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Expression of 6-Cys Gene Superfamily Defines Babesia bovis Sexual Stage Development within Rhipicephalus microplus

View Article: PubMed Central - PubMed

ABSTRACT

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.

No MeSH data available.


Immunoblot analysis for expression of the B. bovis 6-Cys A and B proteins in blood, midgut, and hemolymph stages. A. Immunoblot analysis of uninfected RBC and cultured B. bovis infected RBC with rap-1 mAb BABB75, rabbit polyclonal sera against 6-Cys A-specific synthetic peptide and 6-Cys B-specific synthetic peptide. Synthetic A and B peptides were used as positive controls indicated by the black arrow. The size of peptide A is 21.6 D and peptide B is 24 D. B. Immunoblot analysis of non-infected and B. bovis infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys A-specific synthetic peptide, and pre-immune rabbit sera (PI). C. Immunoblot analysis of non-infected and B. bovis-infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys B-specific synthetic peptide, and pre-immune rabbit sera. Size markers (M) in kDa are indicated at the left of each panel.
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pone.0163791.g005: Immunoblot analysis for expression of the B. bovis 6-Cys A and B proteins in blood, midgut, and hemolymph stages. A. Immunoblot analysis of uninfected RBC and cultured B. bovis infected RBC with rap-1 mAb BABB75, rabbit polyclonal sera against 6-Cys A-specific synthetic peptide and 6-Cys B-specific synthetic peptide. Synthetic A and B peptides were used as positive controls indicated by the black arrow. The size of peptide A is 21.6 D and peptide B is 24 D. B. Immunoblot analysis of non-infected and B. bovis infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys A-specific synthetic peptide, and pre-immune rabbit sera (PI). C. Immunoblot analysis of non-infected and B. bovis-infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys B-specific synthetic peptide, and pre-immune rabbit sera. Size markers (M) in kDa are indicated at the left of each panel.

Mentions: Transcriptional data demonstrate that Bbo 6-Cys genes A and B are constitutively transcribed in tick midgut and hemolymph during the parasite’s life cycle. To investigate if the Bbo 6-Cys genes A and B are translated in these stages, monospecific polyclonal antibodies against peptide cocktails derived from both proteins were used in western blot analysis. RAP-1 monoclonal antibodies were used as a positive and negative control in these western analyses and as expected, RAP-1 was detected infected blood but not hemolymph. Synthetic peptides specific to proteins A and B were also included as controls in the western blots and as expected, reacted positively with antibodies against peptide cocktails specific to proteins A and B (Fig 5A). In contrast, the Bbo 6-Cys proteins A and B were not detected in cultured red blood cells infected with B. bovis in identical immunoblots (Fig 5A). However, the 6-Cys A and B proteins were detected in both midgut and hemolymph stages of B. bovis. Faint bands of approximately 66 kDa which correspond to both proteins A and B were observed in the immunoblots (Fig 5B and 5C). Collectively, these data support the differential expression of the 6-Cys A and B proteins in midgut and hemolymph stages of the parasite.


Expression of 6-Cys Gene Superfamily Defines Babesia bovis Sexual Stage Development within Rhipicephalus microplus
Immunoblot analysis for expression of the B. bovis 6-Cys A and B proteins in blood, midgut, and hemolymph stages. A. Immunoblot analysis of uninfected RBC and cultured B. bovis infected RBC with rap-1 mAb BABB75, rabbit polyclonal sera against 6-Cys A-specific synthetic peptide and 6-Cys B-specific synthetic peptide. Synthetic A and B peptides were used as positive controls indicated by the black arrow. The size of peptide A is 21.6 D and peptide B is 24 D. B. Immunoblot analysis of non-infected and B. bovis infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys A-specific synthetic peptide, and pre-immune rabbit sera (PI). C. Immunoblot analysis of non-infected and B. bovis-infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys B-specific synthetic peptide, and pre-immune rabbit sera. Size markers (M) in kDa are indicated at the left of each panel.
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pone.0163791.g005: Immunoblot analysis for expression of the B. bovis 6-Cys A and B proteins in blood, midgut, and hemolymph stages. A. Immunoblot analysis of uninfected RBC and cultured B. bovis infected RBC with rap-1 mAb BABB75, rabbit polyclonal sera against 6-Cys A-specific synthetic peptide and 6-Cys B-specific synthetic peptide. Synthetic A and B peptides were used as positive controls indicated by the black arrow. The size of peptide A is 21.6 D and peptide B is 24 D. B. Immunoblot analysis of non-infected and B. bovis infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys A-specific synthetic peptide, and pre-immune rabbit sera (PI). C. Immunoblot analysis of non-infected and B. bovis-infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys B-specific synthetic peptide, and pre-immune rabbit sera. Size markers (M) in kDa are indicated at the left of each panel.
Mentions: Transcriptional data demonstrate that Bbo 6-Cys genes A and B are constitutively transcribed in tick midgut and hemolymph during the parasite’s life cycle. To investigate if the Bbo 6-Cys genes A and B are translated in these stages, monospecific polyclonal antibodies against peptide cocktails derived from both proteins were used in western blot analysis. RAP-1 monoclonal antibodies were used as a positive and negative control in these western analyses and as expected, RAP-1 was detected infected blood but not hemolymph. Synthetic peptides specific to proteins A and B were also included as controls in the western blots and as expected, reacted positively with antibodies against peptide cocktails specific to proteins A and B (Fig 5A). In contrast, the Bbo 6-Cys proteins A and B were not detected in cultured red blood cells infected with B. bovis in identical immunoblots (Fig 5A). However, the 6-Cys A and B proteins were detected in both midgut and hemolymph stages of B. bovis. Faint bands of approximately 66 kDa which correspond to both proteins A and B were observed in the immunoblots (Fig 5B and 5C). Collectively, these data support the differential expression of the 6-Cys A and B proteins in midgut and hemolymph stages of the parasite.

View Article: PubMed Central - PubMed

ABSTRACT

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.

No MeSH data available.