The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum

ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport. Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Cecilia P. Sanchez [verfasserIn] Erin D. T. Manson [verfasserIn] Sonia Moliner Cubel [verfasserIn] Luis Mandel [verfasserIn] Stefan K. Weidt [verfasserIn] Michael P. Barrett [verfasserIn] Michael Lanzer [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2022

Schlagwörter:	PfCRT oligopeptides metabolomics malaria P. falciparum membrane transport

Übergeordnetes Werk:	In: Microbiology Spectrum - American Society for Microbiology, 2022, 10(2022), 4
Übergeordnetes Werk:	volume:10 ; year:2022 ; number:4

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1128/spectrum.01101-22

Katalog-ID:	DOAJ031377645

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520			\|a ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport.
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allfields	10.1128/spectrum.01101-22 doi (DE-627)DOAJ031377645 (DE-599)DOAJd2073372fc434f7889e68c2ee165d4ee DE-627 ger DE-627 rakwb eng QR1-502 Cecilia P. Sanchez verfasserin aut The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport. PfCRT oligopeptides metabolomics malaria P. falciparum membrane transport Microbiology Erin D. T. Manson verfasserin aut Sonia Moliner Cubel verfasserin aut Luis Mandel verfasserin aut Stefan K. Weidt verfasserin aut Michael P. Barrett verfasserin aut Michael Lanzer verfasserin aut In Microbiology Spectrum American Society for Microbiology, 2022 10(2022), 4 (DE-627)816693293 (DE-600)2807133-5 21650497 nnns volume:10 year:2022 number:4 https://doi.org/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/article/d2073372fc434f7889e68c2ee165d4ee kostenfrei https://journals.asm.org/doi/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/toc/2165-0497 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2022 4
spelling	10.1128/spectrum.01101-22 doi (DE-627)DOAJ031377645 (DE-599)DOAJd2073372fc434f7889e68c2ee165d4ee DE-627 ger DE-627 rakwb eng QR1-502 Cecilia P. Sanchez verfasserin aut The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport. PfCRT oligopeptides metabolomics malaria P. falciparum membrane transport Microbiology Erin D. T. Manson verfasserin aut Sonia Moliner Cubel verfasserin aut Luis Mandel verfasserin aut Stefan K. Weidt verfasserin aut Michael P. Barrett verfasserin aut Michael Lanzer verfasserin aut In Microbiology Spectrum American Society for Microbiology, 2022 10(2022), 4 (DE-627)816693293 (DE-600)2807133-5 21650497 nnns volume:10 year:2022 number:4 https://doi.org/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/article/d2073372fc434f7889e68c2ee165d4ee kostenfrei https://journals.asm.org/doi/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/toc/2165-0497 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2022 4
allfields_unstemmed	10.1128/spectrum.01101-22 doi (DE-627)DOAJ031377645 (DE-599)DOAJd2073372fc434f7889e68c2ee165d4ee DE-627 ger DE-627 rakwb eng QR1-502 Cecilia P. Sanchez verfasserin aut The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport. PfCRT oligopeptides metabolomics malaria P. falciparum membrane transport Microbiology Erin D. T. Manson verfasserin aut Sonia Moliner Cubel verfasserin aut Luis Mandel verfasserin aut Stefan K. Weidt verfasserin aut Michael P. Barrett verfasserin aut Michael Lanzer verfasserin aut In Microbiology Spectrum American Society for Microbiology, 2022 10(2022), 4 (DE-627)816693293 (DE-600)2807133-5 21650497 nnns volume:10 year:2022 number:4 https://doi.org/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/article/d2073372fc434f7889e68c2ee165d4ee kostenfrei https://journals.asm.org/doi/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/toc/2165-0497 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2022 4
allfieldsGer	10.1128/spectrum.01101-22 doi (DE-627)DOAJ031377645 (DE-599)DOAJd2073372fc434f7889e68c2ee165d4ee DE-627 ger DE-627 rakwb eng QR1-502 Cecilia P. Sanchez verfasserin aut The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport. PfCRT oligopeptides metabolomics malaria P. falciparum membrane transport Microbiology Erin D. T. Manson verfasserin aut Sonia Moliner Cubel verfasserin aut Luis Mandel verfasserin aut Stefan K. Weidt verfasserin aut Michael P. Barrett verfasserin aut Michael Lanzer verfasserin aut In Microbiology Spectrum American Society for Microbiology, 2022 10(2022), 4 (DE-627)816693293 (DE-600)2807133-5 21650497 nnns volume:10 year:2022 number:4 https://doi.org/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/article/d2073372fc434f7889e68c2ee165d4ee kostenfrei https://journals.asm.org/doi/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/toc/2165-0497 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2022 4
