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Kittichotirat W, Patumcharoenpol P, Rujirawat T, Tangphatsornruang S, Yurayart C, Krajaejun T. Pins Gene Table v2.0: An Online Genome Database of 37 Pythium insidiosum Strains for Gene Content Exploration and Phylogenomic Analysis. J Fungi (Basel) 2024; 10:112. [PMID: 38392784 PMCID: PMC10889951 DOI: 10.3390/jof10020112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2023] [Revised: 01/12/2024] [Accepted: 01/15/2024] [Indexed: 02/24/2024] Open
Abstract
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.
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Affiliation(s)
- Weerayuth Kittichotirat
- Bioinformatics and Systems Biology Program, School of Bioresources and Technology and School of Information Technology, King Mongkut's University of Technology Thonburi, Bangkhunthian, Bangkok 10150, Thailand
- Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkhunthin, Bangkok 10150, Thailand
| | - Preecha Patumcharoenpol
- Interdisciplinary Graduate Program in Bioscience, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
| | - Thidarat Rujirawat
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 73170, Thailand
| | - Sithichoke Tangphatsornruang
- National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand
| | - Chompoonek Yurayart
- Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
| | - Theerapong Krajaejun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 73170, Thailand
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Krajaejun T, Patumcharoenpol P, Rujirawat T, Kittichotirat W, Tangphatsornruang S, Lohnoo T, Yingyong W. PacBio long read-assembled draft genome of Pythium insidiosum strain Pi-S isolated from a Thai patient with pythiosis. BMC Res Notes 2023; 16:271. [PMID: 37833791 PMCID: PMC10576409 DOI: 10.1186/s13104-023-06532-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2022] [Accepted: 09/22/2023] [Indexed: 10/15/2023] Open
Abstract
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.
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Affiliation(s)
- Theerapong Krajaejun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
| | - Preecha Patumcharoenpol
- Interdisciplinary Graduate Program in Bioscience, Faculty of Science, Kasetsart University, Bangkok, Thailand
| | - Thidarat Rujirawat
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Weerayuth Kittichotirat
- Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkhuntien, Bangkok, Thailand
| | | | - Tassanee Lohnoo
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Wanta Yingyong
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
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Kittichotirat W, Rujirawat T, Patumcharoenpol P, Krajaejun T. Comparative Genomic Analysis Reveals Gene Content Diversity, Phylogenomic Contour, Putative Virulence Determinants, and Potential Diagnostic Markers within Pythium insidiosum Traits. J Fungi (Basel) 2023; 9:jof9020169. [PMID: 36836284 PMCID: PMC9962146 DOI: 10.3390/jof9020169] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Revised: 01/11/2023] [Accepted: 01/17/2023] [Indexed: 01/31/2023] Open
Abstract
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.
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Affiliation(s)
- Weerayuth Kittichotirat
- Bioinformatics and Systems Biology Program, School of Bioresources and Technology and School of Information Technology, King Mongkut’s University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand
- Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand
| | - Thidarat Rujirawat
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
| | - Preecha Patumcharoenpol
- Interdisciplinary Graduate Program in Bioscience, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
| | - Theerapong Krajaejun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, 270 Rama 6 Road, Bangkok 10400, Thailand
- Correspondence: ; Tel.: +(662)-201-1452
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Secretome Profiling by Proteogenomic Analysis Shows Species-Specific, Temperature-Dependent, and Putative Virulence Proteins of Pythium insidiosum. J Fungi (Basel) 2022; 8:jof8050527. [PMID: 35628782 PMCID: PMC9144242 DOI: 10.3390/jof8050527] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2022] [Revised: 05/15/2022] [Accepted: 05/16/2022] [Indexed: 02/04/2023] Open
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.
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Mar Htun Z, Laikul A, Pathomsakulwong W, Yurayart C, Lohnoo T, Yingyong W, Kumsang Y, Payattikul P, Sae-Chew P, Rujirawat T, Jittorntam P, Jaturapaktrarak C, Chongtrakool P, Krajaejun T. Identification and Biotyping of Pythium insidiosum Isolated from Urban and Rural Areas of Thailand by Multiplex PCR, DNA Barcode, and Proteomic Analyses. J Fungi (Basel) 2021; 7:242. [PMID: 33804838 PMCID: PMC8063814 DOI: 10.3390/jof7040242] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 03/13/2021] [Accepted: 03/15/2021] [Indexed: 12/20/2022] Open
Abstract
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.
