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. 2021 May 17:12:623492.
doi: 10.3389/fimmu.2021.623492. eCollection 2021.

Erythrocyte Adhesion of Merozoite Surface Antigen 2c1 Expressed During Extracellular Stages of Babesia orientalis

Zheng Nie  1   2 , Yangsiqi Ao  1   2 , Sen Wang  1   2 , Xiang Shu  1   2 , Muxiao Li  1   2 , Xueyan Zhan  1   2 , Long Yu  1   2 , Xiaomeng An  1   2 , Yali Sun  1   2 , Jiaying Guo  1   2 , Yangnan Zhao  1   2 , Lan He  1   2   3 , Junlong Zhao  1   2   3
Affiliations

Erythrocyte Adhesion of Merozoite Surface Antigen 2c1 Expressed During Extracellular Stages of Babesia orientalis

Zheng Nie et al. Front Immunol. .

Abstract

Babesia orientalis, a major infectious agent of water buffalo hemolytic babesiosis, is transmitted by Rhipicephalus haemaphysaloides. However, no effective vaccine is available. Essential antigens that are involved in parasite invasion of host red blood cells (RBCs) are potential vaccine candidates. Therefore, the identification and the conduction of functional studies of essential antigens are highly desirable. Here, we evaluated the function of B. orientalis merozoite surface antigen 2c1 (BoMSA-2c1), which belongs to the variable merozoite surface antigen (VMSA) family in B. orientalis. We developed a polyclonal antiserum against the purified recombinant (r)BoMSA-2c1 protein. Immunofluorescence staining results showed that BoMSA-2c1 was expressed only on extracellular merozoites, whereas the antigen was undetectable in intracellular parasites. RBC binding assays suggested that BoMSA-2c1 specifically bound to buffalo erythrocytes. Cytoadherence assays using a eukaryotic expression system in vitro further verified the binding and inhibitory ability of BoMSA-2c1. We found that BoMSA-2c1 with a GPI domain was expressed on the surface of HEK293T cells that bound to water buffalo RBCs, and that the anti-rBoMSA2c1 antibody inhibited this binding. These results indicated that BoMSA-2c1 was involved in mediating initial binding to host erythrocytes of B. orientalis. Identification of the occurrence of binding early in the invasion process may facilitate understanding of the growth characteristics, and may help in formulating strategies for the prevention and control of this parasite.

Keywords: Babesia orientalis; MSA-2c1; adhesion; invasion; merozoite surface antigen.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Expression and purification of rBoMSA-2c1. Expression of rBoMSA-2c1 in E. coli BL21 (DE3) assessed by SDS-PAGE. Lysates of pE-sumo-BoMSA-2c1 without (lane 1) and with (lane 2) IPTG induction. Purified product of rBoMSA-2c1 (lane 3). Arrows indicate the corresponding bands. IPTG, isopropyl-β-d-thiogalactopyranoside.
Figure 2
Figure 2
Western blots of BoMSA-2c1. (A) Recombinant BoMSA-2c1 subjected to probing with water buffalo serum infected with (lane 1) and without (lane 2) B orientalis. (B) Immunoblots of the native BoMSA-2c1 protein in parasite lysates. Lane 1, lysate of the uninfected buffalo erythrocytes does not exhibit reaction with polyclonal BoMSA-2c1 antibody. Lane 2, a specific 37-kDa band in parasite lysate subjected to probing with rBoMSA-2c1 antiserum.
Figure 3
Figure 3
Location of BoMSA-2c1 in B orientalis determined by immunofluorescence staining. BoMSA-2c1 is located only on the extracellular parasite membrane. Reactivity of anti-rBoMSA-2c1 serum with parasite during invasion (A), early-egress (B), and intracellular (C) stages. Anti-TRAP2 has been used as the positive control for the intracellular parasite (D), and pre-immune serum has been used as the negative control for validation of intracellular and extracellular parasites (E, F). PcAb-BoMSA-2c1 and PcAb-BoTRAP2 (blue) exhibited reactions with native proteins on merozoites. Nuclei are indicated in blue with Hoechst counterstain. Scale bars: 1 μm.
Figure 4
Figure 4
Western blots of RBC binding to rBoMSA-2c1. Water buffalo RBCs (100 µL) binding to 1.0 mg/mL (lane 1) and (lane 2) 0 mg/mL of rBoMSA-2c1, and His-sumo (lane 3). Lanes 4–9, buffalo RBCs binding to rBoMSA-2c1 (1.6, 1.2, 0.8, 0.6, 0.4, and 0.2 mg/mL), respectively. His-sumo has been used as the negative control. All lanes exhibited reactions with anti-His antibodies.
Figure 5
Figure 5
Rosette assays of binding and inhibition of BoMSA-2c1. (A) Rosettes formed by HEK 293T cells transfected with BoMSA-2c1. Positive reaction has been defined as the observation of coverage of > 50% of the transfected cell surface with erythrocytes. Negative controls include empty pEGFP-NI vector/non-transfected cells (GFP/NT). (B) Numbers of rosettes formed by HEK 293T cells transfected with genes encoding BoMSA-2c1. Positive reaction has been defined as the clustering of ≥ 5 RBCs with the transfected cells, and rosettes were scored in 10 consecutive fields at 200x magnification in each construct. Cells were subsequently incubated with anti-rBoMSA-2c1 rabbit sera at two dilutions before the addition of water buffalo erythrocytes. Pre-immune rabbit sera diluted 1:100, empty pEGFP-NI vector (GFP), and non-transfected (NT) cells were used as controls. Data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison tests. Results are shown as mean ± SD (shown as error bars). NS, not significant at P > 0.05. Significant at ***P < 0.001. Scale bars: 40 µm.
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