Hap2, a novel gene in Babesia bigemina is expressed in tick stages, and specific antibodies block zygote formation

Abstract

BackgroundBovine babesiosis is a tick-borne disease caused by the protozoan parasites of the genus Babesia. In their host vector, Babesia spp. undergo sexual reproduction. Therefore, the development of sexual stages and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle. HAP2/GCS1 is a protein identified in plants, protozoan parasites and other organisms that has an important role during membrane fusion in fertilization processes. The identification and characterization of HAP-2 protein in Babesia would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future.ResultsTo isolate and sequence the hap2 gene DNA from an infected bovine with Babesia bigemina was purified. The hap2 gene was amplified, cloned and sequenced. The sequences of hap2 from four geographically different strains showed high conservation at the amino acid level, including the typical structure with a signal peptide and the HAP2/GSC domain. Antisera anti-HAP2 against the conserved extracellular region of the HAP2 amino acid sequence were obtained from rabbits. The expression of hap2 in the host and vector tissues was analyzed by using semi-quantitative RT-PCR, and the protein was examined by western blot and immunofluorescence. Based on the RT-PCR and WB results, HAP2 is expressed in both, sexual stages induced in vitro, and in infected ticks as well. We did not detect any expression in asexual erythrocytic stages of B. bigemina, relevantly anti-HAP2 specific antibodies were able to block zygotes formation in vitro.Conclusion Babesia bigemina HAP2 is expressed only in tick-infecting stages, and specific antibodies block zygote formation. Further studies regarding the function of HAP2 during tick infection may provide new insights into the molecular mechanisms of sexual reproduction of the parasite.Electronic supplementary materialThe online version of this article (10.1186/s13071-017-2510-0) contains supplementary material, which is available to authorized users.

Identification of a putative methyltransferase gene of Babesia bigemina as a novel molecular biomarker uniquely expressed in parasite tick stages

“…As a result, in vivo induction of sexual stages would be expected to occur non-synchronously and the rise and fall of ccp expression would be masked. In addition to ccp2 and ccp3 , expression of hap2 , a gene found to be important in B. bovis sexual stage development [ 16 ], was upregulated at 6 h but, unlike the expression pattern of the ccp genes, remained elevated at 15 and 24 h after TCEP induction (Additional file 2 : Figure S2), consistent with a previous study [ 13 ].…”

“…The results represent the means of three experiments, each containing three technical replicates. In addition to the ccp genes, we examined the expression of B bigemina hap2 (BBBOND_0307410) in TCEP-induced parasites and found it was upregulated at 6, 15 and 24 h (Additional file 2 : Figure S2) as previously demonstrated [ 13 ]. Additionally, we confirmed polypeptide expression of CCp2 and CCp3 by IFA using a set of previously characterized polyclonal antibodies [ 12 ].…”

“…One of the challenges in studying Babesia gametocytes is the lack of in vitro techniques to consistently induce gametogenesis which restricts evaluation of drugs or vaccines targeting parasite sexual stage proteins. Two studies have successfully induced B. bigemina sexual stages using xanthurenic acid, but both studies required ex vivo blood stages as starting materials [ 13 , 19 ]. However, malaria gametogenesis can be induced from in vitro adapted parasites.…”

An experimental genetically attenuated live vaccine to prevent transmission of Toxoplasma gondii by cats

“…Amongst numerous putative microgamete-specific transcripts in our data, the one coding for a microgamete-specific gamete fusion protein, HAP2, particularly drew our attention because of its relatively well-defined localisation in microgametes, its role in membrane fusion leading to fertilisation and zygote formation in Plasmodium 41,42 and Babesia 43,44 , and its prominence in the microgamete transcriptome of E . tenella 15 , all of which underscores its vital, conserved function in sexual reproduction in the Apicomplexa 45 .…”

Identification of proteins expressed by Babesia bigemina kinetes

“…Bohaliga et al [11] identified BBBOND_0204030 as a gene uniquely transcribed by B. bigemina tick stages and by in vitro induced B. bigemina sexual stages. The B. bigemina HAP2 protein, that is associated with the development of gametes in apicomplexan parasites [12], was found to be expressed by parasite stages developing in the midgut of infected ticks at day 3 post-repletion of female R . microplus ticks.…”