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Affiliation(s)
- Zin Mar Htun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand;
- Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand;
- Department of Microbiology, University of Medicine, Mandalay 05024, Myanmar
| | - Aree Laikul
- Department of Large Animal and Wildlife Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom 73140, Thailand;
| | | | - Chompoonek Yurayart
- Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand;
| | - Tassanee Lohnoo
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; (T.L.); (W.Y.); (Y.K.); (P.P.); (P.S.-C.); (T.R.); (P.J.); (C.J.)
| | - Wanta Yingyong
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; (T.L.); (W.Y.); (Y.K.); (P.P.); (P.S.-C.); (T.R.); (P.J.); (C.J.)
| | - Yothin Kumsang
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; (T.L.); (W.Y.); (Y.K.); (P.P.); (P.S.-C.); (T.R.); (P.J.); (C.J.)
| | - Penpan Payattikul
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; (T.L.); (W.Y.); (Y.K.); (P.P.); (P.S.-C.); (T.R.); (P.J.); (C.J.)
| | - Pattarana Sae-Chew
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; (T.L.); (W.Y.); (Y.K.); (P.P.); (P.S.-C.); (T.R.); (P.J.); (C.J.)
| | - Thidarat Rujirawat
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; (T.L.); (W.Y.); (Y.K.); (P.P.); (P.S.-C.); (T.R.); (P.J.); (C.J.)
| | - Paisan Jittorntam
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; (T.L.); (W.Y.); (Y.K.); (P.P.); (P.S.-C.); (T.R.); (P.J.); (C.J.)
| | - Chalisa Jaturapaktrarak
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; (T.L.); (W.Y.); (Y.K.); (P.P.); (P.S.-C.); (T.R.); (P.J.); (C.J.)
| | - Piriyaporn Chongtrakool
- Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand;
| | - Theerapong Krajaejun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand;
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Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete Pythium insidiosum. mSystems 2020; 5:5/3/e00196-20. [PMID: 32398276 PMCID: PMC7219551 DOI: 10.1128/msystems.00196-20] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
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. 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.
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The Repurposed Drug Disulfiram Inhibits Urease and Aldehyde Dehydrogenase and Prevents In Vitro Growth of the Oomycete Pythium insidiosum. Antimicrob Agents Chemother 2019; 63:AAC.00609-19. [PMID: 31138572 DOI: 10.1128/aac.00609-19] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2019] [Accepted: 05/18/2019] [Indexed: 11/20/2022] Open
Abstract
Pythium insidiosum is an oomycete microorganism that causes a life-threatening infectious disease, called pythiosis, in humans and animals. The disease has been increasingly reported worldwide. Conventional antifungal drugs are ineffective against P. insidiosum Treatment of pythiosis requires the extensive removal of infected tissue (i.e., eye and leg), but inadequate surgery and recurrent infection often occur. A more effective treatment is needed for pythiosis patients. Drug repurposing is a promising strategy for the identification of a U.S. Food and Drug Administration-approved drug for the control of P. insidiosum Disulfiram has been approved to treat alcoholism, but it exhibits antimicrobial activity against various pathogens. In this study, we explored whether disulfiram possesses an anti-P. insidiosum activity. A total of 27 P. insidiosum strains, isolated from various hosts and geographic areas, were susceptible to disulfiram in a dose-dependent manner. The MIC range of disulfiram against P. insidiosum (8 to 32 mg/liter) was in line with that of other pathogens. Proteogenomic analysis indicated that several potential targets of disulfiram (i.e., aldehyde dehydrogenase and urease) were present in P. insidiosum By homology modeling and molecular docking, disulfiram can bind the putative aldehyde dehydrogenase and urease of P. insidiosum at low energies (i.e., -6.1 and -4.0 Kcal/mol, respectively). Disulfiram diminished the biochemical activities of these enzymes. In conclusion, disulfiram can inhibit the growth of many pathogenic microorganisms, including P. insidiosum The drug can bind and inactivate multiple proteins of P. insidiosum, which may contribute to its broad antimicrobial property. Drug repurposing of disulfiram could be a new treatment option for pythiosis.
