1.Identification and genetic characterization of the TRAP family in Babesia and Theileria
For the identification of the BbiTRAP-1 coding sequence, a TBLASTN search was performed in the genome of B. bigemina (BOND strain) using the predicted amino acid sequence of the B. bovis TRAP-1 gene as a query.
This search resulted in the identification of an orthologous TRAP-1 gene of 3186 bp that encodes a 1061 aa protein (BBBOND_0202740). This gene has 5 exons and is located in chromosome II, as in B. bovis (Figure 1.A). At the genome level, BbiTRAP-1 as well as downstream and upstream surrounding genes are syntenic in comparison with B. bovis, B. divergens, B. microti and B. ovata (Figure 2).
The BbiTRAP-1 protein has a signal peptide at the N- terminus, the adhesive domains vWFA (approx. 200 residues) and TSP-1 (approx. 60 residues and also known as TRS) and the characteristic transmembrane domain (Figure 1.A). A metal ion-dependent adhesion site (MIDAS) motif is also present in the vWFA domain. This motif in BbiTRAP-1 is identical to the reported consensus sequence (5) and is composed of five non-contiguous amino acids, Asp-Xaa-Ser-Xaa-Ser (where ‘Xaa’ can be any amino acid), that are brought together to accommodate a divalent cation.
Even though the amino acid sequence of the TRAP-1 orthologs in B. bovis and B. bigemina share an amino acid identity of only 49,62%, the modular composition and domain order in both species is the same as in its more distant ortholog, P. falciparum (Figure 1.B)
Previous studies in Plasmodium sp. show that TRAP proteins belong to a family with at least 6 members (22). As in Apicomplexa the genes encoding for these proteins are expanded in a lineage-specific fashion, we searched for the presence of other members of the TRAP gene family in the Babesia and Theileria genera. We first searched on PiroplasmaDB for all the genes that coded for proteins containing the TSP-1 or vWFA domain since these functional domains are present in all TRAP proteins characterized so far (5). Our criteria to assign a protein to the TRAP family, includes the presence of an acidic CTD with a conserved tryptophan residue near the C-terminal end of the protein, other typical features of canonical apicomplexan TRAP proteins (12).
The results of this search, based on available current information, are shown in supplementary Table 1. All species of Babesia and Theileria contain at least two TRAP genes in their genome. In general, the percentages of amino acid identity of TRAP proteins were between 18.43-66.25% for TRAP-1 in Babesia spp. and 42.66-82.91% for TRAP-1 in Theileria spp.
B. bovis is the species with the largest number of TRAP genes with four members, meanwhile B. canis has only one TRAP-2 gene. Particularly, in B. bigemina, TRAP-2 has the same domain architecture as TRAP-1 whereas TRAP-3 has two TSP-1 domains and vWA domains respectively (Figure 1.C).
A second search focused on identifying encoded proteins containing just the TSP-1 domain led us to also identify three new members of another group of TRAP-related proteins (Figure 1.C and Supplementary Table 2). These were named TRP after the report of these orthologous proteins in Plasmodium berghei (12). Unlike TRAP, these TRP proteins lack the vWFA domain but contain a variable number of TSP-1 domains and a non-acidic CTD where the conserved tryptophan residue is absent.
2. Phylogenetic analysis
Phylogenetic analysis of sequences of the TRAP family in B. bigemina with paralogs and orthologs of available related species was performed using Maximum Likelihood (Figure 3), Neighbor Joining analysis retrieved a tree with similar topology (data not shown). This analysis, based on TRAP-1, showed that B. ovata is the species with the closest genetic relationship with B. bigemina. Both parasites are classified as a large-type Babesia that infect cattle. A further subclade including B. bovis and B. orientalis is also recognized with high bootstrap value (98%). In general, all TRAP-1 orthologs cluster together with the exception of B. microti, which is considered as a sensu lato Babesia sp. In contrast, TRAP-2 and TRAP-3 protein sequences are grouped with their respective paralogs.
