Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei
Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named c...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-07-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2023.1204707/full |
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| author | Sabine Bachmaier Matthew K. Gould Eleni Polatoglou Radoslaw Omelianczyk Ana E. Brennand Maha A. Aloraini Jane C. Munday David Horn Michael Boshart Harry P. de Koning |
| author_facet | Sabine Bachmaier Matthew K. Gould Eleni Polatoglou Radoslaw Omelianczyk Ana E. Brennand Maha A. Aloraini Jane C. Munday David Horn Michael Boshart Harry P. de Koning |
| author_sort | Sabine Bachmaier |
| collection | DOAJ |
| description | Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology. |
| first_indexed | 2024-03-13T01:14:49Z |
| format | Article |
| id | doaj.art-a23c0cd921084cd7b7799b4ed7628644 |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-03-13T01:14:49Z |
| publishDate | 2023-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-a23c0cd921084cd7b7799b4ed76286442023-07-05T12:07:37ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882023-07-011310.3389/fcimb.2023.12047071204707Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma bruceiSabine Bachmaier0Matthew K. Gould1Eleni Polatoglou2Radoslaw Omelianczyk3Ana E. Brennand4Maha A. Aloraini5Jane C. Munday6David Horn7Michael Boshart8Harry P. de Koning9Faculty of Biology, Genetics, Ludwig-Maximillians University Munich (LMU), Martinsried, GermanyFaculty of Biology, Genetics, Ludwig-Maximillians University Munich (LMU), Martinsried, GermanyFaculty of Biology, Genetics, Ludwig-Maximillians University Munich (LMU), Martinsried, GermanyFaculty of Biology, Genetics, Ludwig-Maximillians University Munich (LMU), Martinsried, GermanyFaculty of Biology, Genetics, Ludwig-Maximillians University Munich (LMU), Martinsried, GermanyInstitute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United KingdomInstitute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United KingdomThe Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United KingdomFaculty of Biology, Genetics, Ludwig-Maximillians University Munich (LMU), Martinsried, GermanyInstitute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United KingdomCyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.https://www.frontiersin.org/articles/10.3389/fcimb.2023.1204707/fullTrypanosoma bruceicAMPCARPflagellumadenylate cyclaseRNAi screening |
| spellingShingle | Sabine Bachmaier Matthew K. Gould Eleni Polatoglou Radoslaw Omelianczyk Ana E. Brennand Maha A. Aloraini Jane C. Munday David Horn Michael Boshart Harry P. de Koning Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei Frontiers in Cellular and Infection Microbiology Trypanosoma brucei cAMP CARP flagellum adenylate cyclase RNAi screening |
| title | Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei |
| title_full | Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei |
| title_fullStr | Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei |
| title_full_unstemmed | Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei |
| title_short | Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei |
| title_sort | novel kinetoplastid specific camp binding proteins identified by rnai screening for camp resistance in trypanosoma brucei |
| topic | Trypanosoma brucei cAMP CARP flagellum adenylate cyclase RNAi screening |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2023.1204707/full |
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