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Agarwal S, Iyer G, Srinivasan B, Benurwar S, Agarwal M, Narayanan N, Lakshmipathy M, Radhika N, Rajagopal R, Krishnakumar S, K LT. Clinical profile, risk factors and outcome of medical, surgical and adjunct interventions in patients with Pythium insidiosum keratitis. Br J Ophthalmol 2018; 103:296-300. [PMID: 30206158 DOI: 10.1136/bjophthalmol-2017-311804] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2017] [Revised: 06/05/2018] [Accepted: 08/20/2018] [Indexed: 11/04/2022]
Abstract
PURPOSE To report clinical profile and compare management options for Pythium keratitis. METHOD Retrospective interventional study of 46 patients diagnosed as Pythium keratitis by PCR DNA sequencing from January 2014 to July 2017. Interventions were categorised into medical management (MM) (topical azithromycin and linezolid with oral azithromycin at presentation), surgery (S) (therapeutic penetrating keratoplasty, TPK), surgical adjunct (SA) (cryotherapy±alcohol with TPK) and medical adjunct (MA) (MM after TPK). RESULTS Primary treatment included MM (1 eye), SA (3 eyes) and S (42 eyes). Recurrence occurred in 27/43 eyes (MM+S group). Second surgery (S) was required in 11 eyes (TPK-2), with additional procedures (SA) in 10 eyes and evisceration in five eyes. 8/43 eyes received MA after TPK-1. One eye required TPK-3. Recurrence occured in all eyes that received MA (100%) and in 28 of 54 TPKs (51.8%) (TPK 1+2+3) in 42 eyes. Recurrence was noted in 1/14 (7.1%) that underwent SA. CONCLUSION The currently available and recommended treatment for Pythium keratitis is surgical by means of a TPK and in worse cases evisceration. In our study, MM/MA measures showed no benefit with recurrence or worsening of infection requiring resurgery. Almost 50% of TPKs had a recurrence requiring resurgery. However, adjunctive procedures during TPK appear to have additional benefit with low risk of recurrence and could be included as routine care.
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Affiliation(s)
- Shweta Agarwal
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - Geetha Iyer
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - Bhaskar Srinivasan
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - Saket Benurwar
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - Mamta Agarwal
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - Niveditha Narayanan
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - Meena Lakshmipathy
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - N Radhika
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - Rama Rajagopal
- CJ Shah Cornea Services, Dr G Sitalakshmi Memorial Clinic for Ocular Surface Disorders, Chennai, Tamil Nadu, India
| | - S Krishnakumar
- L&T Ophthalmic Pathology Department, Vision Research Foundation, Chennai, Tamil Nadu, India
| | - Lily Therese K
- L&T Microbiology Research Centre, Vision Research Foundation, Chennai, Tamil Nadu, India
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Liu Y, Lan X, Song S, Yin L, Dry IB, Qu J, Xiang J, Lu J. In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire. FRONTIERS IN PLANT SCIENCE 2018; 9:286. [PMID: 29706971 PMCID: PMC5908963 DOI: 10.3389/fpls.2018.00286] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/04/2017] [Accepted: 02/19/2018] [Indexed: 05/20/2023]
Abstract
Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate "JL-7-2" genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria.
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Affiliation(s)
- Yunxiao Liu
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
- Center for Viticulture and Enology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
| | - Xia Lan
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
- Center for Viticulture and Enology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
| | - Shiren Song
- Center for Viticulture and Enology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
| | - Ling Yin
- Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Guangxi Academy of Agricultural Sciences, Nanning, China
| | - Ian B. Dry
- CSIRO Agriculture & Food, Urrbrae, SA, Australia
| | - Junjie Qu
- Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Guangxi Academy of Agricultural Sciences, Nanning, China
| | - Jiang Xiang
- Center for Viticulture and Enology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
| | - Jiang Lu
- Center for Viticulture and Enology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
- Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Guangxi Academy of Agricultural Sciences, Nanning, China
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10
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Rujirawat T, Patumcharoenpol P, Lohnoo T, Yingyong W, Kumsang Y, Payattikul P, Tangphatsornruang S, Suriyaphol P, Reamtong O, Garg G, Kittichotirat W, Krajaejun T. Probing the Phylogenomics and Putative Pathogenicity Genes of Pythium insidiosum by Oomycete Genome Analyses. Sci Rep 2018. [PMID: 29515152 PMCID: PMC5841299 DOI: 10.1038/s41598-018-22540-1] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
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.
