Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking
ABSTRACT Malaria parasites increase their host erythrocyte’s permeability to various nutrients, fueling intracellular pathogen development and replication. The plasmodial surface anion channel (PSAC) mediates this uptake and is linked to the parasite-encoded RhopH complex, consisting of CLAG3, RhopH...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2020-10-01
|
| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.01354-20 |
| _version_ | 1818434418363072512 |
|---|---|
| author | Moaz Ahmad Javier Manzella-Lapeira Gagandeep Saggu Daisuke Ito Joseph A. Brzostowski Sanjay A. Desai |
| author_facet | Moaz Ahmad Javier Manzella-Lapeira Gagandeep Saggu Daisuke Ito Joseph A. Brzostowski Sanjay A. Desai |
| author_sort | Moaz Ahmad |
| collection | DOAJ |
| description | ABSTRACT Malaria parasites increase their host erythrocyte’s permeability to various nutrients, fueling intracellular pathogen development and replication. The plasmodial surface anion channel (PSAC) mediates this uptake and is linked to the parasite-encoded RhopH complex, consisting of CLAG3, RhopH2, and RhopH3. While interactions between these subunits are well established, it is not clear whether they remain associated from their synthesis in developing merozoites through erythrocyte invasion and trafficking to the host membrane. Here, we explored protein-protein interactions between RhopH subunits using live-cell imaging and Förster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated single- and double-tagged parasite lines for fluorescence measurements. While CLAG3-mCerulean served as an efficient FRET donor for RhopH2-mVenus within rhoptry organelles, mCerulean targeted to this organelle via a short signal sequence produced negligible FRET. Upon merozoite egress and reinvasion, these tagged RhopH subunits were deposited into the new host cell’s parasitophorous vacuole; these proteins were then exported and trafficked to the erythrocyte membrane, where CLAG3 and RhopH2 remained fully associated. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. These studies establish live-cell FRET imaging in human malaria parasites, reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation. IMPORTANCE Malaria parasites grow within circulating red blood cells and uptake nutrients through a pore on their host membrane. Here, we used gene editing to tag CLAG3 and RhopH2, two proteins linked to the nutrient pore, with fluorescent markers and tracked these proteins in living infected cells. After their synthesis in mature parasites, imaging showed that both proteins are packaged into membrane-bound rhoptries. When parasites ruptured their host cells and invaded new red blood cells, these proteins were detected within a vacuole around the parasite before they migrated and inserted in the surface membrane of the host cell. Using simultaneous labeling of CLAG3 and RhopH2, we determined that these proteins interact tightly during migration and after surface membrane insertion. Red blood cells infected with two parasites had twice the protein at their surface and a parallel increase in the number of nutrient pores. Our work suggests that these proteins directly facilitate parasite nutrient uptake from human plasma. |
| first_indexed | 2024-12-14T16:36:40Z |
| format | Article |
| id | doaj.art-4efcaa676c4942f39b89dfc626ceb3f7 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-14T16:36:40Z |
| publishDate | 2020-10-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-4efcaa676c4942f39b89dfc626ceb3f72022-12-21T22:54:26ZengAmerican Society for MicrobiologymBio2150-75112020-10-0111510.1128/mBio.01354-20Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous TraffickingMoaz Ahmad0Javier Manzella-Lapeira1Gagandeep Saggu2Daisuke Ito3Joseph A. Brzostowski4Sanjay A. Desai5Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USALaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USALaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USALaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USALaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USALaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USAABSTRACT Malaria parasites increase their host erythrocyte’s permeability to various nutrients, fueling intracellular pathogen development and replication. The plasmodial surface anion channel (PSAC) mediates this uptake and is linked to the parasite-encoded RhopH complex, consisting of CLAG3, RhopH2, and RhopH3. While interactions between these subunits are well established, it is not clear whether they remain associated from their synthesis in developing merozoites through erythrocyte invasion and trafficking to the host membrane. Here, we explored protein-protein interactions between RhopH subunits using live-cell imaging and Förster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated single- and double-tagged parasite lines for fluorescence measurements. While CLAG3-mCerulean served as an efficient FRET donor for RhopH2-mVenus within rhoptry organelles, mCerulean targeted to this organelle via a short signal sequence produced negligible FRET. Upon merozoite egress and reinvasion, these tagged RhopH subunits were deposited into the new host cell’s parasitophorous vacuole; these proteins were then exported and trafficked to the erythrocyte membrane, where CLAG3 and RhopH2 remained fully associated. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. These studies establish live-cell FRET imaging in human malaria parasites, reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation. IMPORTANCE Malaria parasites grow within circulating red blood cells and uptake nutrients through a pore on their host membrane. Here, we used gene editing to tag CLAG3 and RhopH2, two proteins linked to the nutrient pore, with fluorescent markers and tracked these proteins in living infected cells. After their synthesis in mature parasites, imaging showed that both proteins are packaged into membrane-bound rhoptries. When parasites ruptured their host cells and invaded new red blood cells, these proteins were detected within a vacuole around the parasite before they migrated and inserted in the surface membrane of the host cell. Using simultaneous labeling of CLAG3 and RhopH2, we determined that these proteins interact tightly during migration and after surface membrane insertion. Red blood cells infected with two parasites had twice the protein at their surface and a parallel increase in the number of nutrient pores. Our work suggests that these proteins directly facilitate parasite nutrient uptake from human plasma.https://journals.asm.org/doi/10.1128/mBio.01354-20FRETPlasmodium falciparumfluorescencemalarianutrient transportpatch-clamp |
| spellingShingle | Moaz Ahmad Javier Manzella-Lapeira Gagandeep Saggu Daisuke Ito Joseph A. Brzostowski Sanjay A. Desai Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking mBio FRET Plasmodium falciparum fluorescence malaria nutrient transport patch-clamp |
| title | Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking |
| title_full | Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking |
| title_fullStr | Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking |
| title_full_unstemmed | Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking |
| title_short | Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking |
| title_sort | live cell fret reveals that malaria nutrient channel proteins clag3 and rhoph2 remain associated throughout their tortuous trafficking |
| topic | FRET Plasmodium falciparum fluorescence malaria nutrient transport patch-clamp |
| url | https://journals.asm.org/doi/10.1128/mBio.01354-20 |
| work_keys_str_mv | AT moazahmad livecellfretrevealsthatmalarianutrientchannelproteinsclag3andrhoph2remainassociatedthroughouttheirtortuoustrafficking AT javiermanzellalapeira livecellfretrevealsthatmalarianutrientchannelproteinsclag3andrhoph2remainassociatedthroughouttheirtortuoustrafficking AT gagandeepsaggu livecellfretrevealsthatmalarianutrientchannelproteinsclag3andrhoph2remainassociatedthroughouttheirtortuoustrafficking AT daisukeito livecellfretrevealsthatmalarianutrientchannelproteinsclag3andrhoph2remainassociatedthroughouttheirtortuoustrafficking AT josephabrzostowski livecellfretrevealsthatmalarianutrientchannelproteinsclag3andrhoph2remainassociatedthroughouttheirtortuoustrafficking AT sanjayadesai livecellfretrevealsthatmalarianutrientchannelproteinsclag3andrhoph2remainassociatedthroughouttheirtortuoustrafficking |