{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/377487"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/377487","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"New approaches to understanding the interactions between Plasmodium falciparum merozoites and human erythrocytes during invasion","abstract":"Malaria is responsible for the death of over 600,000 people annually, with the majority of mortality caused by Plasmodium falciparum parasites. The blood stage of infection is responsible for all the clinical symptoms of malaria. Malaria pathogenesis depends on parasite multiplication, which in turn is dependent on the parasite stage that is released from infected erythrocytes (merozoite), being able to invade a human erythrocyte and muliply. Invasion is a complex process involving multiple receptor-ligand interactions and multiple steps. It begins with attachment of the parasite to the erythrocyte, followed by reorientation to position the parasite to invade, then strong membrane wrapping occurs, and finally, a tight junction is formed, which passes around the merozoite, forming the parasitophorous vacuole. This process has been studied in detail for decades and as a result, we know a lot about the molecular components involved. However, dissecting the precise role of individual proteins has, to date, been limited by the availability of quantifiable phenotypic assays. Almost all assays to date have been carried out in static conditions, whereas a major proportion of invasion in the body is likely to occur under the flow of blood circulation. The thesis presented here, therefore, aims to investigate the molecular interactions of invasion using more dynamic assays. The first assay employed allowed direct quantification of the attachment interactions between merozoites and erythrocytes by using optical tweezers to directly manipulate recently expressed P. falciparum merozoites and erythrocytes. This allows the measurement of the strength of attachment, as well as the frequency with which such attachments occur. I collected over >4000 individual merozoite-erythrocyte interactions in combination with a range of inhibitors, antibodies, and genetically modified strains, many of which I generated directly for this work. This enabled me to quantitate the contribution of individual P. falciparum proteins and host receptors to these merozoite-erythrocyte attachment interactions. I showed that the disruption of interactions involved early in invasion affects attachment strength in a manner that correlates with invasion efficiency. Conditional deletion of the major P. falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. Demonstrating the importance of attachment strength to invasion efficiency led to an interest in the effect on parasite growth in more dynamic culture conditions, where the strength of attachment interactions may be more crucial. Previous literature shows parasite growth can change when cultures are kept under suspension, but the relationship between the orbital rotation speed used to keep the culture in suspension and growth has not been systematically investigated. The observed relationship between orbital shaking speed and growth was unexpected. As orbital shaking speed was increased, growth rates first decreased and then increased relative to static conditions, and culture volumes/flask 3 type and haematocrit all changed how a given shaking speed affected growth. Furthermore, shaking conditions that were mildly determinantal to growth in wild-type lines had a more detrimental effect on several PfEBA and PfRH knock-out lines. This demonstrated the need for an assay that allowed quantification of the effect of physiological blood flow rates on invasion. I have, therefore, developed a custom microfluidic device in which invasion can occur in channels that match the dimensions and flow rates previously measured in the human microvasculature. Invasion is monitored using a video microscope and quantified using an automated image analysis pipeline. The population of erythrocytes, late-stage infected erythrocytes (schizonts), newly invaded erythrocytes (rings), and merozoites are tracked, and their flow rate is monitored, which allows invasion rates to be calculated. In summary, this thesis describes the development and application of three assays to explore P. falciparum invasion in more detail and provides more insight into the function of the PfEBA and PfRH invasion ligand families in particular.","abstract_html":"Malaria is responsible for the death of over 600,000 people annually, with the majority of mortality caused by Plasmodium falciparum parasites. The blood stage of infection is responsible for all the clinical symptoms of malaria. Malaria pathogenesis depends on parasite multiplication, which in turn is dependent on the parasite stage that is released from infected erythrocytes (merozoite), being able to invade a human erythrocyte and muliply. Invasion is a complex process involving multiple receptor-ligand interactions and multiple steps. It begins with attachment of the parasite to the erythrocyte, followed by reorientation to position the parasite to invade, then strong membrane wrapping occurs, and finally, a tight junction is formed, which passes around the merozoite, forming the parasitophorous vacuole. This process has been studied in detail for decades and as a result, we know a lot about the molecular components involved. However, dissecting the precise role of individual proteins has, to date, been limited by the availability of quantifiable phenotypic assays. Almost all assays to date have been carried out in static conditions, whereas a major proportion of invasion in the body is likely to occur under the flow of blood circulation. The thesis presented here, therefore, aims to investigate the molecular interactions of invasion using more dynamic assays. The first assay employed allowed direct quantification of the attachment interactions between merozoites and erythrocytes by using optical tweezers to directly manipulate recently expressed P. falciparum merozoites and erythrocytes. This allows the measurement of the strength of attachment, as well as the frequency with which such attachments occur. I collected over >4000 individual merozoite-erythrocyte interactions in combination with a range of inhibitors, antibodies, and genetically modified strains, many of which I generated directly for this work. This enabled me to quantitate the contribution of individual P. falciparum proteins and host receptors to these merozoite-erythrocyte attachment interactions. I showed that the