Pins Gene Table v2.0: An Online Genome Database of 37 Pythium insidiosum Strains for Gene Content Exploration and Phylogenomic Analysis.

Unlike most pathogenic oomycetes, Pythium insidiosum infects humans and animals instead of plants. P. insidiosum has three clinically relevant genotypes/clades that cause a severe disease called pythiosis. To develop strategies for infection control, it is necessary to understand the biology and pathogenesis of this pathogen. Investigating the evolutionary mechanisms behind the host-specific adaptation is vital, and comparative genomic analysis can help with this. To facilitate genomic analysis, an online bioinformatics tool called P. insidiosum (Pins) Gene Table v2.0 was developed. This tool includes genomic data from 37 genetically diverse P. insidiosum strains and four related species. The database contains 732,686 genes, grouped into 80,061 unique clusters and further divided into core and variable categories at genus, species, and genotype levels. A high-resolution phylogenomic relationship among P. insidiosum strains and other oomycetes was projected through hierarchical clustering and core gene analyses. 3156 P. insidiosum-specific genes were shared among all genotypes and may be responsible for causing disease in humans and animals. After comparing these species-specific genes to the MvirDB database, 112 had significant matches with 66 known virulence proteins, some of which might be involved in vascular occlusion, which is a pathological feature of pythiosis. The correlation of genotypes, geographic origins, and affected hosts of P. insidiosum suggests that clade-I strains are more specific to animals, while clade-II/III strains are more specific to humans. The clade-specific genes might link to host preference. In summary, Pins Gene Table v2.0 is a comprehensive genome database accessible to users with minimal bioinformatics experience for the analysis of P. insidiosum genomes.

Potential anti-Pythium insidiosum therapeutics identified through screening of agricultural fungicides.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Clinical manifestations of pythiosis include an eye, blood vessel, skin, or gastrointestinal tract infection. Pythiosis has been increasingly reported worldwide, with an overall mortality rate of 28%. Radical surgery is required to save patients' lives due to the limited efficacy of antimicrobial drugs. Effective medical treatments are urgently needed for pythiosis. This study aims to find anti-P. insidiosum agents by screening 17 agricultural fungicides that inhibit plant-pathogenic oomycetes and validating their efficacy and safety. Cyazofamid outperformed other fungicides as it can potently inhibit genetically diverse P. insidiosum isolates while exhibiting minimal cellular toxicities. The calculated therapeutic scores determined that the concentration of cyazofamid causing significant cellular toxicities was eight times greater than the concentration of the drug effectively inhibiting P. insidiosum. Furthermore, other studies showed that cyazofamid exhibits low-to-moderate toxicities in animals. The mechanism of cyazofamid action is likely the inhibition of cytochrome b, an essential component in ATP synthesis. Molecular docking and dynamic analyses depicted a stable binding of cyazofamid to the Qi site of the P. insidiosum's cytochrome b orthologous protein. In conclusion, our search for an effective anti-P. insidiosum drug indicated that cyazofamid is a promising candidate for treating pythiosis. With its high efficacy and low toxicity, cyazofamid is a potential chemical for treating pythiosis, reducing the need for radical surgeries, and improving recovery rates. Our findings could pave the way for the development of new and effective treatments for pythiosis.IMPORTANCEPythiosis is a severe infection caused by Pythium insidiosum. The disease is prevalent in tropical/subtropical regions. This infectious condition is challenging to treat with antifungal drugs and often requires surgical removal of the infected tissue. Pythiosis can be fatal if not treated promptly. There is a need for a new treatment that effectively inhibits P. insidiosum. This study screened 17 agricultural fungicides that target plant-pathogenic oomycetes and found that cyazofamid was the most potent in inhibiting P. insidiosum. Cyazofamid showed low toxicity to mammalian cells and high affinity to the P. insidiosum's cytochrome b, which is involved in energy production. Cyazofamid could be a promising candidate for the treatment of pythiosis, as it could reduce the need for surgery and improve the survival rate of patients. This study provides valuable insights into the biology and drug susceptibility of P. insidiosum and opens new avenues for developing effective therapies for pythiosis.

MGI short-read genome assemblies of Pythium insidiosum (reclassified as Pythium periculosum) strains Pi057C3 and Pi050C3.