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Affiliation(s)
- Thidarat Rujirawat
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.,Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.,Molecular Medicine Program, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Preecha Patumcharoenpol
- Department of Biomedical Informatics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA.,Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkok, Thailand
| | - Tassanee Lohnoo
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Wanta Yingyong
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Yothin Kumsang
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Penpan Payattikul
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Sithichoke Tangphatsornruang
- Genomic Research Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand
| | - Prapat Suriyaphol
- Bioinformatics and Data Management for Research, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Onrapak Reamtong
- Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
| | - Gagan Garg
- CSIRO Agriculture and Food, Centre for Environment and Life Sciences, Floreat, WA, Australia
| | - Weerayuth Kittichotirat
- Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkok, Thailand.
| | - Theerapong Krajaejun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
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11
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Ascunce MS, Huguet-Tapia JC, Ortiz-Urquiza A, Keyhani NO, Braun EL, Goss EM. Phylogenomic analysis supports multiple instances of polyphyly in the oomycete peronosporalean lineage. Mol Phylogenet Evol 2017. [DOI: 10.1016/j.ympev.2017.06.013] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
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12
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Sun J, Gao Z, Zhang X, Zou X, Cao L, Wang J. Transcriptome analysis of Phytophthora litchii reveals pathogenicity arsenals and confirms taxonomic status. PLoS One 2017; 12:e0178245. [PMID: 28570700 PMCID: PMC5453482 DOI: 10.1371/journal.pone.0178245] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2017] [Accepted: 05/10/2017] [Indexed: 12/17/2022] Open
Abstract
Litchi downy blight, caused by Peronophythora litchii, is one of the major diseases of litchi and has caused severe economic losses. P. litchii has the unique ability to produce downy mildew like sporangiophores under artificial culture. The pathogen had been placed in a new family Peronophytophthoraceae by some authors. In this study, the whole transcriptome of P. litchii from mycelia, sporangia, and zoospores was sequenced for the first time. A set of 23637 transcripts with an average length of 1284 bp was assembled. Using six open reading frame (ORF) predictors, 19267 representative ORFs were identified and were annotated by searching against several public databases. There were 4666 conserved gene families and various sets of lineage-specific genes among P. litchii and other four closely related oomycetes. In silico analyses revealed 490 pathogen-related proteins including 128 RXLR and 22 CRN effector candidates. Based on the phylogenetic analysis of 164 single copy orthologs from 22 species, it is validated that P. litchii is in the genus Phytophthora. Our work provides valuable data to elucidate the pathogenicity basis and ascertain the taxonomic status of P. litchii.
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Affiliation(s)
- Jinhua Sun
- The Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, PR China
| | - Zhaoyin Gao
- The Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, PR China
| | - Xinchun Zhang
- The Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, PR China
| | - Xiaoxiao Zou
- The Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, PR China
| | - Lulu Cao
- The Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, PR China
| | - Jiabao Wang
- The Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, PR China
- * E-mail:
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13
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Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance. Antimicrob Agents Chemother 2017; 61:AAC.02352-16. [PMID: 28115356 DOI: 10.1128/aac.02352-16] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2016] [Accepted: 01/19/2017] [Indexed: 11/20/2022] Open
Abstract
Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.
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14
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Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis. J Clin Microbiol 2016; 54:43-8. [PMID: 26719582 DOI: 10.1128/jcm.02113-15] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Pythiosis is an emerging and life-threatening infectious disease of humans and animals living in tropical and subtropical countries and is caused by the fungus-like organism Pythium insidiosum. Antifungals are ineffective against this pathogen. Most patients undergo surgical removal of the infected organ, and many die from advanced infections. Early and accurate diagnosis leads to prompt management and promotes better prognosis for affected patients. Immunohistochemical assays (IHCs) have been developed using rabbit antibodies raised against P. insidiosum crude extract, i.e., culture filtrate antigen (CFA), for the histodiagnosis of pythiosis, but cross-reactivity with pathogenic fungi compromises the diagnostic performance of the IHC. Therefore, there is a need to improve detection specificity. Recently, the elicitin protein, ELI025, was identified in P. insidiosum, but it was not identified in other human pathogens, including true fungi. The ELI025-encoding gene was successfully cloned and expressed as a recombinant protein in Escherichia coli. This study aims to develop a new IHC using the rabbit anti-ELI025 antibody (anti-ELI) and to compare its performance with the previously reported anti-CFA-based IHC. Thirty-eight P. insidiosum histological sections stained positive by anti-ELI-based and anti-CFA-based IHCs indicating 100% detection sensitivity for the two assays. The anti-ELI antibody stained negative for all 49 negative-control sections indicating 100% detection specificity. In contrast, the anti-CFA antibody stained positive for one of the 49 negative controls (a slide prepared from Fusarium-infected tissue) indicating 98% detection specificity. In conclusion, the anti-ELI based IHC is sensitive and specific for the histodiagnosis of pythiosis and is an improvement over the anti-CFA-based assay.
