaround time for conventional PCR was 10 h, whilst that for RT-PCR was 7.5 h. The lowest amounts of genomic DNA template required for successful amplification by conventional and RT-PCR were 1 and 1 × 10(-4) ng, respectively. In conclusion, the RT-PCR assay retained 100% sensitivity and 100% specificity for detection of P. insidiosum. It showed a substantially improved analytical sensitivity and turnaround time that could improve diagnosis of pythiosis. The assay could also facilitate quantitative DNA analysis and epidemiological studies of P. insidiosum.

Efficiency comparison of three methods for extracting genomic DNA of the pathogenic oomycete Pythium insidiosum.

BACKGROUND: The fungus-like organism Pythium insidiosum is the causative agent of a life-threatening tropical infectious disease, pythiosis, which has high rates of morbidity and mortality. A lack of reliable diagnostic tools and effective treatments for pythiosis presents a major challenge to healthcare professionals. Unfortunately, surgical removal of infected organs remains the default treatment for pythiosis. P. insidiosum is an understudied organism. In-depth study of the pathogen at the molecular level could lead to better means of infection control High quality genomic DNA (gDNA) is needed for molecular biology-based research and application development, such as: PCR-assisted diagnosis, population studies, phylogenetic analysis, and molecular genetics assays. OBJECTIVE: To evaluate quality and quantity of the P. insidiosum gDNA extracted by three separate protocols intended for fungal gDNA preparation. MATERIAL AND METHOD: Seven P. insidiosum isolates were subjected to gDNA extraction by using conventional-extraction, rapid-extraction, and salt-extraction protocols. RESULTS: The conventional protocol offered the best gDNA in terms of quality and quantity, and could be scaled up. The rapid-extraction protocol had a short turnaround time, but the quality and quantity of the gDNA obtained were limited. The salt-extraction protocol was simple, rapid, and efficient, making it appealing for high throughput preparation of small-scale gDNA samples. CONCLUSION: Compared to rapid-extraction protocol, both conventional-extraction and salt-extraction protocols provided a better quality and quantity of gDNA, suitable for molecular studies of P. insidiosum. In contrast to the other two methods, the salt-extraction protocol does not require the use of hazardous and expensive materials such as phenol, chloroform, or liquid nitrogen.

A peptide ELISA to detect antibodies against Pythium insidiosum based on predicted antigenic determinants of exo-1,3-beta-glucanase.

Human pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Diagnosis of pythiosis relies on culture identification, serodiagnosis, and molecular-based assay. Preparation of a serodiagnostic test requires culture filtrate antigen (CFA) extracted from the live pathogen. A 74-kDa immunoreactive protein of P. insidiosum, is encoded by the exo-1,3-beta-glucanase gene (PinsEXO1). PinsEXO1 protein is recognized by sera from pythiosis patients but not by sera from uninfected patients; therefore, this protein could be used to detect anti-P. insidiosum antibodies. In this study we aimed to: identify, synthesize, and evaluate an antigenic determinant (epitope) of PinsEXO1 to be used to serodiagnose pythiosis based on peptide ELISA, and to compare the diagnostic performance of that test with the current CFA-based ELISA. Two antigenic determinants of PinsEXO1 (Peptide-A and -B) were predicted using the PREDITOP program. The sera from 34 pythiosis patients and 92 control subjects were evaluated. Peptide-A, Peptide-B, and CFA-based ELISAs all had a specificity of 100%. Peptide-B ELISA had a sensitivity of 91% and an accuracy of 98% and both Peptide-A and CFA-based ELISAs had a sensitivity of 100% and an accuracy of 100%. Peptide-A is a more efficient epitope than Peptide-B, and can be used as an alternative antigen to develop a serodiagnostic assay for pythiosis.

The 74-kilodalton immunodominant antigen of the pathogenic oomycete Pythium insidiosum is a putative exo-1,3-beta-glucanase.

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ss-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ss-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.

Evaluation of an in-house immunoperoxidase staining assay for histodiagnosis of human pythiosis.

Pythiosis, a life-threatening infectious disease of humans and animals in tropical and subtropical countries, is caused by the fungus-like organism Pythium insidiosum. As diagnosis of pythiosis is difficult, delayed diagnosis of pythiosis leads to poor prognosis. We developed an immunoperoxidase staining assay using rabbit anti-P. insidiosum antibodies to detect P. insidiosum directly in infected tissues of 19 patients with vascular (n = 11), ocular (n = 7) or cutaneous (n = 1) pythiosis. Tissue sections from 31 patients with various fungal infections were included as controls. Tissue sections from all pythiosis patients and 2 patients with Fusarium infections were stained positive, whereas the other 29 control sections were stained negative. Sensitivity and specificity of the assay was 100% and 94%, respectively. Based on the prevalence of human pythiosis (2%), calculated positive predictive value and negative predictive value was 24% and 100%, respectively. Thus, the diagnostic value of this assay is for ruling out pythiosis. The assay requires routine laboratory equipments and can easily be performed by pathologists in rural hospitals where the disease is more prevalent.