Babesia bovis merozoite surface antigen 2 proteins are expressed on the merozoite and sporozoite surface, and specific antibodies inhibit attachment and invasion of erythrocytes

Abstract

The Babesia bovis merozoite surface antigen 2 (MSA-2) locus encodes four proteins, MSA-2a(1), -2a(2), -2b, and -2c. With the use of specific antibodies, each MSA-2 protein was shown to be expressed on the surface of live extracellular merozoites and coexpression on single merozoites was confirmed. Individual antisera against MSA-2a, MSA-2b, and MSA-2c significantly inhibited merozoite invasion of bovine erythrocytes. As tick-derived sporozoites also directly invade erythrocytes, expression of each MSA-2 protein on the sporozoite surface was examined and verified. Finally, statistically significant inhibition of sporozoite binding to the erythrocytes was demonstrated by using antisera specific for MSA-2a, MSA-2b, and MSA-2c. These results indicate an important role for MSA-2 proteins in the initial binding and invasion of host erythrocytes and support the hypothesis that sporozoites and merozoites use common surface molecules in erythrocyte invasion.

FIG. 1.

Specificity of anti-MSA-2 murine and rabbit sera used in expression analysis. Normal, uninfected erythrocytes (lanes 1, 3, and 5) or B. bovis-infected erythrocytes (lanes 2, 4, and 6) were electrophoresed on sodium dodecyl sulfate-containing polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with 1:500 dilutions of murine sera specific for MSA-2a, -2b, or -2c (A) or rabbit sera specific for MSA-2b or -2c (B). Molecular size markers are on the left of each panel.

FIG. 2.

Expression of MSA-2 proteins on the surface of live B. bovis merozoites. Merozoites were incubated with antisera for MSA-2a (A), -2b (B), or 2c (C) and then with a secondary antibody labeled with tetramethyl rhodamine isothiocyanate, which emits a red fluorescence. Merozoite viability was determined with CFDA, which emits a green fluorescence on live cells. Labeling was observed with the positive-control monoclonal antibody 23/10.36.18 against MSA-1 (D) but not with antisera against A. marginale (E). Bars = 5 μm.

FIG. 3.

Coexpression of MSA-2 proteins on the surface of live B. bovis merozoites. Merozoites were incubated with mouse anti-MSA-2a plus rabbit anti-MSA-2b (A to D), mouse anti-MSA-2a plus rabbit anti-MSA-2c (E to H), or mouse anti-MSA-2c plus rabbit anti-MSA-2b (I to L), and then with a rhodamine-labeled anti-murine IgG antibody (red fluorescence) and an Alexa Fluor 350-labeled anti-rabbit IgG antibody (blue fluorescence). Merozoite viability was determined with CFDA, which emits a green fluorescence on live cells. Images are phase contrast and the following channels: individual channel for rhodamine (A, E, and I), individual channel for Alexa Fluor 350 (B, F, and J); combined channels for rhodamine and Alexa Fluor 350 (C, G, and K); and combined channels for rhodamine, Alexa Fluor 350, and fluorescein (D, H, L, and M). Labeling was not observed when merozoites were incubated with a combination of a mouse antiserum against A. marginale recombinant MSP-2 OpAG3 plus a rabbit antiserum against MSP-1 (M). Bars = 5 μm.

FIG. 4.

Specificity of anti-MSA-2 bovine sera used for neutralization of merozoites and sporozoites. Normal, uninfected erythrocytes (lanes 1 and 3) or B. bovis-infected erythrocytes (lanes 2 and 4) were electrophoresed on sodium dodecyl sulfate-containing polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with a 1:500 dilution of sera from calves immunized with recombinant MSA-2a (A), -2b (B), or -2c (C). Molecular size markers are indicated by arrows.

FIG. 5.

Antibody-mediated inhibition of merozoite invasion. Shown are the total numbers of infected erythrocytes from 2,000 cells counted at 5 h after merozoite addition. Merozoites were incubated with M199 medium alone; with negative-control bovine antiserum against ovalbumin; with positive-control bovine antiserum against MSA-1; with two different antisera against MSA-2a, -2b, and -2c; or with the combined antisera (MSA-1, -2a, -2b, and -2c). Error bars indicate standard deviations of the results from triplicate cultures.

FIG. 6.

Expression of MSA-2 proteins by sporozoites at the time of attachment to erythrocytes. Smears of erythrocyte cultures initiated with sporozoites were incubated with specific mouse antiserum against MSA-2a (A), MSA-2b (B), MSA-2c (C), monoclonal antibody 23/10.36.18 against MSA-1 (D), or negative-control mouse antiserum against A. marginale MSP-2 OpAG3 (E). Erythrocyte cultures treated identically but with the use of extracts from uninfected larvae were incubated with specific mouse antiserum against each MSA-2 protein; anti-MSA-2a is shown in panel F (magnification, ×63,000). The reaction was visualized with AEC, which results in brown staining. Bars = 10 μm.

FIG. 7.

Antibody-mediated inhibition of sporozoite attachment to erythrocytes. The total number of parasites attached to 2,000 erythrocytes was counted at 5 h after sporozoite addition. Sporozoites were incubated with M199 medium alone; with negative-control bovine antiserum against ovalbumin; with positive-control bovine antiserum against MSA-1; or with two different antisera against MSA-2a, -2b, or -2c. Error bars indicate standard deviations of the results from triplicate cultures.