A CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasion
<p>More than 2 billion people are at risk of contracting malaria, and around half a million die every year. Malaria is transmitted by sporozoites from Anopheles female mosquitoes to human host during a blood meal. From the host dermal layer, sporozoites travel to the liver, traverse through mu...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2021
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| _version_ | 1797051307863834624 |
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| author | Lee, J |
| author2 | Douglas, A |
| author_facet | Douglas, A Lee, J |
| author_sort | Lee, J |
| collection | OXFORD |
| description | <p>More than 2 billion people are at risk of contracting malaria, and around half a million die every year. Malaria is transmitted by sporozoites from Anopheles female mosquitoes to human host during a blood meal. From the host dermal layer, sporozoites travel to the liver, traverse through multiple hepatocytes until stopping in one last cell. They then establish a productive cell invasion, followed by a parasitophorous vacuole formation. Inside this vacuole, sporozoites develop into thousands of merozoites which are later released by hepatocyte rupture. Then, merozoites travel to the blood vessel to invade erythrocytes where malarial clinical symptoms appear– as such, preventing the liver stage infection can protect infectees from clinical onset. However, the liver stage remains a poorly understood malaria stage today owing to its complex molecular cell invasion mechanism. Therefore, in order to understand how sporozoites establish a hepatocyte invasion and which host cell surface receptor-sporozoite surface ligand interaction is involved in this event, CRISPR-Cas9 screen technology was applied in this study. 480 genes were knocked out, and their impact on the sporozoite invasion was assessed. Initially, results showed ITGAV, RPN1, ATP2B1, TMEM30A, FCGR2B, ITGB5, SLC35A2, MGAT1, EMC1 and APOH genes as hits. Amongst these, ITGAV and ITGB5 (αVβ5 integrin) were investigated as αV-integrin is a known binding partner of TRAP. However, further experiments revealed that ITGAV and ITGB5 hits were false positives induced by a lack of cell adhesion due to integrin disruptions rather than a direct binding activity with the sporozoites. Nevertheless, the CRISPR-Cas9 screen method used for this study remains a useful tool in studying hepatocyte-sporozoite protein interactions due to its success in identifying hepatocyte surface proteins, SR-B1 and ApoH, which are known to be important for the malaria liver stage. Therefore, this study presents an effective method to study parasite-host protein relationships by incorporating a sporozoite invasion assay with a CRISPR-Cas9 screen.</p> |
| first_indexed | 2024-03-06T18:17:38Z |
| format | Thesis |
| id | oxford-uuid:0529d567-4a65-4abe-9783-5f917abc9aca |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:17:38Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | oxford-uuid:0529d567-4a65-4abe-9783-5f917abc9aca2022-03-26T08:55:37ZA CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasionThesishttp://purl.org/coar/resource_type/c_bdccuuid:0529d567-4a65-4abe-9783-5f917abc9acaEnglishHyrax Deposit2021Lee, JDouglas, ASpencer, A<p>More than 2 billion people are at risk of contracting malaria, and around half a million die every year. Malaria is transmitted by sporozoites from Anopheles female mosquitoes to human host during a blood meal. From the host dermal layer, sporozoites travel to the liver, traverse through multiple hepatocytes until stopping in one last cell. They then establish a productive cell invasion, followed by a parasitophorous vacuole formation. Inside this vacuole, sporozoites develop into thousands of merozoites which are later released by hepatocyte rupture. Then, merozoites travel to the blood vessel to invade erythrocytes where malarial clinical symptoms appear– as such, preventing the liver stage infection can protect infectees from clinical onset. However, the liver stage remains a poorly understood malaria stage today owing to its complex molecular cell invasion mechanism. Therefore, in order to understand how sporozoites establish a hepatocyte invasion and which host cell surface receptor-sporozoite surface ligand interaction is involved in this event, CRISPR-Cas9 screen technology was applied in this study. 480 genes were knocked out, and their impact on the sporozoite invasion was assessed. Initially, results showed ITGAV, RPN1, ATP2B1, TMEM30A, FCGR2B, ITGB5, SLC35A2, MGAT1, EMC1 and APOH genes as hits. Amongst these, ITGAV and ITGB5 (αVβ5 integrin) were investigated as αV-integrin is a known binding partner of TRAP. However, further experiments revealed that ITGAV and ITGB5 hits were false positives induced by a lack of cell adhesion due to integrin disruptions rather than a direct binding activity with the sporozoites. Nevertheless, the CRISPR-Cas9 screen method used for this study remains a useful tool in studying hepatocyte-sporozoite protein interactions due to its success in identifying hepatocyte surface proteins, SR-B1 and ApoH, which are known to be important for the malaria liver stage. Therefore, this study presents an effective method to study parasite-host protein relationships by incorporating a sporozoite invasion assay with a CRISPR-Cas9 screen.</p> |
| spellingShingle | Lee, J A CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasion |
| title | A CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasion |
| title_full | A CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasion |
| title_fullStr | A CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasion |
| title_full_unstemmed | A CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasion |
| title_short | A CRISPR-Cas9 screen for hepatocyte receptors for malaria parasite invasion |
| title_sort | crispr cas9 screen for hepatocyte receptors for malaria parasite invasion |
| work_keys_str_mv | AT leej acrisprcas9screenforhepatocytereceptorsformalariaparasiteinvasion AT leej crisprcas9screenforhepatocytereceptorsformalariaparasiteinvasion |