OBJECTIVES: Pythium insidiosum causes a difficult-to-treat infectious condition called pythiosis, with high morbidity and mortality. So far, genome data of at least 10 strains of P. insidiosum, primarily classified in the phylogenetic clades I and II, have been sequenced using various next-generation sequencing platforms. The MGI short-read platform was employed to obtain genome data of 2 clade-III strains of P. insidiosum (recently reclassified as Pythium periculosum) from patients in Thailand and the United States. This work is a part of our attempt to generate a comprehensive genome database from diverse pathogen strains. DATA DESCRIPTION: A 150-bp paired-end library was prepared from a gDNA sample of P. insidiosum (P. periculosum) strains Pi057C3 and Pi050C3 (also known as ATCC90586) to generate draft genome sequences using an MGISEQ-2000RS sequencer. As a result, for the strain Pi057C3, we obtained a 42.5-Mb assembled genome (164x coverage) comprising 14,134 contigs, L50 of 241, N50 of 45,748, 57.6% CG content, and 12,147 ORFs. For the strain Pi050C3, we received a 43.3-Mb draft genome (230x coverage) containing 14,511 contigs, L50 of 245, N50 of 45,208, 57.7% CG content, and 12,249 ORFs. The genome sequences have been deposited in the NCBI/DDBJ databases under the accession numbers JAKCXM000000000.1 (strain Pi057C3) and JAKCXL000000000.1 (strain Pi050C3).

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

OBJECTIVES: Pythium insidiosum is the causative agent of pythiosis, a difficult-to-treat condition, in humans and animals worldwide. Biological information about this filamentous microorganism is sparse. Genomes of several P. insidiosum strains were sequenced using the Illumina short-read NGS platform, producing incomplete genome sequence data. PacBio long-read platform was employed to obtain a better-quality genome of Pythium insidiosum. The obtained genome data could promote basic research on the pathogen's biology and pathogenicity. DATA DESCRIPTION: gDNA sample was extracted from the P. insidiosum strain Pi-S for whole-genome sequencing by PacBio long-read NGS platform. Raw reads were assembled using CANU (v2.1), polished using ARROW (SMRT link version 5.0.1), aligned with the original raw PacBio reads using pbmm2 (v1.2.1), consensus sequence checked using ARROW, and gene predicted using Funannotate pipeline (v1.7.4). The genome completion was assessed using BUSCO (v4.0.2). As a result, 840 contigs (maximum length: 1.3 Mb; N50: 229.9 Kb; L50: 70) were obtained. Sequence assembly showed a genome size of 66.7 Mb (178x coverage; 57.2% G-C content) that contained 20,375 ORFs. A BUSCO-based assessment revealed 85.5% genome completion. All assembled contig sequences have been deposited in the NCBI database under the accession numbers BBXB02000001 - BBXB02000840.

Generation of protoplasts provides a powerful experimental research tool for biological and pathogenicity studies of Pythium insidiosum.

INTRODUCTION: Pythiosis is a high-mortality infectious condition in humans and animals. The etiologic agent is Pythium insidiosum. Patients present with an ocular, vascular, cutaneous/subcutaneous, or gastrointestinal infection. Antifungal medication often fails to fight against P. insidiosum. The effective treatment is limited to radical surgery, resulting in organ loss. Fatal outcomes are observed in advanced cases. Pythiosis needs to be studied to discover novel methods for disease control. Genome data of P. insidiosum is publicly available. However, information on P. insidiosum biology and pathogenicity is still limited due to the lack of a cost-effective animal model and molecular tools. MATERIALS AND METHODS: We aimed to develop a high-efficiency protocol for generating P. insidiosum protoplast, and used it to set up an animal model, in vitro drug susceptibility assay, and DNA transformation for this pathogen. RESULTS: P. insidiosum protoplast was successfully generated to establish a feasible pythiosis model in embryonic chicken eggs and an efficient in vitro drug susceptibility assay. DNA transformation is a critical method for gene manipulation necessary for functional genetic studies in pathogens. Attempts to establish a DNA transformation method for P. insidiosum using protoplast were partly successful. Significant work needs to be done for genetically engineering a more robust selection marker to generate stable transformants at increased efficiency. CONCLUSION: This study is the first to report an efficient P. insidiosum protoplast production for clinical and research applications. Such advances are crucial to speeding up the pathogen's biology and pathogenicity exploration.

Comparative Genomic Analysis Reveals Gene Content Diversity, Phylogenomic Contour, Putative Virulence Determinants, and Potential Diagnostic Markers within Pythium insidiosum Traits.

