title: Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1.
abstract: The intraerythrocytic apicomplexan Babesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-length B. microti AMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including other Babesia and Theileria species, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed in Escherichia coli reacted with a mouse anti-BmAMA1 antibody using Western blotting. In vitro binding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation of B. microti parasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Prasun
authors.initials: P
authors.lastname: Moitra
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Zheng
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Vivek
authors.initials: V
authors.lastname: Anantharaman
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Rajdeep
authors.initials: R
authors.lastname: Banerjee
authors.affiliation: Microscopy and Imaging Core Facility, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Takeda
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kozakai
authors.affiliation: Nantucket Cottage Hospital, Nantucket, Massachusetts, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Lepore
authors.affiliation: Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Krause
authors.affiliation: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: L
authors.initials: L
authors.lastname: Aravind
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA Sanjai.Kumar@fda.hhs.gov.
authors.forename: Sanjai
authors.initials: S
authors.lastname: Kumar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D041001:Babesia microti / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001404:Babesiosis / Q000276:immunology / Q000469:parasitology*; D005260:Female; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000953:Antigens, Protozoan; D015800:Protozoan Proteins