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15
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Che J, Shi J, Gao Z, Zhang Y. Transcriptome Analysis Reveals the Genetic Basis of the Resveratrol Biosynthesis Pathway in an Endophytic Fungus (Alternaria sp. MG1) Isolated from Vitis vinifera. Front Microbiol 2016; 7:1257. [PMID: 27588016 PMCID: PMC4988973 DOI: 10.3389/fmicb.2016.01257] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2015] [Accepted: 07/29/2016] [Indexed: 12/19/2022] Open
Abstract
Alternaria sp. MG1, an endophytic fungus previously isolated from Merlot grape, produces resveratrol from glucose, showing similar metabolic flux to the phenylpropanoid biosynthesis pathway, currently found solely in plants. In order to identify the resveratrol biosynthesis pathway in this strain at the gene level, de novo transcriptome sequencing was conducted using Illumina paired-end sequencing. A total of 22,954,434 high-quality reads were assembled into contigs and 18,570 unigenes were identified. Among these unigenes, 14,153 were annotated in the NCBI non-redundant protein database and 5341 were annotated in the Swiss-Prot database. After KEGG mapping, 2701 unigenes were mapped onto 115 pathways. Eighty-four unigenes were annotated in major pathways from glucose to resveratrol, coding 20 enzymes for glycolysis, 10 for phenylalanine biosynthesis, 4 for phenylpropanoid biosynthesis, and 4 for stilbenoid biosynthesis. Chalcone synthase was identified for resveratrol biosynthesis in this strain, due to the absence of stilbene synthase. All the identified enzymes indicated a reasonable biosynthesis pathway from glucose to resveratrol via glycolysis, phenylalanine biosynthesis, phenylpropanoid biosynthesis, and stilbenoid pathways. These results provide essential evidence for the occurrence of resveratrol biosynthesis in Alternaria sp. MG1 at the gene level, facilitating further elucidation of the molecular mechanisms involved in this strain's secondary metabolism.
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Affiliation(s)
- Jinxin Che
- College of Food Science and Engineering, Northwest A & F UniversityYangling, China
| | - Junling Shi
- Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical UniversityXi'an, China
| | - Zhenhong Gao
- College of Food Science and Engineering, Northwest A & F UniversityYangling, China
| | - Yan Zhang
- College of Food Science and Engineering, Northwest A & F UniversityYangling, China
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16
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Jo Y, Choi H, Cho WK. Genome Sequence of Dengue virus 3 from the Pythium insidiosum Transcriptomes. Front Microbiol 2016; 7:926. [PMID: 27379056 PMCID: PMC4908670 DOI: 10.3389/fmicb.2016.00926] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2016] [Accepted: 05/31/2016] [Indexed: 12/25/2022] Open
Affiliation(s)
- Yeonhwa Jo
- Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University Seoul, Republic of Korea
| | - Hoseong Choi
- Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University Seoul, Republic of Korea
| | - Won K Cho
- Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National UniversitySeoul, Republic of Korea; The Taejin Genome InstituteHoengseong, Republic of Korea
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17
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Anderson RG, Deb D, Fedkenheuer K, McDowell JM. Recent Progress in RXLR Effector Research. MOLECULAR PLANT-MICROBE INTERACTIONS : MPMI 2015; 28:1063-72. [PMID: 26125490 DOI: 10.1094/mpmi-01-15-0022-cr] [Citation(s) in RCA: 101] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/20/2023]
Abstract
Some of the most devastating oomycete pathogens deploy effector proteins, with the signature amino acid motif RXLR, that enter plant cells to promote virulence. Research on the function and evolution of RXLR effectors has been very active over the decade that has transpired since their discovery. Comparative genomics indicate that RXLR genes play a major role in virulence for Phytophthora and downy mildew species. Importantly, gene-for-gene resistance against these oomycete lineages is based on recognition of RXLR proteins. Comparative genomics have revealed several mechanisms through which this resistance can be broken, most notably involving epigenetic control of RXLR gene expression. Structural studies have revealed a core fold that is present in the majority of RXLR proteins, providing a foundation for detailed mechanistic understanding of virulence and avirulence functions. Finally, functional studies have demonstrated that suppression of host immunity is a major function for RXLR proteins. Host protein targets are being identified in a variety of plant cell compartments. Some targets comprise hubs that are also manipulated by bacteria and fungi, thereby revealing key points of vulnerability in the plant immune network.