Pythium insidiosum has successfully evolved into a human/animal filamentous pathogen, causing pythiosis, a life-threatening disease, worldwide. The specific rDNA-based genotype of P. insidiosum (clade I, II, or III) is associated with the different hosts and disease prevalence. Genome evolution of P. insidiosum can be driven by point mutations, pass vertically to the offspring, and diverge into distinct lineages, leading to different virulence, including the ability to be unrecognized by the host. We conducted comprehensive genomic comparisons of 10 P. insidiosum strains and 5 related Pythium species using our online "Gene Table" software to investigate the pathogen's evolutionary history and pathogenicity. In total, 245,378 genes were found in all 15 genomes and grouped into 45,801 homologous gene clusters. Gene contents among P. insidiosum strains varied by as much as 23%. Our results showed a strong agreement between the phylogenetic analysis of 166 core genes (88,017 bp) identified across all genomes and the hierarchical clustering analysis of gene presence/absence profiles, suggesting divergence of P. insidiosum into two groups, clade I/II and clade III strains, and the subsequent segregation of clade I and clade II. A stringent gene content comparison using the Pythium Gene Table provided 3263 core genes exclusively presented in all P. insidiosum strains but no other Pythium species, which could involve host-specific pathogenesis and serve as biomarkers for diagnostic purposes. More studies focusing on characterizing the biological function of the core genes (including the just-identified putative virulence genes encoding hemagglutinin/adhesin and reticulocyte-binding protein) are needed to explore the biology and pathogenicity of this pathogen.

Selection of an Appropriate In Vitro Susceptibility Test for Assessing Anti-Pythium insidiosum Activity of Potassium Iodide, Triamcinolone Acetonide, Dimethyl Sulfoxide, and Ethanol.

The orphan but highly virulent pathogen Pythium insidiosum causes pythiosis in humans and animals. Surgery is a primary treatment aiming to cure but trading off losing affected organs. Antimicrobial drugs show limited efficacy in treating pythiosis. Alternative drugs effective against the pathogen are needed. In-house drug susceptibility tests (i.e., broth dilution, disc diffusion, and radial growth assays) have been established, some of which adapted the standard protocols (i.e., CLSI M38-A2 and CLSI M51) designed for fungi. Hyphal plug, hyphal suspension, and zoospore are inocula commonly used in the drug susceptibility assessment for P. insidiosum. A side-by-side comparison demonstrated that each method had advantages and limitations. Minimum inhibitory and cidal concentrations of a drug varied depending on the selected method. Material availability, user experience, and organism and drug quantities determined which susceptibility assay should be used. We employed the hyphal plug and a combination of broth dilution and radial growth methods to screen and validate the anti-P. insidiosum activities of several previously reported chemicals, including potassium iodide, triamcinolone acetonide, dimethyl sulfoxide, and ethanol, in which data on their anti-P. insidiosum efficacy are limited. We tested each chemical against 29 genetically diverse isolates of P. insidiosum. These chemicals possessed direct antimicrobial effects on the growth of the pathogen in a dose- and time-dependent manner, suggesting their potential application in pythiosis treatment. Future attempts should focus on standardizing these drug susceptibility methods, such as determining susceptibility/resistant breakpoints, so healthcare workers can confidently interpret a result and select an effective drug against P. insidiosum.

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

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.

Severe skin and soft tissue pythiosis acquired in a hot spring in the southwestern United States, a case report and review of North American cases.

BACKGROUND: Human pythiosis, caused primarily by the aquatic oomycete, Pythium insidiosum, is an emerging but uncommon infection in North America. The infection is frequently life-threatening and is often initially unrecognized due to its rarity and similar presentation to certain fungal infections. METHODS: We report a case of skin and soft tissue pythiosis in a patient without significant underlying comorbidities acquired in a New Mexico hot spring and review its successful treatment. We also review all reported pythiosis cases in North America. RESULTS: Eleven confirmed cases of human pythiosis acquired in North America were identified. The majority of cases occurred in children (64%), ten of eleven cases were acquired in the southern U.S., Mexico, Central America or the Caribbean and four of the eleven individuals succumbed to the infection. CONCLUSIONS: With recognition and aggressive surgical and medical treatment good clinical outcomes can be achieved when treating human pythiosis.

Global Distribution and Clinical Features of Pythiosis in Humans and Animals.

