Coactosin Phosphorylation Controls <named-content content-type="genus-species">Entamoeba histolytica</named-content> Cell Membrane Protrusions and Cell Motility
ABSTRACT Invasion of the colon wall by Entamoeba histolytica during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix. Transcriptional silencing of the E. histolytica surface metalloprotease EhMSP-1 produces hyperadherent less-motile trop...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2020-08-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mBio.00660-20 |
| _version_ | 1818346806212296704 |
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| author | Muhammad M. Hasan José E. Teixeira Ying-Wai Lam Christopher D. Huston |
| author_facet | Muhammad M. Hasan José E. Teixeira Ying-Wai Lam Christopher D. Huston |
| author_sort | Muhammad M. Hasan |
| collection | DOAJ |
| description | ABSTRACT Invasion of the colon wall by Entamoeba histolytica during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix. Transcriptional silencing of the E. histolytica surface metalloprotease EhMSP-1 produces hyperadherent less-motile trophozoites that are deficient in forming invadosomes. Reversible protein phosphorylation is often implicated in regulation of cell motility and invadosome formation. To identify such intermediaries of the EhMSP-1-silenced phenotype, here we compared the phosphoproteomes of EhMSP-1-silenced and vector control trophozoites by using quantitative tandem mass spectrometry-based proteomics. Six proteins were found to be differentially phosphorylated in EhMSP-1-silenced and control cells, including EhCoactosin, a member of the ADF/cofilin family of actin-binding proteins, which was more frequently phosphorylated at serine 147. Regulated overexpression of wild-type, phosphomimetic, and nonphosphorylatable EhCoactosin variants was used to test if phosphorylation functions in control of E. histolytica actin dynamics. Each of the overexpressed proteins colocalized with F-actin during E. histolytica phagocytosis. Nonetheless, trophozoites overexpressing an EhCoactosin phosphomimetic mutant formed more and poorly coordinated cell membrane protrusions compared to those in control or cells expressing a nonphosphorylatable mutant, while trophozoites overexpressing nonphosphorylatable EhCoactosin were significantly more motile within a model of mammalian extracellular matrix. Therefore, although EhCoactosin’s actin-binding ability appeared unaffected by phosphorylation, EhCoactosin phosphorylation helps to regulate amoebic motility. These data help to understand the mechanisms underlying altered adherence and motility in EhMSP-1-silenced trophozoites and lay the groundwork for identifying kinases and phosphatases critical for control of amoebic invasiveness. IMPORTANCE Invasive amoebiasis, caused by the intestinal parasite Entamoeba histolytica, causes life-threatening diarrhea and liver abscesses, but, for unknown reasons, only approximately 10% of E. histolytica infections become symptomatic. A key requirement of invasion is the ability of the parasite to migrate through tissue layers. Here, we systematically looked for differences in protein phosphorylation between control parasites and a previously identified hyperadherent E. histolytica cell line that has reduced motility. We identified EhCoactosin, an actin-binding protein not previously known to be phosphoregulated, as one of the differentially phosphorylated proteins in E. histolytica and demonstrated that EhCoactosin phosphorylation functions in control of cell membrane dynamics and amoebic motility. This and the additional differentially phosphorylated proteins reported lay the groundwork for identifying kinases and phosphatases that regulate tissue invasiveness. |
| first_indexed | 2024-12-13T17:24:07Z |
| format | Article |
| id | doaj.art-cde7775a75114ada93f1cfd3282f8755 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-13T17:24:07Z |
| publishDate | 2020-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-cde7775a75114ada93f1cfd3282f87552022-12-21T23:37:14ZengAmerican Society for MicrobiologymBio2150-75112020-08-0111410.1128/mBio.00660-20Coactosin Phosphorylation Controls <named-content content-type="genus-species">Entamoeba histolytica</named-content> Cell Membrane Protrusions and Cell MotilityMuhammad M. Hasan0José E. Teixeira1Ying-Wai Lam2Christopher D. Huston3Department of Medicine, University of Vermont, Larner College of Medicine, Burlington, Vermont, USADepartment of Medicine, University of Vermont, Larner College of Medicine, Burlington, Vermont, USAProteomics Facility, Vermont Genetics Network, University of Vermont, Burlington, Vermont, USADepartment of Medicine, University of