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Affiliation(s)
- Ryan G Anderson
- Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA, U.S.A
| | - Devdutta Deb
- Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA, U.S.A
| | - Kevin Fedkenheuer
- Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA, U.S.A
| | - John M McDowell
- Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA, U.S.A
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18
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Tangphatsornruang S, Ruang-Areerate P, Sangsrakru D, Rujirawat T, Lohnoo T, Kittichotirat W, Patumcharoenpol P, Grenville-Briggs LJ, Krajaejun T. Comparative mitochondrial genome analysis of Pythium insidiosum and related oomycete species provides new insights into genetic variation and phylogenetic relationships. Gene 2015; 575:34-41. [PMID: 26299654 DOI: 10.1016/j.gene.2015.08.036] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2015] [Revised: 07/25/2015] [Accepted: 08/17/2015] [Indexed: 01/18/2023]
Abstract
Oomycetes are eukaryotic microorganisms, which are phylogenetically distinct from the true-fungi, which they resemble morphologically. While many oomycetes are pathogenic to plants, Pythium insidiosum is capable of infecting humans and animals. Mitochondrial (mt) genomes are valuable genetic resources for exploring the evolution of eukaryotes. During the course of 454-based nuclear genome sequencing, we identified a complete 54.9 kb mt genome sequence, containing 2 large inverted repeats, from P. insidiosum. It contains 65 different genes (including 2 ribosomal RNA genes, 25 transfer RNA genes and 38 genes encoding NADH dehydrogenases, cytochrome b, cytochrome c oxidases, ATP synthases, and ribosomal proteins). Thirty-nine of the 65 genes have two copies, giving a total of 104 genes. A set of 30 conserved protein-coding genes from the mt genomes of P. insidiosum, 11 other oomycetes, and 2 diatoms (outgroup) were used for phylogenetic analyses. The oomycetes can be classified into 2 phylogenetic groups, in relation to their taxonomic lineages: Saprolegnialean and Peronosporalean. P. insidiosum is more closely related to Pythium ultimum than other oomycetes. In conclusion, the complete mt genome of P. insidiosum was successfully sequenced, assembled, and annotated, providing a useful genetic resource for exploring the biology and evolution of P. insidiosum and other oomycetes.
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Affiliation(s)
- Sithichoke Tangphatsornruang
- Genomic Research Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand
| | - Panthita Ruang-Areerate
- Genomic Research Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand
| | - Duangjai Sangsrakru
- Genomic Research Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand
| | - Thidarat Rujirawat
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Molecular Medicine Program, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Tassanee Lohnoo
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Weerayuth Kittichotirat
- Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkhuntien, Bangkok, Thailand
| | - Preecha Patumcharoenpol
- Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkhuntien, Bangkok, Thailand
| | - Laura J Grenville-Briggs
- Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden
| | - Theerapong Krajaejun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
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19
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Yang X, Hu H, Yu D, Sun Z, He X, Zhang J, Chen Q, Tian R, Fan J. Candidate Resistant Genes of Sand Pear (Pyrus pyrifolia Nakai) to Alternaria alternata Revealed by Transcriptome Sequencing. PLoS One 2015; 10:e0135046. [PMID: 26292286 PMCID: PMC4546377 DOI: 10.1371/journal.pone.0135046] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Accepted: 07/17/2015] [Indexed: 11/18/2022] Open
Abstract
Pear black spot (PBS) disease, which is caused by Alternaria alternata (Aa), is one of the most serious diseases affecting sand pear (Pyrus pyrifolia Nakai) cultivation worldwide. To investigate the defense mechanisms of sand pear in response to Aa, the transcriptome of a sand pear germplasm with differential resistance to Aa was analyzed using Illumina paired-end sequencing. Four libraries derived from PBS-resistant and PBS-susceptible sand pear leaves were characterized through inoculation or mock-inoculation. In total, 20.5 Gbp of sequence data and 101,632,565 reads were generated, representing 44717 genes. Approximately 66% of the genes or sequenced reads could be aligned to the pear reference genome. A large number (5213) of differentially expressed genes related to PBS resistance were obtained; 34 microsatellites were detected in these genes, and 28 genes were found to be closely related to PBS resistance. Using a transcriptome analysis in response to PBS inoculation and comparison analysis to the PHI database, 4 genes (Pbr039001, Pbr001627, Pbr025080 and Pbr023112) were considered to be promising candidates for sand pear resistance to PBS. This study provides insight into changes in the transcriptome of sand pear in response to PBS infection, and the findings have improved our understanding of the resistance mechanism of sand pear to PBS and will facilitate future gene discovery and functional genome studies of sand pear.