allfieldsSound	10.1128/spectrum.01101-22 doi (DE-627)DOAJ031377645 (DE-599)DOAJd2073372fc434f7889e68c2ee165d4ee DE-627 ger DE-627 rakwb eng QR1-502 Cecilia P. Sanchez verfasserin aut The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport. PfCRT oligopeptides metabolomics malaria P. falciparum membrane transport Microbiology Erin D. T. Manson verfasserin aut Sonia Moliner Cubel verfasserin aut Luis Mandel verfasserin aut Stefan K. Weidt verfasserin aut Michael P. Barrett verfasserin aut Michael Lanzer verfasserin aut In Microbiology Spectrum American Society for Microbiology, 2022 10(2022), 4 (DE-627)816693293 (DE-600)2807133-5 21650497 nnns volume:10 year:2022 number:4 https://doi.org/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/article/d2073372fc434f7889e68c2ee165d4ee kostenfrei https://journals.asm.org/doi/10.1128/spectrum.01101-22 kostenfrei https://doaj.org/toc/2165-0497 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2022 4
language	English
source	In Microbiology Spectrum 10(2022), 4 volume:10 year:2022 number:4
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topic_facet	PfCRT oligopeptides metabolomics malaria P. falciparum membrane transport Microbiology
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container_title	Microbiology Spectrum
authorswithroles_txt_mv	Cecilia P. Sanchez @@aut@@ Erin D. T. Manson @@aut@@ Sonia Moliner Cubel @@aut@@ Luis Mandel @@aut@@ Stefan K. Weidt @@aut@@ Michael P. Barrett @@aut@@ Michael Lanzer @@aut@@
publishDateDaySort_date	2022-01-01T00:00:00Z
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issn	21650497
topic_title	QR1-502 The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum PfCRT oligopeptides metabolomics malaria P. falciparum membrane transport
topic	misc QR1-502 misc PfCRT misc oligopeptides misc metabolomics misc malaria misc P. falciparum misc membrane transport misc Microbiology
topic_unstemmed	misc QR1-502 misc PfCRT misc oligopeptides misc metabolomics misc malaria misc P. falciparum misc membrane transport misc Microbiology
topic_browse	misc QR1-502 misc PfCRT misc oligopeptides misc metabolomics misc malaria misc P. falciparum misc membrane transport misc Microbiology
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title	The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum
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title_full	The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum
author_sort	Cecilia P. Sanchez
journal	Microbiology Spectrum
journalStr	Microbiology Spectrum
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author_browse	Cecilia P. Sanchez Erin D. T. Manson Sonia Moliner Cubel Luis Mandel Stefan K. Weidt Michael P. Barrett Michael Lanzer
container_volume	10
class	QR1-502
format_se	Elektronische Aufsätze
author-letter	Cecilia P. Sanchez
doi_str_mv	10.1128/spectrum.01101-22
author2-role	verfasserin
title_sort	knock-down of the chloroquine resistance transporter pfcrt is linked to oligopeptide handling in plasmodium falciparum
callnumber	QR1-502
title_auth	The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum
abstract	ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport.
abstractGer	ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport.
abstract_unstemmed	ABSTRACT The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport.
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container_issue	4
title_short	The Knock-Down of the Chloroquine Resistance Transporter PfCRT Is Linked to Oligopeptide Handling in Plasmodium falciparum
url	https://doi.org/10.1128/spectrum.01101-22 https://doaj.org/article/d2073372fc434f7889e68c2ee165d4ee https://journals.asm.org/doi/10.1128/spectrum.01101-22 https://doaj.org/toc/2165-0497
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author2	Erin D. T. Manson Sonia Moliner Cubel Luis Mandel Stefan K. Weidt Michael P. Barrett Michael Lanzer
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