3. Structural analysis of TRAP family
To further confirm the conservation of BbiTRAP1-3 proteins with the already reported orthologs, the 3D structure of the 3 proteins was predicted in silico (Supplementary Figure S1). Homology modelling was performed upon the structure of a fragment of Plasmodium vivax sporozoite surface protein 2 (PDB code: 4hql.2.A) (also referred as TRAP) (23) which was the candidate with the highest GMQE scores (0.60; 0.49 and 0.5 for TRAP-1, TRAP-2 and TRAP-3, respectively).
The predicted 3D structures of the canonical vWFA and TSP-1 domains contain eight α-helices (α1 to α8), eight β-strands (β1to β8) and the MIDAS site (Figure 4). While the degree of amino acid conservation between BbiTRAP-1, 2 and 3 and sporozoite surface protein 2 is low (Seq. Similarity 0.34; 0.30 and 0.30 for TRAP-1, TRAP-2 and TRAP-3 respectively) their overall predicted domain structures are well conserved.
4. Sequence analysis of BiTRAP-1 repeats
The TRF software was used to screen the BbiTRAP-1 gene for the presence of tandem repeats based on previous findings of these repeats in the central part of the TRAP-1 gene in B. bovis (16).
The BbiTRAP-1 protein contains four blocks of long amino acid repeats in the central portion of the protein. Two of these blocks have complete repeat modules of 84 amino acids each, while the last two are truncated (comprised of 60 and 73 amino acids, respectively).
The tandem repeat motifs in TRAP-1 proteins were also analyzed in different B. bigemina strains obtained either from translated genomic data or from sequenced PCR products. Comparative analysis among strains from Argentina, Australia, Puerto Rico, Brazil and Mexico determined that the tandem repeat modules varied in number and sequence among distinct isolates (Table 1). In order to facilitate the analysis of the variation of the repeat modules, a code number was assigned to each repeat. An alignment of all the repeats identified in this work is shown in Figure 5.
Table 1: Variability of the TRAP-1 protein sequence of different B. bigemina strains. A number was assigned to each repeat module. R I-R IV: the four repeat blocks shown in Fig.1B and 1C for the BOND strain.
Strain
|
R I
|
R II
|
R III
|
R IV
|
Origin
|
Source
|
S3P
|
1
|
2
|
3
|
4
|
Argentina
|
Genome database (10)
|
S2A
|
13
|
14
|
3
|
15
|
Argentina
|
Own results
|
S1A
|
21
|
14
|
3
|
15
|
Argentina
|
Own results
|
M1A
|
22
|
14
|
3
|
15
|
Argentina
|
Own results
|
38
|
16
|
17
|
3
|
4
|
Argentina
|
Own results
|
S2P
|
16
|
12
|
23
|
11
|
Argentina
|
Own results
|
Brazil
|
1
|
14
|
3
|
18
|
Brazil
|
Own results
|
JG29
|
10
|
2
|
-
|
11
|
Mexico
|
Genome database (10)
|
Mexico_seed
|
19
|
20
|
-
|
11
|
Mexico
|
Own results
|
Nayarit
|
24
|
-
|
25
|
26
|
Mexico
|
Own results
|
PR
|
9
|
-
|
-
|
4
|
Puerto Rico
|
Genome database (10)
|
BOND
|
5
|
6
|
7
|
8
|
Australia
|
Genome database (10)
|
The number and sequence of the repeats among strains is rather variable. The Australian BOND strain has four unique repeat units as well as the other South American strains. Strains from Puerto Rico and Mexico have a shorter repeat region with only two and three repeat blocks, respectively. Some of the repeat modules are present in strains from a specific region, such as repeats 1, 3, 14 and 15 that were only found in Argentina and Brazil. Remarkably, all Argentinean attenuated strains analyzed here (S1A, S2A and M1A) show a relatively conserved repeat pattern where the first repeat module is variable but the last three ones (repeats 14, 3, 15) are exactly the same. The sequences of the Australian repeats are not present in any other strain analyzed here.
Regarding BbiTRAP-2 and 3, four blocks of tandem repeats were found in BiTRAP-2, between amino acids 71-178. Two of these blocks have 27 amino acids each, while the last two have 25 and 28 amino acids, respectively. No repeats were identified in BiTRAP-3.