Vermont, Larner College of Medicine, Burlington, Vermont, USAABSTRACT Invasion of the colon wall by Entamoeba histolytica during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix. Transcriptional silencing of the E. histolytica surface metalloprotease EhMSP-1 produces hyperadherent less-motile trophozoites that are deficient in forming invadosomes. Reversible protein phosphorylation is often implicated in regulation of cell motility and invadosome formation. To identify such intermediaries of the EhMSP-1-silenced phenotype, here we compared the phosphoproteomes of EhMSP-1-silenced and vector control trophozoites by using quantitative tandem mass spectrometry-based proteomics. Six proteins were found to be differentially phosphorylated in EhMSP-1-silenced and control cells, including EhCoactosin, a member of the ADF/cofilin family of actin-binding proteins, which was more frequently phosphorylated at serine 147. Regulated overexpression of wild-type, phosphomimetic, and nonphosphorylatable EhCoactosin variants was used to test if phosphorylation functions in control of E. histolytica actin dynamics. Each of the overexpressed proteins colocalized with F-actin during E. histolytica phagocytosis. Nonetheless, trophozoites overexpressing an EhCoactosin phosphomimetic mutant formed more and poorly coordinated cell membrane protrusions compared to those in control or cells expressing a nonphosphorylatable mutant, while trophozoites overexpressing nonphosphorylatable EhCoactosin were significantly more motile within a model of mammalian extracellular matrix. Therefore, although EhCoactosin’s actin-binding ability appeared unaffected by phosphorylation, EhCoactosin phosphorylation helps to regulate amoebic motility. These data help to understand the mechanisms underlying altered adherence and motility in EhMSP-1-silenced trophozoites and lay the groundwork for identifying kinases and phosphatases critical for control of amoebic invasiveness. IMPORTANCE Invasive amoebiasis, caused by the intestinal parasite Entamoeba histolytica, causes life-threatening diarrhea and liver abscesses, but, for unknown reasons, only approximately 10% of E. histolytica infections become symptomatic. A key requirement of invasion is the ability of the parasite to migrate through tissue layers. Here, we systematically looked for differences in protein phosphorylation between control parasites and a previously identified hyperadherent E. histolytica cell line that has reduced motility. We identified EhCoactosin, an actin-binding protein not previously known to be phosphoregulated, as one of the differentially phosphorylated proteins in E. histolytica and demonstrated that EhCoactosin phosphorylation functions in control of cell membrane dynamics and amoebic motility. This and the additional differentially phosphorylated proteins reported lay the groundwork for identifying kinases and phosphatases that regulate tissue invasiveness.https://journals.asm.org/doi/10.1128/mBio.00660-20ADF/cofilincoactosinEntamoeba histolyticaamoebiasiscell motilityphosphoproteomics |
| spellingShingle | Muhammad M. Hasan José E. Teixeira Ying-Wai Lam Christopher D. Huston Coactosin Phosphorylation Controls <named-content content-type="genus-species">Entamoeba histolytica</named-content> Cell Membrane Protrusions and Cell Motility mBio ADF/cofilin coactosin Entamoeba histolytica amoebiasis cell motility phosphoproteomics |
| title | Coactosin Phosphorylation Controls <named-content content-type="genus-species">Entamoeba histolytica</named-content> Cell Membrane Protrusions and Cell Motility |
| title_full | Coactosin Phosphorylation Controls <named-content content-type="genus-species">Entamoeba histolytica</named-content> Cell Membrane Protrusions and Cell Motility |
| title_fullStr | Coactosin Phosphorylation Controls <named-content content-type="genus-species">Entamoeba histolytica</named-content> Cell Membrane Protrusions and Cell Motility |
| title_full_unstemmed | Coactosin Phosphorylation Controls <named-content content-type="genus-species">Entamoeba histolytica</named-content> Cell Membrane Protrusions and Cell Motility |
| title_short | Coactosin Phosphorylation Controls <named-content content-type="genus-species">Entamoeba histolytica</named-content> Cell Membrane Protrusions and Cell Motility |
| title_sort | coactosin phosphorylation controls named content content type genus species entamoeba histolytica named content cell membrane protrusions and cell motility |
| topic | ADF/cofilin coactosin Entamoeba histolytica amoebiasis cell motility phosphoproteomics |
| url | https://journals.asm.org/doi/10.1128/mBio.00660-20 |
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