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Affiliation(s)
- Xiaoping Yang
- School of Life Sciences, Wuhan University, Wuhan, Hubei, 430072, P. R. China
- Research Institute of Fruit and Tea, Hubei Academy of Agricultural Science, Wuhan, Hubei, 430064, P. R. China
| | - Hongju Hu
- Research Institute of Fruit and Tea, Hubei Academy of Agricultural Science, Wuhan, Hubei, 430064, P. R. China
| | - Dazhao Yu
- School of Life Sciences, Wuhan University, Wuhan, Hubei, 430072, P. R. China
- Hubei Laboratory of Crop Diseases, Insect Pests and Weeds Control, Wuhan, Hubei, 430064, P. R. China
- * E-mail: (DZY); (ZHS)
| | - Zhonghai Sun
- Research Institute of Fruit and Tea, Hubei Academy of Agricultural Science, Wuhan, Hubei, 430064, P. R. China
- * E-mail: (DZY); (ZHS)
| | - Xiujuan He
- Research Institute of Fruit and Tea, Hubei Academy of Agricultural Science, Wuhan, Hubei, 430064, P. R. China
| | - Jingguo Zhang
- Research Institute of Fruit and Tea, Hubei Academy of Agricultural Science, Wuhan, Hubei, 430064, P. R. China
| | - Qiliang Chen
- Research Institute of Fruit and Tea, Hubei Academy of Agricultural Science, Wuhan, Hubei, 430064, P. R. China
| | - Rui Tian
- Research Institute of Fruit and Tea, Hubei Academy of Agricultural Science, Wuhan, Hubei, 430064, P. R. China
| | - Jing Fan
- Research Institute of Fruit and Tea, Hubei Academy of Agricultural Science, Wuhan, Hubei, 430064, P. R. China
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20
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Keeratijarut A, Lohnoo T, Rujirawat T, Yingyong W, Kalambaheti T, Miller S, Phuntumart V, Krajaejun T. The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity. PLoS One 2015; 10:e0135239. [PMID: 26263509 PMCID: PMC4532416 DOI: 10.1371/journal.pone.0135239] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2015] [Accepted: 07/20/2015] [Indexed: 01/08/2023] Open
Abstract
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.
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Affiliation(s)
- Angsana Keeratijarut
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
- Molecular Medicine Program, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Tassanee Lohnoo
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Thidarat Rujirawat
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Wanta Yingyong
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Thareerat Kalambaheti
- Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
| | - Shannon Miller
- Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America
| | - Vipaporn Phuntumart
- Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America
| | - Theerapong Krajaejun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
- * E-mail:
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21
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Draft Genome Sequence of the Pathogenic Oomycete Pythium insidiosum Strain Pi-S, Isolated from a Patient with Pythiosis. GENOME ANNOUNCEMENTS 2015; 3:3/3/e00574-15. [PMID: 26089407 PMCID: PMC4472884 DOI: 10.1128/genomea.00574-15] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Pythium insidiosum is an oomycete that causes a life-threatening infectious disease called pythiosis in humans and animals living in tropical and subtropical countries. Here, we report the first draft genome sequence of P. insidiosum. The genome of P. insidiosum is 53.2 Mb and contains 14,962 open reading frames.