disruption of interactions involved early in invasion affects attachment strength in a manner that correlates with invasion efficiency. Conditional deletion of the major P. falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. Demonstrating the importance of attachment strength to invasion efficiency led to an interest in the effect on parasite growth in more dynamic culture conditions, where the strength of attachment interactions may be more crucial. Previous literature shows parasite growth can change when cultures are kept under suspension, but the relationship between the orbital rotation speed used to keep the culture in suspension and growth has not been systematically investigated. The observed relationship between orbital shaking speed and growth was unexpected. As orbital shaking speed was increased, growth rates first decreased and then increased relative to static conditions, and culture volumes/flask 3 type and haematocrit all changed how a given shaking speed affected growth. Furthermore, shaking conditions that were mildly determinantal to growth in wild-type lines had a more detrimental effect on several PfEBA and PfRH knock-out lines. This demonstrated the need for an assay that allowed quantification of the effect of physiological blood flow rates on invasion. I have, therefore, developed a custom microfluidic device in which invasion can occur in channels that match the dimensions and flow rates previously measured in the human microvasculature. Invasion is monitored using a video microscope and quantified using an automated image analysis pipeline. The population of erythrocytes, late-stage infected erythrocytes (schizonts), newly invaded erythrocytes (rings), and merozoites are tracked, and their flow rate is monitored, which allows invasion rates to be calculated. In summary, this thesis describes the development and application of three assays to explore P. falciparum invasion in more detail and provides more insight into the function of the PfEBA and PfRH invasion ligand families in particular.","abstract_has_math":false,"creators":["Kals, Emma"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Rayner, Julian"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-06-08","date_published":"2024-06-08","updated_at":"2026-07-22T22:24:13Z","subjects":["Flow rate","Invasion","Malaria","Microfludics","Optical tweezers","Orbital shaking","PfEBA","PfRH","Plasmodium falciparum","Wall Shear Stress"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/af99004a-2487-4963-885f-cd1dd7115493/download","https://creativecommons.org/licenses/by/4.0/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.114296","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Rayner, Julian"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Wellcome Trust"]},{"key":"dc:creator","label":"Author","values":["Kals, Emma"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-06-08"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/377487"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Flow rate","Invasion","Malaria","Microfludics","Optical tweezers","Orbital shaking","PfEBA","PfRH","Plasmodium falciparum","Wall Shear Stress"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/af99004a-2487-4963-885f-cd1dd7115493/download","https://creativecommons.org/licenses/by/4.0/"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2025-12-17"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.114296"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/44f69637-be76-466d-b6af-cfb0a3dba3d0/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Malaria is responsible for the death of over 600,000 people annually, with the majority of mortality caused by Plasmodium falciparum parasites. The blood stage of infection is responsible for all the clinical symptoms of malaria. Malaria pathogenesis depends on parasite multiplication, which in turn is dependent on the parasite stage that is released from infected erythrocytes (merozoite), being able to invade a human erythrocyte and muliply. Invasion is a complex process involving multiple receptor-ligand interactions and multiple steps. It begins with attachment of the parasite to the erythrocyte, followed by reorientation to position the parasite to invade, then strong membrane wrapping occurs, and finally, a tight junction is formed, which passes around the merozoite, forming the parasitophorous vacuole. This process has been studied in detail for decades and as a result, we know a lot about the molecular components involved. However, dissecting the precise role of individual proteins has, to date, been limited by the availability of quantifiable phenotypic assays. Almost all assays to date have been carried out in static conditions, whereas a major proportion of invasion in the body is likely to occur under the flow of blood circulation. The thesis presented here, therefore, aims to investigate the molecular interactions of invasion using more dynamic assays. The first assay employed allowed direct quantification of the attachment interactions between merozoites and erythrocytes by using optical tweezers to directly manipulate recently expressed P. falciparum merozoites and erythrocytes. This allows the measurement of the strength of attachment, as well as the frequency with which such attachments occur. I collected over >4000 individual merozoite-erythrocyte interactions in combination with a range of inhibitors, antibodies, and genetically modified strains, many of which I generated directly for this work. This enabled me to quantitate the contribution of individual P. falciparum proteins and host receptors to these merozoite-erythrocyte attachment interactions. I showed that the disruption of interactions involved early in invasion affects attachment strength in a manner that correlates with invasion efficiency. Conditional deletion of the major P. falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. Demonstrating the importance of attachment strength to invasion efficiency led to an interest in the effect on parasite growth in more dynamic culture conditions, where the strength of attachment interactions may be more crucial. Previous literature shows parasite growth can change when cultures are kept under suspension, but the relationship between the orbital rotation speed used to keep the culture in suspension and growth has not been systematically investigated. The observed relationship between orbital shaking speed and growth was unexpected. As orbital shaking speed was increased, growth rates first decreased and then increased relative to static conditions, and culture volumes/flask 