Pythiosis is a difficult-to-treat infectious disease caused by Pythium insidiosum. The condition is unfamiliar among healthcare workers. Manifestation of pythiosis is similar to other fungal infections, leading to misdiagnosis and delayed treatment. The geographical extent of pythiosis at a global scale is unclear. This study aimed to analyze the clinical information recorded in the scientific literature to comprehensively project epidemiological characteristics, clinical features, and future trends of pythiosis. From 1980 to 2021, 4203 cases of pythiosis in humans (n = 771; 18.3%) and animals (primarily horse, dog, and cow; n = 3432; 81.7%), with an average of 103 cases/year, were recruited. Pythiosis case reports significantly increased in the last decade. Pythiosis spanned 23 tropical, subtropical, and temperate countries worldwide. Some patients acquired pythiosis from a trip to an endemic country. Strikingly, 94.3% of human cases were in India and Thailand, while 79.2% of affected animals were in the U.S.A. and Brazil. Clinical features of pythiosis varied. Vascular and ocular pythiosis were only observed in humans, whereas cutaneous/subcutaneous and gastrointestinal infections were predominant in animals. Mortality depended on host species and clinical forms: for example, none in patients with ocular pythiosis, 0.7% in cows with a cutaneous lesion, 26.8% in humans with vascular disease, 86.4% in dogs with gastrointestinal pathology, and 100% in several animals with disseminated infection. In summary, this study reports up-to-date epidemiological and clinical features of pythiosis in humans and animals. It increases awareness of this life-threatening disease, as the illness or outbreak can exist in any country, not limited to the endemic areas.

Nucleic Acid-Based Detection of Pythium insidiosum: A Systematic Review.

Pythiosis, a life-threatening infectious condition caused by Pythium insidiosum, has been increasingly reported in humans and animals worldwide. Antifungal drugs usually fail to control the pathogen. The surgical removal of an infected organ is the treatment of choice. Many affected patients die due to advanced infection. A timely and accurate diagnosis could lead to a better prognosis in pythiosis patients and save their lives. Although a standard culture method is available in microbiological laboratories, it is time-consuming, laborious, and insensitive for P. insidiosum identification. Immunological assays have been developed to improve the diagnosis of pythiosis. However, immunological methods are commercially unavailable and primarily detect anti-P. insidiosum antibodies, which constitute indirect evidence of pythiosis, making it challenging to differentiate a past from a recent infection. Moreover, such immunological tests cannot diagnose patients with a local infection, such as in the eye. Nucleic acid-based tests (NATs) are efficient for the direct and rapid detection of P. insidiosum DNA in trace-amount or culture-negative specimens. The reagents and equipment required for NATs are usually available in molecular diagnostic laboratories. Herein, we provide a systematic review to comprehensively present the principal and clinical usages, advantages, and limitations of such NATs in the detection of P. insidiosum. Various NATs have been established to detect P. insidiosum, which can be classified into amplification-based (i.e., PCR assays, isothermal tests, and next-generation sequencing methods) and non-amplification-based (i.e., DNA hybridization) techniques. This concise review on NATs constitutes an up-to-date reference with which healthcare professionals can learn about and decide upon which detection method is suitable for their respective laboratory environments.

History and Perspective of Immunotherapy for Pythiosis.

The fungus-like microorganism Pythium insidiosum causes pythiosis, a life-threatening infectious disease increasingly reported worldwide. Antimicrobial drugs are ineffective. Radical surgery is an essential treatment. Pythiosis can resume post-surgically. Immunotherapy using P. insidiosum antigens (PIA) has emerged as an alternative treatment. This review aims at providing up-to-date information of the immunotherapeutic PIA, with the focus on its history, preparation, clinical application, outcome, mechanism, and recent advances, in order to promote the proper use and future development of this treatment modality. P. insidiosum crude extract is the primary source of immunotherapeutic antigens. Based on 967 documented human and animal (mainly horses) pythiosis cases, PIA immunotherapy reduced disease morbidity and mortality. Concerning clinical outcomes, 19.4% of PIA-immunized human patients succumbed to vascular pythiosis instead of 41.0% in unimmunized cases. PIA immunotherapy may not provide an advantage in a local P. insidiosum infection of the eye. Both PIA-immunized and unimmunized horses with pythiosis showed a similar survival rate of ~70%; however, demands for surgical intervention were much lesser in the immunized cases (22.8% vs. 75.2%). The proposed PIA action involves switching the non-protective T-helper-2 to protective T-helper-1 mediated immunity. By exploring the available P. insidiosum genome data, synthetic peptides, recombinant proteins, and nucleic acids are potential sources of the immunotherapeutic antigens worth investigating. The PIA therapeutic property needs improvement for a better prognosis of pythiosis patients.

