scholarly journals CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

2021 ◽  
Vol 9 ◽  
Author(s):  
Raghavendra Yadavalli ◽  
Kousuke Umeda ◽  
Hannah A. Waugh ◽  
Adrienne N. Tracy ◽  
Asha V. Sidhu ◽  
...  
Keyword(s):  
Heterologous Gene Expression ◽  
Protozoan Parasite ◽  
Close Relative ◽  
Perkinsus Marinus ◽  
Post Transfection ◽  
Expression Platform ◽  
C Terminus ◽  
Gene Expression Platform ◽  
Optimized Protocol ◽  
Dermo Disease

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of pPmMOE[MOE1]:GFP and 25.0 × 106P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

scholarly journals A rapid phenotype change in the pathogen Perkinsus marinus was associated with a historically significant marine disease emergence in the eastern oyster

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ryan B. Carnegie ◽  
Susan E. Ford ◽  
Rita K. Crockett ◽  
Peter R. Kingsley-Smith ◽  
Lydia M. Bienlien ◽  
...  
Keyword(s):  
South Carolina ◽  
Climatic Factors ◽  
Disease Outbreaks ◽  
Protozoan Parasite ◽  
Eastern Oyster ◽  
Perkinsus Marinus ◽  
Novel Ecosystem ◽  
The Usa ◽  
Exotic Pathogen ◽  
Dermo Disease

AbstractThe protozoan parasite Perkinsus marinus, which causes dermo disease in Crassostrea virginica, is one of the most ecologically important and economically destructive marine pathogens. The rapid and persistent intensification of dermo in the USA in the 1980s has long been enigmatic. Attributed originally to the effects of multi-year drought, climatic factors fail to fully explain the geographic extent of dermo’s intensification or the persistence of its intensified activity. Here we show that emergence of a unique, hypervirulent P. marinus phenotype was associated with the increase in prevalence and intensity of this disease and associated mortality. Retrospective histopathology of 8355 archival oysters from 1960 to 2018 spanning Chesapeake Bay, South Carolina, and New Jersey revealed that a new parasite phenotype emerged between 1983 and 1990, concurrent with major historical dermo disease outbreaks. Phenotypic changes included a shortening of the parasite’s life cycle and a tropism shift from deeper connective tissues to digestive epithelia. The changes are likely adaptive with regard to the reduced oyster abundance and longevity faced by P. marinus after rapid establishment of exotic pathogen Haplosporidium nelsoni in 1959. Our findings, we hypothesize, illustrate a novel ecosystem response to a marine parasite invasion: an increase in virulence in a native parasite.

Host oyster tissue extracts modulate in vitro protease expression and cellular differentiation in the protozoan parasite, Perkinsus marinus

Parasitology ◽  
2003 ◽  
Vol 126 (4) ◽  
pp. 293-302 ◽  
Author(s):  
E. A. MACINTYRE ◽  
C. G. EARNHART ◽  
S. L. KAATTARI
Keyword(s):  
Serine Proteases ◽  
Protozoan Parasite ◽  
Eastern Oyster ◽  
Defined Medium ◽  
Tissue Homogenate ◽  
Oyster Tissue ◽  
Perkinsus Marinus ◽  
Tissue Extracts

Perkinsus marinus is responsible for a chronic disease (Dermo) of the Eastern oyster, Crassostrea virginica. In order to simulate the in vivo environment more closely, a chemically defined medium (JL-ODRP-3) was supplemented with tissue homogenate extracts or plasma from oysters possessing varying degrees of susceptibility to P. marinus infection. In media supplemented with extracts from highly susceptible oysters (C. virginica), P. marinus cells secreted elevated amounts of a set of low molecular weight serine proteases (LMP: 30–45 kDa) as assessed by enhanced digestion within gelatin-substrate SDS–PAGE gels. Oyster species of low susceptibility (C. gigas and C. ariakensis) did not exhibit this ability to upregulate P. marinus LMP expression. Oyster extract supplementation also led to pronounced changes in P. marinus cellular morphology, such that the cells were comparable to those observed within naturally infected oysters.

scholarly journals Pheo-Type: A Diagnostic Gene-expression Assay for the Classification of Pheochromocytoma and Paraganglioma

2016 ◽  
Vol 101 (3) ◽  
pp. 1034-1043 ◽  
Author(s):  
Aidan Flynn ◽  
Trisha Dwight ◽  
Jessica Harris ◽  
Diana Benn ◽  
Li Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Classification Accuracy ◽  
Microarray Dataset ◽  
Support Vector ◽  
Gene Expression Assay ◽  
Expression Platform ◽  
Gene Expression Platform ◽  
Surveillance Programs ◽  
Sporadic Cases ◽  
Type Gene

Abstract Context: Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers. Objective: This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology. Design: A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential. Results: A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1–3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application. Conclusions: The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.

Transfection of the protozoan parasite Perkinsus marinus

2008 ◽  
Vol 157 (1) ◽  
pp. 44-53 ◽  
Author(s):  
José A. Fernández-Robledo ◽  
Zhuoer Lin ◽  
Gerardo R. Vasta
Keyword(s):  
Protozoan Parasite ◽  
Perkinsus Marinus

scholarly journals Downy Mildew effector HaRxL21 interacts with the transcriptional repressor TOPLESS to promote pathogen susceptibility

2020 ◽  
Author(s):  
Sarah Harvey ◽  
Priyanka Kumari ◽  
Dmitry Lapin ◽  
Thomas Griebel ◽  
Richard Hickman ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Transcriptional Repression ◽  
Plant Immunity ◽  
Effector Proteins ◽  
Host Susceptibility ◽  
Close Relative ◽  
C Terminus ◽  
Rxlr Effector ◽  
Oomycete Pathogen ◽  
Hyaloperonospora Arabidopsidis

AbstractHyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen causing Arabidopsis downy mildew. Effector proteins secreted from the pathogen into the plant play key roles in promoting infection by suppressing plant immunity and manipulating the host to the pathogen’s advantage. One class of oomycete effectors share a conserved ‘RxLR’ motif critical for their translocation into the host cell. Here we characterize the interaction between an RxLR effector, HaRxL21 (RxL21), and the Arabidopsis transcriptional co-repressor Topless (TPL). We establish that RxL21 and TPL interact via an EAR motif at the C-terminus of the effector, mimicking the host plant mechanism for recruiting TPL to sites of transcriptional repression. We show that this motif, and hence interaction with TPL, is necessary for the virulence function of the effector. Furthermore, we provide evidence that RxL21 uses the interaction with TPL, and its close relative TPL-related 1, to repress plant immunity and enhance host susceptibility to both biotrophic and necrotrophic pathogens.

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