{"doi":"10.1101/2025.06.26.658380","title":"Glycosylation of <i>Plasmodium falciparum</i> TRAP supports sporozoite motility and invasion","abstract":"ABSTRACT The human malaria parasite Plasmodium falciparum ( Pf ) expresses ten different thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. Pf TRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr 256 and C-mannosylated at Trp 250 . We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two Pf TRAP glyco-null mutant parasites, Pf TRAP_T256A and Pf TRAP_W250F, and assessed the fitness of these mutant parasites across the life cycle compared to the wild-type NF54 line as well as a Pf TRAP knockout line. The Pf TRAP glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. Pf TRAP abundance in these mutants was significantly decreased despite normal transcript levels. Biophysical assays with recombinant proteins confirmed that glycosylation of the Pf TRAP TSR stabilizes the domain and is likely required for its folding and secretion. These findings demonstrate that glycosylation of Pf TRAP’s TSR is critical for its proper expression and function, and underscore the importance of TSR glycosylation in the mosquito stage of the life cycle. IMPORTANCE Malaria is a mosquito-borne disease caused by Plasmodium parasites, of which P. falciparum is the deadliest. Plasmodium has ten proteins bearing thrombospondin type 1 repeats (TSRs), protein folds that aid cell-cell recognition and binding. Each of Plasmodium ’s ten TSR-bearing proteins is important for invading tissues in the mosquito vector and human host. TSRs are decorated with sugar molecules, a modification termed glycosylation. To better understand the importance of TSR glycosylation in Plasmodium , we investigated the P. falciparum protein TRAP, which is only expressed in mosquito-stage parasite forms called sporozoites. When Pf TRAP was mutated to prevent glycosylation, abundance of the protein significantly decreased and parasites were unable to colonize the mosquito salivary glands. Furthermore, these mutant sporozoites were unable to move or to invade human liver cells. Our study reveals how TSR glycosylation can support the function of proteins that are required for parasite virulence.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":569280,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9632,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":286694,"name":"Vladimir Vigdorovich","orcid":"0000-0003-4195-4858","position":1,"is_corresponding":false},{"id":1267073,"name":"Nastaran Rezakhani","orcid":null,"position":2,"is_corresponding":false},{"id":1474523,"name":"Lucia Pazzagli","orcid":null,"position":3,"is_corresponding":false},{"id":992668,"name":"Hardik Patel","orcid":"0000-0003-0716-4510","position":4,"is_corresponding":false},{"id":640226,"name":"Gigliola Zanghì","orcid":"0000-0002-9656-9642","position":5,"is_corresponding":false},{"id":971406,"name":"Mohd Kamil","orcid":"0000-0001-9429-2662","position":6,"is_corresponding":false},{"id":992669,"name":"Alexander Watson","orcid":"0000-0003-2135-4556","position":7,"is_corresponding":false},{"id":579030,"name":"Nelly Camargo","orcid":null,"position":8,"is_corresponding":false},{"id":1474215,"name":"Erin M. Knutson","orcid":"0000-0002-2637-7688","position":9,"is_corresponding":false},{"id":286695,"name":"D. Noah Sather","orcid":"0000-0002-9128-172X","position":10,"is_corresponding":false},{"id":447460,"name":"Ashley M. Vaughan","orcid":"0000-0001-5815-756X","position":11,"is_corresponding":false},{"id":237003,"name":"Kristian E. Swearingen","orcid":"0000-0002-6756-4471","position":14,"is_corresponding":false},{"id":1474214,"name":"Priya Gupta","orcid":"0000-0001-6192-7051","position":0,"is_corresponding":true}],"reference_count":88,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:56:59.652443Z","pmid":"40667256","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}