Immunological Cross-Reactivity of Proteins Extracted from the Oomycete Pythium insidiosum and the Fungus Basidiobolus ranarum Compromises the Detection Specificity of Immunodiagnostic Assays for Pythiosis.

Pythiosis, a life-threatening disease caused by Pythium insidiosum, has been increasingly diagnosed worldwide. A recently developed immunochromatographic test (ICT) enables the rapid diagnosis of pythiosis. During the 3-year clinical implementation of ICT in Thailand, we collected the laboratory reports of 38 animals with suspected pythiosis and detected ICT false-positive results in three horses and a dog with basidiobolomycosis. P. insidiosum and Basidiobolus ranarum cause infections with indistinguishable clinical and microscopic features. This study investigated cross-reactive antibodies by probing P. insidiosum and B. ranarum crude extracts and cell-free synthesized I06 protein (encoded in P. insidiosum genome, not other fungi) against a panel of pythiosis, basidiobolomycosis, rabbit anti-I06 peptide, and control sera by Western blot analyses. ICT false-positive results occurred from the cross-reactivity of anti-B. ranarum antibodies to the 15, 50, 60, and 120 kDa proteins of P. insidiosum, not double infections caused by both pathogens. Notably, ICT could help to screen pythiosis, and the positive test requires confirmation by culture or molecular method. The detection specificity of ICT requires improvement. The crude extract containing multispecies antigens needs replacement with a refined P. insidiosum-specific protein. We proposed that the 55 kDa I06 protein is an excellent candidate for developing a more specific serodiagnostic test for pythiosis.

Prospecting Biomarkers for Diagnostic and Therapeutic Approaches in Pythiosis.

Pythiosis, whose etiological agent is the oomycete Pythium insidiosum, is a life-threatening disease that occurs mainly in tropical and subtropical countries, affecting several animal species. It is frequently found in horses in Brazil and humans in Thailand. The disease is difficult to diagnose because the pathogen's hyphae are often misdiagnosed as mucoromycete fungi in histological sections. Additionally, there is no specific antigen to use for rapid diagnosis, the availability of which could improve the prognosis in different animal species. In this scenario, we investigated which P. insidiosum antigens are recognized by circulating antibodies in horses and humans with pythiosis from Brazil and Thailand, respectively, using 2D immunoblotting followed by mass spectrometry for the identification of antigens. We identified 23 protein spots, 14 recognized by pooled serum from horses and humans. Seven antigens were commonly recognized by both species, such as the heat-shock cognate 70 KDa protein, the heat-shock 70 KDa protein, glucan 1,3-beta-glucosidase, fructose-bisphosphate aldolase, serine/threonine-protein phosphatase, aconitate hydratase, and 14-3-3 protein epsilon. These results demonstrate that there are common antigens recognized by the immune responses of horses and humans, and these antigens may be studied as biomarkers for improving diagnosis and treatment.

Genome data of four Pythium insidiosum strains from the phylogenetically-distinct clades I, II, and III.

OBJECTIVES: We employed the Illumina NGS platform to sequence genomes of 4 different strains of the pathogenic oomycete Pythium insidiosum, the causative agent of pythiosis. These strains were isolated from humans in Thailand (n = 3) and the United States (n = 1), and phylogenetically classified into clade-I, -II, and -III. Our study augmented the completeness of the P. insidiosum genome database for exploration of the biology, evolution, and pathogenesis of the pathogen. DATA DESCRIPTION: One paired-end library (180-bp insert) was prepared from a gDNA sample of P. insidiosum strains ATCC200269 (clade-I), Pi19 (clade-II), MCC18 (clade-II), and SIMI4763 (clade-III) for whole-genome sequencing by Illumina HiSeq2000/HiSeq2500 NGS platform. A range of 28.4-59.4 million raw reads, accounted for 3.0-7.3 Gb, were obtained and assembled into the genome sizes of 47.1 Mb (15,153 contigs; 85% completeness; 19,329 open reading frames [ORFs]) for strain ATCC200269, 35.4 Mb (14,576 contigs; 83% completeness; 13,895 ORFs) for strain Pi19, 34.5 Mb (11,084 contigs; 84% completeness; 13,249 ORFs) for strain MCC18, and 47.1 Mb (15,162 contigs; 85% completeness; 19,340 ORFs) for strain SIMI4763. The genome data can be downloaded from the NCBI/DDBJ databases under the accessions BCFN00000000.1 (ATCC200269), BCFS00000000.1 (Pi19), BCFT00000000.1 (MCC18), and BCFU00000000.1 (SIMI4763).