5. Expression of recombinant BbiTRAP-1
For further characterization of TRAP-1 in B. bigemina, we generated a recombinant form of this protein containing only predicted extracellular regions and a His tag in order to facilitate expression and purification. The truncated recombinant BbiTRAP-1 protein (rBbiTRAP-1) was obtained at high yield and it could be purified under native conditions. After SDS-PAGE analysis (Figure 6.A, lane 1), a unique band of ~107 kDa, was observed. Even though the molecular weight of recombinant BbiTRAP-1 was higher than predicted, positive Western blot results with the anti His antibody confirmed the identity of the purified protein (Figure 6.B, lane 1).
6. Evaluation of antibody reactivity against recombinant and native BbiTRAP-1
6.1 BbiTRAP-1 is recognized by antibodies from naturally infected cattle
In order to determine if antibodies present in the serum of B. bigemina infected cattle would react with rBbiTRAP-1, a Western blot analysis was performed. Sera from these bovines specifically recognized the same band of ~107 kDa protein that was obtained with the anti-His antibody (Figure 7.A, lanes 4, 5 and 6). No reaction was observed with sera from uninfected cattle (Figure 7.A, lanes 7 and 8), or from a bovine infected with B. bovis (Figure 7.A, lanes 1, 2 and 3).
To further characterize the expression of the BbiTRAP-1 protein in merozoites, we initially tested the ability of sera from mice and bovines immunized with recombinant BbiTRAP-1 to recognize the native protein in immunoblot assays. In both animal models, antisera reacted with a single band of approximately 111 kDa in parasite lysates derived from B. bigemina-infected erythrocytes (Figure 7.B. lane 2 for mice and Figure 7.C. lane 1 for bovine). Accordingly, mice or bovine pre-immune sera did not react with any B. bigemina antigen (Figure 7.B. lane 1 and Figure 7.C. lane 2). Altogether, the data confirms expression of BbiTRAP-1 in blood stages of the parasite.
6.2 Truncated BbiTRAP-1 is not immunodominant in B. bigemina infected bovines
An in house indirect ELISA was developed to further assess the immunogenicity of BbiTRAP-1 using sera from B. bigemina infected animals. For this purpose, a set of 69 sera from B. bigemina-infected herds from 2 different geographic origins (Misiones and Santa Fe, Argentina) was used. The results showed that 29.62% from experimentally-infected and 42.85% of naturally-infected bovines recognized BbiTRAP-1 in this ELISA. The specificity of the test was optimal (100%) meanwhile the sensitivity had the same values as above since no false negative results were obtained. This pattern of reactivity suggests that rBbiTRAP-1 is not an immunodominant antigen, and would not be an optimal candidate for developing novel serological diagnostic assays.
6.3 BbiTRAP 1 contains neutralization sensitive epitopes
To determine whether BbiTRAP-1 has neutralization-sensitive B-cell epitopes, an in vitro invasion assay was performed using anti recombinant BbiTRAP-1 murine and bovine hyperimmune sera. After 72 h of the initiation of the cultures, antibodies against BbiTRAP-1 present in both sera neutralized invasion by 39.94% in mice and 46.92% in bovine samples. As expected, B. bigemina cultures containing pre-immunization serum as a negative control (considered as 100% of infection) exhibited normal parasitemias similar to those observed in control cultures without addition of any serum. Confirmation of the presence of in vitro neutralization sensitive epitopes in BbiTRAP-1 suggests that this protein may also be exposed to neutralizing antibodies during infection, and could be targeted as a possible component of a subunit vaccine against B. bigemina.
6.4 BbiTRAP-1 is expressed in intraerythrocytic merozoites
Finally, we have analyzed the localization of BbiTRAP-1 in intracellular merozoites. The pattern of reactivity of bovine serum raised against recombinant BbiTRAP-1 was tested by IFAT using fixed smears of B. bigemina infected erythrocytes. The green fluorescence of the FITC-anti bovine conjugate localized with a strong focal signal in the wider region of the pear-shaped merozoites compared with the rest of the parasite’s body (Figure 8, g). This positive signal was not observed when infected red blood cells were incubated with a B. bigemina negative bovine serum (Figure 8, d). Both anti BbiTRAP-1 and anti B. bigemina antisera showed a very similar staining pattern including a weak fluorescence signal associated with the erythrocyte membrane.