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22
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Lerksuthirat T, Lohnoo T, Inkomlue R, Rujirawat T, Yingyong W, Khositnithikul R, Phaonakrop N, Roytrakul S, Sullivan TD, Krajaejun T. The elicitin-like glycoprotein, ELI025, is secreted by the pathogenic oomycete Pythium insidiosum and evades host antibody responses. PLoS One 2015; 10:e0118547. [PMID: 25793767 PMCID: PMC4368664 DOI: 10.1371/journal.pone.0118547] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2014] [Accepted: 01/20/2015] [Indexed: 12/31/2022] Open
Abstract
Pythium insidiosum is a unique oomycete that can infect humans and animals. Patients with a P. insidiosum infection (pythiosis) have high rates of morbidity and mortality. The pathogen resists conventional antifungal drugs. Information on the biology and pathogenesis of P. insidiosum is limited. Many pathogens secrete proteins, known as effectors, which can affect the host response and promote the infection process. Elicitins are secretory proteins and are found only in the oomycetes, primarily in Phytophthora and Pythium species. In plant-pathogenic oomycetes, elicitins function as pathogen-associated molecular pattern molecules, sterol carriers, and plant defense stimulators. Recently, we reported a number of elicitin-encoding genes from the P. insidiosum transcriptome. The function of elicitins during human infections is unknown. One of the P. insidiosum elicitin-encoding genes, ELI025, is highly expressed and up-regulated at body temperature. This study aims to characterize the biochemical, immunological, and genetic properties of the elicitin protein, ELI025. A 12.4-kDa recombinant ELI025 protein (rELI025) was expressed in Escherichia coli. Rabbit anti-rELI025 antibodies reacted strongly with the native ELI025 in P. insidiosum’s culture medium. The detected ELI025 had two isoforms: glycosylated and non-glycosylated. ELI025 was not immunoreactive with sera from pythiosis patients. The region near the transcriptional start site of ELI025 contained conserved oomycete core promoter elements. In conclusion, ELI025 is a small, abundant, secreted glycoprotein that evades host antibody responses. ELI025 is a promising candidate for development of diagnostic and therapeutic targets for pythiosis.
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Affiliation(s)
- Tassanee Lerksuthirat
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
- Molecular Medicine Program, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Tassanee Lohnoo
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Ruchuros Inkomlue
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Thidarat Rujirawat
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
- Molecular Medicine Program, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Wanta Yingyong
- Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Rommanee Khositnithikul
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Narumon Phaonakrop
- Proteomics Research Laboratory, Genome Institute, National Science and Technology Development Agency, Pathum Thani, Thailand
| | - Sittiruk Roytrakul
- Proteomics Research Laboratory, Genome Institute, National Science and Technology Development Agency, Pathum Thani, Thailand
| | - Thomas D. Sullivan
- Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, United States of America
| | - Theerapong Krajaejun
- Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
- * E-mail:
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Loreto &ES, Tondolo JSM, Zanette RA, Alves SH, Santurio JM. Update on pythiosis immunobiology and immunotherapy. World J Immunol 2014; 4:88-97. [DOI: 10.5411/wji.v4.i2.88] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/27/2014] [Revised: 05/06/2014] [Accepted: 06/11/2014] [Indexed: 02/05/2023] Open
Abstract
Pythiosis is an invasive, ulcerative, pyogranulomatous disease caused by Pythium insidiosum, a fungus-like oomycete that has been reported to affect humans, horses, dogs, and other mammals mainly in tropical and subtropical areas of the world. The disease is characterized by an eosinophilic granulomatous and a Th2 immune response which in turn helps to protect the fungus from the host cells. Pythiosis can present clinically in subcutaneous, gastrointestinal, and vascular tissues or in a systemically disseminated form depending on the species and site of infection. Changes in iron metabolism and anemia are commonly observed. The diagnosis is accomplished through clinical and pathological features, laboratory characteristics of cultures, serological and molecular tests. Treatment includes radical surgery, antimicrobial drugs, immunotherapy or a combination of these treatments. Immunotherapy is a practical and non-invasive alternative for treating pythiosis which is believed to promote a switch from a Th2 to Th1 immune response, resulting in a favorable clinical response. This therapy has demonstrated cure rates above 70% and 55% in horses and humans but low cure rates in dogs and cats. Despite the curative properties of this type of immunotherapy, the antibodies that are produced do not prevent host reinfection. Thus, development of effective adjuvants and new diagnostic techniques for early disease diagnosis are of utmost importance. The aim of this review was to promote pythiosis awareness and to provide an update about the immunotherapy and immunobiology of this disease.
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