Identification and Biotyping of Pythium insidiosum Isolated from Urban and Rural Areas of Thailand by Multiplex PCR, DNA Barcode, and Proteomic Analyses.

Pythium insidiosum causes pythiosis, a fatal infectious disease of humans and animals worldwide. Prompt diagnosis and treatment are essential to improve the clinical outcome of pythiosis. Diagnosis of P. insidiosum relies on immunological, molecular, and proteomic assays. The main treatment of pythiosis aims to surgically remove all affected tissue to prevent recurrent infection. Due to the marked increase in case reports, pythiosis has become a public health concern. Thailand is an endemic area of human pythiosis. To obtain a complete picture of how the pathogen circulates in the environment, we surveyed the presence of P. insidiosum in urban (Bangkok) and rural areas of Thailand. We employed the hair-baiting technique to screen for P. insidiosum in 500 water samples. Twenty-seven culture-positive samples were identified as P. insidiosum by multiplex PCR, multi-DNA barcode (rDNA, cox1, cox2), and mass spectrometric analyses. These environmental strains of P. insidiosum fell into Clade-II and -III genotypes and exhibited a close phylogenetic/proteomic relationship with Thai clinical strains. Biodiversity of the environmental strains also existed in a local habitat. In conclusion, P. insidiosum is widespread in Thailand. A better understanding of the ecological niche of P. insidiosum could lead to the effective prevention and control of this pathogen.

Loop-mediated Isothermal Amplification (LAMP) for Identification of Pythium insidiosum.

OBJECTIVE: Pythium insidiosum causes a life-threatening condition called pythiosis. High morbidity and mortality of pythiosis are consequences of delayed diagnosis. We aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of P. insidiosum for use in remote areas, where pythiosis is prevalent. METHODS: We designed four LAMP primers to amplify the rDNA sequence. A side-by-side comparison evaluated performances of LAMP and the previously-established multiplex PCR (M-PCR), using gDNA samples extracted from colonies of P. insidiosum (n = 28) and other fungi (n = 54), and tissues of animals with (n = 16) or without (n = 13) pythiosis. RESULTS: LAMP demonstrated a 50% shorter assay duration (1.5 h) and a 10-fold lower limit of detection (10-4 ng) than did M-PCR. Based on colony-extracted gDNAs, LAMP and M-PCR correctly reported P. insidiosum in all 28 samples, providing 100% sensitivity. While M-PCR did not amplify all fungal controls (100% specificity), LAMP falsely detected one organism (98% specificity). Based on the clinical samples, LAMP and M-PCR provided an equivalently-high specificity (100%). However, LAMP showed a markedly-higher sensitivity than that of M-PCR (88% vs. 56%). CONCLUSIONS: LAMP is a simple, useful, efficient assay for the detection of P. insidiosum in clinical specimens and pure cultures in resource-limited laboratories.

Draft genome sequence of the oomycete Pythium destruens strain ATCC 64221 from a horse with pythiosis in Australia.

OBJECTIVES: Genome sequences are a vital resource for accelerating the biological exploration of an organism of interest. Pythium destruens (a synonym of Pythium insidiosum) causes a difficult-to-treat infectious disease called pythiosis worldwide. Detection and management of pythiosis are challenging. Basic knowledge of the disease is lacking. Genomes of this organism isolated from different continents (i.e., Asia and the Americas) have been sequenced and publicly available. Here, we sequenced the genome of an Australian isolate of P. destruens. Genome data will facilitate the comparative analysis of this and related species at the molecular level. DATA DESCRIPTION: Genomic DNA of the P. destruens strain ATCC 64221, isolated from a horse with pythiosis in Australia, was used to prepare one paired-end library (with 180-bp insert) for next-generation sequencing, using the Illumina HiSeq 2500 short-read platform. Raw reads were cleaned and assembled by several bioinformatics tools. A total of 20,860,454 processed reads, accounted for 2,614,890,553 total bases, can be assembled into a 37.8-Mb genome, consisting 13,060 contigs (average length: 2896 bases; range: 300-142,967), N50 of 11,370 bases, and 2.9% 'N' composition. The genome was determined 85.9% completeness, contained 14,424 predicted genes, and can be retrieved online at the NCBI/DDBJ databases under the accession number BCFQ01000000.1.

