Erythrocyte invasion by Babesia bovis merozoites is inhibited by polyclonal antisera directed against peptides derived from a homologue of Plasmodium falciparum apical membrane antigen 1

Abstract

Apical membrane antigen 1 (AMA-1) is a micronemal protein secreted to the surface of merozoites of Plasmodium species and Toxoplasma gondii tachyzoites in order to fulfill an essential but noncharacterized function in host cell invasion. Here we describe cloning and characterization of a Babesia bovis AMA-1 homologue designated BbAMA-1. The overall level of similarity of BbAMA-1 to P. falciparum AMA-1 was low (18%), but characteristic features like a transmembrane domain near the C terminus, a predicted short cytoplasmic C-terminal sequence with conserved sequence properties, and an extracellular domain containing 14 conserved cysteine residues putatively involved in disulfide bridge formation are typical of AMA-1. Rabbit polyclonal antisera were raised against three synthetic peptides derived from the N-terminal region and domains II and III of the putative extracellular domain and were shown to recognize specifically recombinant BbAMA-1 expressed in Escherichia coli. Immunofluorescence microscopy showed that there was labeling of the apical half of merozoites with these antisera. Preincubation of free merozoites with all three antisera reduced the efficiency of invasion of erythrocytes by a maximum of 65%. Antisera raised against the N-terminal peptide detected a 82-kDa protein on Western blots and a 69-kDa protein in the supernatant that was harvested after in vitro invasion, suggesting that proteolytic processing and secretion take place during or shortly after invasion. A combination of two-dimensional Western blotting and metabolic labeling allowing direct identification of spots reacting with the BbAMA-1 peptide antisera together with the very low silver staining intensity of these spots indicated that very low levels of BbAMA-1 are present in Babesia merozoites.

FIG. 1.

Multiple-sequence alignment of AMA-1 proteins of B. bovis (Bb), T. gondii (Tg), P. vivax (Pv), and P. falciparum (Pf). Similar and identical residues are shaded. Black shading indicates similarity in all four species, and gray shading indicates similarity in three species. Synthetic peptides 1, 2, and 3 are indicated by black bars. The signal peptide cleavage site is indicated by an arrow, and the transmembrane region is indicated by a grey bar. Cysteine residues that form disulfide bonds in P. falciparum AMA-1 are indicated by domain (I, II, and III) and bond (a, b, and c) designations.

FIG. 2.

Western blots of recombinant BbAMA-1 probed with polyclonal rabbit antisera against synthetic peptides: Direct Blue-stained strips of polyvinylidene difluoride membrane obtained after blotting of display E. coli lysates of uninduced cells (lane 1) and induced cells (lanes 2 and 11) expressing BbAMA-1 (lane 2) and B. bovis rab5 (lane 11). Immunoblots of recombinant BbAMA-1 (lanes 3 to 9) were incubated with preimmune serum (lanes 3, 5, and 7), with immune serum against peptide 1 (lane 4), peptide 2 (lane 6), or peptide 3 (lane 8), or with B. bovis rab5 antiserum (lane 9). Lane 10 is an immunoblot of B. bovis recombinant protein rab5 with AMA-1 antisera. Molecular masses are indicated on the left.

FIG. 3.

Immunofluorescence reactivity of antiserum against BbAMA-1 incubated with acetone-fixed B. bovis-infected bovine erythrocytes. (A) Incubation with preimmune sera. (B) Incubation with immune sera against peptide 1 (p1), peptide 2 (p2), and peptide 3 (p3), as indicated on the right. Panels in the left column show anti-AMA-1 staining; panels in the middle column show DAPI staining; and panels in the right column are overlay images. The images in the bottom row of panel B are enlargements of duplicated B. bovis merozoites reacting with anti-peptide 3.

FIG. 4.

Inhibition of erythrocyte invasion by B. bovis merozoites with antisera raised against four different synthetic peptides. The black bar represents the value for invasion of B. bovis merozoites that were directly added to medium after liberation, and this value was considered the 100% value with which all incubation data were compared. The open bars indicate the values for invasion of erythrocytes by B. bovis merozoites after preincubation with immune sera against peptide 1, peptide 2, peptide 3, and peptide C, whereas the grey bars indicate the values for invasion of B. bovis merozoites after preincubation with preimmune serum. Each bar indicates the average value for six individual experiments, and the error bars indicate standard deviations. Data were examined by using the Kruskal-Wallis nonparametric test. Pairwise comparisons of the groups were performed by post hoc analysis as advised by Kruskall-Wallis, and P values are indicated at the top.

FIG. 5.

Western blot analysis of total merozoites and invasion supernatant incubated with sera raised against AMA-1 peptides after 1D SDS-PAGE (A) and 2D SDS-PAGE (B to G). (A) Total merozoites (lane 1) and invasion supernatant (lane 3) were incubated with anti-peptide 1 antiserum. The arrows indicate bands at 82 kDa (lane 1) and 69 kDa (lane 3). Lanes 2 and 4 contained an erythrocyte control incubated with anti-peptide 1 antiserum. (B and D) Western blots of 2D gels loaded with invasion material and incubated with anti-peptide 1 and anti-peptide 3 antisera, respectively. (C and E) Erythrocyte controls incubated with anti-peptide 1 and anti-peptide 3 antisera, respectively. (F and G) Silver-stained image of a 2D gel loaded with invasion supernatant obtained from metabolically labeled B. bovis run in parallel (F) and subsequently exposed to film (G). The arrows indicate BbAMA-1-specific spots in total merozoite and invasion supernatants. Molecular masses are indicated on the left.