Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1

Abstract

eThe 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.

Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti

“…divergens AMA1 antibodies (24) and ϳ80% in vitro inhibition of B. microti has been achieved with anti-B. microti AMA1 antibodies (31), confirming that AMA1 is certainly a key player in Babesia invasion. However, in a controlled human malaria challenge trial, the P. falciparum AMA1 (PfAMA1) vaccine conferred little protection even against the homologous parasite ( [62][63][64], showing that AMA1 alone may not be suitable as a candidate on its own.…”

“…In these parasites, part of the trigger that commits a parasite to invasion is the formation of the moving junction (MJ), key components of which are apical membrane antigen 1 (AMA1), which is initially stored in the micronemes, binding with rhoptry neck protein 2 (RON2) (30). The AMA1 proteins of both B. divergens (24) and B. microti (31) have been identified, and their general structures show high degrees of homology to those of the AMA1 proteins from other parasites. Here we report on the identification and characterization of the RON2 proteins from both major species involved in human babesiosis, B. divergens and B. microti, allowing further characterization of their role in invasion in comparison with the roles of the RON2 homologs of other apicomplexan parasites.…”

Surface Antigen 1 Is a Crucial Secreted Protein That Mediates Babesia microti Invasion Into Host Cells

“…Nonetheless, previous studies have shown a significant difference between in vitro culture and in vivo environment. For example, it has been reported that the antiserum raised against BmAMA1 can effectively block the invasion of B. microti into host cells in an in vitro culture environment (Moitra et al, 2015), but the mice immunized with rBmAMA1 did not show any significant protection after B. microti challenge (Wang et al, 2017). Therefore, more evidence needs to be gathered to support that BmSA1 is important for the invasion of B. microti into host cells in an in vivo environment.…”

Comparison of multiplex PCR hybridization-based and singleplex real-time PCR-based assays for detection of low prevalence pathogens in spiked samples

“…Infected mouse blood was obtained from the Kumar laboratory (FDA/CBER/DETTD, Silver Spring, MD) and detailed procedures were described elsewhere (Moitra et al, 2015). Briefly, DBA/2 mice are injected with a blood sample containing intraerythrocytic Babesia microti (ATCC PRA-99), allowed to infect until microscopic observation of a blood sample reveals 10–20% parasitemia.…”