Expression, purification, and characterization of the recombinant exo-1,3-ß-glucanase (Exo1) of the pathogenic oomycete Pythium insidiosum.

Pythiosis is a deadly infectious disease of humans and animals living in tropical and subtropical countries. The causative agent is the oomycete Pythium insidiosum. Treatment of pythiosis is challenging. The use of antimicrobial agents usually fails in the treatment of pythiosis. Many patients undergo surgical removal of an infected organ (i.e., eye, arm, and leg). The immunotherapeutic vaccine, prepared from the crude extract of P. insidiosum, shows limited efficacy against pythiosis. The fatal outcome occurs in patients with advanced disease. There are urgent needs for an effective therapeutic modality for pythiosis. Recently, the exo-1,3-ß-glucanase (Exo1) has been identified as a conserve immunoreactive protein of P. insidiosum. Exo1 was predicted to reside at the cell membrane and hydrolyze cell wall ß-glucan during cell growth. An Exo1 ortholog is absent in the human genome, making it an appealing target for drug or vaccine development. We attempted to clone and express the codon-optimized exo1 gene of P. insidiosum in E. coli. To solve the inclusion body formation, expression and purification of Exo1 were achievable in the denaturing condition using SDS- and urea-based buffers. Exo1 lacked hydrolytic activity due to the absence of proper protein folding and post-translational modifications. ELISA and Western blot analyses demonstrated the immunoreactivity of Exo1 against pythiosis sera. In conclusion, we successfully expressed and purified the immunoreactive Exo1 protein of P. insidiosum. The recombinant Exo1 can be produced at an unlimited amount and could serve as an extra protein to enhance the effectiveness of the current form of the vaccine against pythiosis.

Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete Pythium insidiosum.

Protein production relies on time-consuming genetic engineering and in vivo expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of in vivo protein biosynthesis by processing in vitro transcription and translation of multiple genes to proteins within hours. We employed an automated CFPS to simultaneously synthesize proteins from 24 genes of the oomycete Pythium insidiosum (which causes the life-threatening disease pythiosis) and screen for a diagnostic and therapeutic target. CFPS successfully synthesized 18 proteins (∼75% success rate). One protein, namely, I06, was explicitly recognized by all pythiosis sera, but not control sera, tested. Py. insidiosum secreted a significant amount of I06. The protein architecture of I06 is compatible with the oligopeptide elicitor (OPEL) of the phylogenetically related plant-pathogenic oomycete Phytophthora parasitica The OPEL-like I06 protein of Py. insidiosum can stimulate host antibody responses, similar to the P. parasitica OPEL that triggers plant defense mechanisms. OPEL-like I06 homologs are present only in the oomycetes. Py. insidiosum contains two OPEL-like I06 homologs, but only one of the two homologs was expressed during hyphal growth. Twenty-nine homologs derived from 15 oomycetes can be phylogenetically divided into two groups. The OPEL-like genes might occur in the common ancestor, before independently undergoing gene gain and loss during the oomycete speciation. In conclusion, CFPS offers a fast in vitro protein synthesis. CFPS simultaneously generated multiple proteins of Py. insidiosum and facilitated the identification of the secretory OPEL-like I06 protein, a potential target for the development of a control measure against the pathogen.IMPORTANCE Technical limitations of conventional biotechnological methods (i.e., genetic engineering and protein synthesis) prevent extensive functional studies of the massive amounts of genetic information available today. We employed a cell-free protein synthesis system to rapidly and simultaneously generate multiple proteins from genetic codes of the oomycete Pythium insidiosum, which causes the life-threatening disease called pythiosis, in humans and animals worldwide. We aimed to screen for potential diagnostic and therapeutic protein targets of this pathogen. Eighteen proteins were synthesized. Of the 18 proteins, one was a secreted immunoreactive protein, called I06, that triggered host immunity and was recognized explicitly by all tested sera from pythiosis patients. It is one of the OPEL proteins; these proteins are present only in the unique group of microorganisms called oomycetes. Here, we demonstrated that cell-free protein synthesis was useful for the production of multiple proteins to facilitate functional studies and identify a potential target for diagnosis and treatment of pythiosis.