3 type and haematocrit all changed how a given shaking speed affected growth. Furthermore, shaking conditions that were mildly determinantal to growth in wild-type lines had a more detrimental effect on several PfEBA and PfRH knock-out lines. This demonstrated the need for an assay that allowed quantification of the effect of physiological blood flow rates on invasion. I have, therefore, developed a custom microfluidic device in which invasion can occur in channels that match the dimensions and flow rates previously measured in the human microvasculature. Invasion is monitored using a video microscope and quantified using an automated image analysis pipeline. The population of erythrocytes, late-stage infected erythrocytes (schizonts), newly invaded erythrocytes (rings), and merozoites are tracked, and their flow rate is monitored, which allows invasion rates to be calculated. In summary, this thesis describes the development and application of three assays to explore P. falciparum invasion in more detail and provides more insight into the function of the PfEBA and PfRH invasion ligand families in particular."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["97c4973708bd3bea81b5eb288213a758","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["New approaches to understanding the interactions between Plasmodium falciparum merozoites and human erythrocytes during invasion"]}]}],"canonical_facts":{"dc:contributor.advisor":["Rayner, Julian"],"dc:contributor.sponsor":["Wellcome Trust"],"dc:creator":["Kals, Emma"],"dc:date.issued":["2024-06-08"],"dc:description.abstract":["Malaria is responsible for the death of over 600,000 people annually, with the majority of mortality caused by Plasmodium falciparum parasites. The blood stage of infection is responsible for all the clinical symptoms of malaria. Malaria pathogenesis depends on parasite multiplication, which in turn is dependent on the parasite stage that is released from infected erythrocytes (merozoite), being able to invade a human erythrocyte and muliply. Invasion is a complex process involving multiple receptor-ligand interactions and multiple steps. It begins with attachment of the parasite to the erythrocyte, followed by reorientation to position the parasite to invade, then strong membrane wrapping occurs, and finally, a tight junction is formed, which passes around the merozoite, forming the parasitophorous vacuole. This process has been studied in detail for decades and as a result, we know a lot about the molecular components involved. However, dissecting the precise role of individual proteins has, to date, been limited by the availability of quantifiable phenotypic assays. Almost all assays to date have been carried out in static conditions, whereas a major proportion of invasion in the body is likely to occur under the flow of blood circulation. The thesis presented here, therefore, aims to investigate the molecular interactions of invasion using more dynamic assays. The first assay employed allowed direct quantification of the attachment interactions between merozoites and erythrocytes by using optical tweezers to directly manipulate recently expressed P. falciparum merozoites and erythrocytes. This allows the measurement of the strength of attachment, as well as the frequency with which such attachments occur. I collected over >4000 individual merozoite-erythrocyte interactions in combination with a range of inhibitors, antibodies, and genetically modified strains, many of which I generated directly for this work. This enabled me to quantitate the contribution of individual P. falciparum proteins and host receptors to these merozoite-erythrocyte attachment interactions. I showed that the disruption of interactions involved early in invasion affects attachment strength in a manner that correlates with invasion efficiency. Conditional deletion of the major P. falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. Demonstrating the importance of attachment strength to invasion efficiency led to an interest in the effect on parasite growth in more dynamic culture conditions, where the strength of attachment interactions may be more crucial. Previous literature shows parasite growth can change when cultures are kept under suspension, but the relationship between the orbital rotation speed used to keep the culture in suspension and growth has not been systematically investigated. The observed relationship between orbital shaking speed and growth was unexpected. As orbital shaking speed was increased, growth rates first decreased and then increased relative to static conditions, and culture volumes/flask 3 type and haematocrit all changed how a given shaking speed affected growth. Furthermore, shaking conditions that were mildly determinantal to growth in wild-type lines had a more detrimental effect on several PfEBA and PfRH knock-out lines. This demonstrated the need for an assay that allowed quantification of the effect of physiological blood flow rates on invasion. I have, therefore, developed a custom microfluidic device in which invasion can occur in channels that match the dimensions and flow rates previously measured in the human microvasculature. Invasion is monitored using a video microscope and quantified using an automated image analysis pipeline. The population of erythrocytes, late-stage infected erythrocytes (schizonts), newly invaded erythrocytes (rings), and merozoites are tracked, and their flow rate is monitored, which allows invasion rates to be calculated. In summary, this thesis describes the development and application of three assays to explore P. falciparum invasion in more detail and provides more insight into the function of the PfEBA and PfRH invasion ligand families in particular."],"dc:format.checksum.md5":["97c4973708bd3bea81b5eb288213a758","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.114296"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/44f69637-be76-466d-b6af-cfb0a3dba3d0/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/377487"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/af99004a-2487-4963-885f-cd1dd7115493/download","https://creativecommons.org/licenses/by/4.0/"],"dc:rights.embargodate":["2025-12-17"],"dc:rights.embargotype":["embargo"],"dc:subject":["Flow rate","Invasion","Malaria","Microfludics","Optical tweezers","Orbital shaking","PfEBA","PfRH","Plasmodium falciparum","Wall Shear Stress"],"dc:title":["New approaches to understanding the interactions between Plasmodium falciparum merozoites and human erythrocytes during invasion"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:13Z"}