Schistosoma mansoni Fibroblast Growth Factor Receptor A Orchestrates Multiple Functions in Schistosome Biology and in the Host-Parasite Interplay

Du, Xiaofeng; McManus, Donald P.; Fogarty, Conor E.; Jones, Malcolm K.; You, Hong

doi:10.3389/fimmu.2022.868077

Cited by 4 publications

(9 citation statements)

References 101 publications

(193 reference statements)

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“…The repression of SmfgfrA in miracidia hatched from SmfgfrA-modulated eggs was further confirmed by their significantly modified movement behavior in water, represented by enhanced turning and circling movement. This re-emphasizes the importance of SmfgfrA in the neuronal functioning and development of schistosomes, a characteristic explored in our previous study showing that SmFGFRA was abundant in the neural mass of mature eggs and miracidia [57]. Together, these outcomes may indicate a long-term effect of CRISPRimediated gene repression in schistosomes, as described for Toxoplasma gondii [44].…”

Section: Discussionsupporting

confidence: 75%

“…A possible reason is that SmfgfrA is more highly expressed in eggs than in adult worms and schistosomula [57], resulting in the chromatin in eggs being more open (euchromatin) and providing greater access to dCas9 binding and functioning [80]. Also, the morphology of eggs is relatively simple with a high stem-like cell content [81], compared with other developmental stages, which may indicate increased potential for manipulating the stem cells and stem cell marker genes (eg.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, we found the CRISPRi-induced gene repression efficiency in adult worms is much higher than that observed in schistosomula, although adult worms and schistosomula present similar level of SmfgfrA transcripts [57]. This might be explained by the more abundant distribution of this molecule in the tegument of adult worms than that in schistosomula [57], which may result in a relatively higher efficacy of SmfgfrA -repression in adult worms as genes with more tegument location may provide more proximity to the external environment containing CRISPR components. In addition, the relatively larger surface area / volume ratio in adult worms than schistosomula [82] may also lead to a higher efficacy of delivering CRISPR components into adult flukes by electroporation.…”

Section: Discussionmentioning

confidence: 99%

“…The membrane was first blocked with Odyssey Blocking Buffer (TBS) (LI-COR Biosciences, Lincoln, Nebraska, USA) for 1 h at room temperature with shaking. Subsequently, the membrane was incubated with a polyclonal mouse anti-rSmFGFRA-L (anti-recombinant SmFGFRA extracellular ligand binding domain) antibody (previously generated in our laboratory [57]) (1:100 diluted in Odyssey buffer with 0.1% (v/v) Tween-20) for 1 h with shaking at room temperature. Following washes (4X) in Tris-Buffered Saline (TBS)/0.1% Tween-20 (TBST), the membrane was incubated with IRDye-labeled 680LT goat anti-mouse IgG antibody (Li-COR Biosciences) (1:15,000 diluted in Odyssey buffer with 0.1% Tween-20 and 0.01% SDS) for 1 h with shaking in a dark chamber.…”

Section: Methodsmentioning

confidence: 99%

“…To unravel the critical roles of stem cells in driving the schistosome life cycle [50][51][52][53][54], characterization of the somatic stem cell marker SmFGFRA has drawn increased attention [50,[52][53][54][55][56]. Our previous study demonstrated SmFGFRA is abundantly expressed in different S. mansoni developmental stages [57]. The distribution pattern of SmFGFRA in embryonic cells of immature eggs, in the neural mass of mature eggs and miracidia, and its co-location with EdU + (thymidine analog 5-ethynyl-2'-deoxyuridine) cells in adult S. mansoni, strongly implicated its important roles in maintaining schistosome stem cells, in development of the nervous and reproductive systems, and in the host-parasite interplay [57].…”

Section: Introductionmentioning

confidence: 99%

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CRISPR interference for Sequence-Specific Regulation of Fibroblast Growth Factor Receptor A in Schistosoma mansoni

McManus

French

et al. 2022

Preprint

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Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A. SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2'-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CRISPR interference for Sequence-Specific Regulation of Fibroblast Growth Factor Receptor A in Schistosoma mansoni

McManus

French

et al. 2022

Preprint

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Lentiviral Transduction-based CRISPR/Cas9 Editing of Schistosoma mansoni Acetylcholinesterase

Du,

McManus,

French

et al. 2023

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Background: Recent studies on CRISPR/Cas9-mediated gene editing in Schistosoma mansoni have shed new light on the study and control of this parasitic helminth. However, the gene editing efficiency in this parasite is modest. Methods: To improve the efficiency of CRISPR/Cas9 genome editing in schistosomes, we used lentivirus, which has been effectively used for gene editing in mammalian cells, to deliver plasmid DNA encoding Cas9 nuclease, a sgRNA targeting acetylcholinesterase (SmAChE) and a mCherry fluorescence marker into schistosomes. Results: MCherry fluorescence was observed in transduced eggs, schistosomula, and adult worms, indicating that the CRISPR components had been delivered into these parasite stages by lentivirus. In addition, clearly changed phenotypes were observed in SmAChE-edited parasites, including decreased SmAChE activity, reduced hatching ability of edited eggs, and altered behavior of miracidia hatched from edited eggs. Next-generation sequencing analysis demonstrated that the lentiviral transduction-based CRISPR/Cas9 gene modifications in SmAChE-edited schistosomes were homology-directed repair predominant but with much lower efficiency than that obtained using electroporation (data previously published by our laboratory) for the delivery of CRISPR components. Conclusion: Taken together, electroporation is more efficient than lentiviral transduction in the delivery of CRISPR/Cas9 into schistosomes for programmed genome editing. The exploration of tactics for enhancing CRISPR/Cas9 gene editing provides the basis for the future improvement of programmed genome editing in S. mansoni.

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CRISPR interference for sequence-specific regulation of fibroblast growth factor receptor A in Schistosoma mansoni

McManus

French

et al. 2023

Front. Immunol.

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Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function studies targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A (FGFRA). SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2’-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.

show abstract

Schistosoma mansoni Fibroblast Growth Factor Receptor A Orchestrates Multiple Functions in Schistosome Biology and in the Host-Parasite Interplay

Cited by 4 publications

References 101 publications

CRISPR interference for Sequence-Specific Regulation of Fibroblast Growth Factor Receptor A in Schistosoma mansoni

CRISPR interference for Sequence-Specific Regulation of Fibroblast Growth Factor Receptor A in Schistosoma mansoni

Lentiviral Transduction-based CRISPR/Cas9 Editing of Schistosoma mansoni Acetylcholinesterase

CRISPR interference for sequence-specific regulation of fibroblast growth factor receptor A in Schistosoma mansoni

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