Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage

Abstract

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle. IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.

Imaging of Virus-Infected Cells with Soft X-ray Tomography

“…Cryo-SXT can provide understanding of the spatial organization of the different cellular organelles during the infection, but to achieve a better comprehension of the complexity of virus-host interactions, it is recommended to combine cryo-SXT data with other imaging techniques that can provide the precise localization or the higher resolution structure of the particular viral or host factors being studied. Correlative workflows combining cryo-SXT with techniques such as cryo-ET, visible light fluorescence microscopy, hard X-ray fluorescence tomography or numerical simulations have already proven their potential in several multi-length scale structural studies [24,[43][44][45][46][75][76][77]. A particularly powerful approach is a correlative cryo-imaging pipeline, in which the same sample is imaged with super-resolution 3D structured illumination fluorescence microscopy (cryo-SIM) and with cryo-SXT, allowing for a comprehensive view of both cellular ultrastructure and the related molecular organization over extended cellular volumes [78,79].…”

Plasmepsin-like Aspartyl Proteases in Babesia

“…In B. microti-the basal piroplasmid species (Babesia microti-like group)-the obscure coiled structure that protrudes from the parasite into the host cytoplasm has been observed and speculated to contain digestive enzymes. Similarly, possible haemoglobin-containing vesicles were also observed from B. divergens trophozoites, indicating-yet not confirming-the endocytic uptake of hemoglobin from the cytoplasm of host erythrocytes [47]. The role of hemoglobin invaginations in trophozoites of different species of Babesia remains unclear, but could be relevant to the biology of piroplasms, e.g., by limited digestion of hemoglobin providing the essential source of heme to these heme auxotrophic organisms.…”

Comparative single-cell transcriptional atlases ofBabesiaspecies reveal conserved and species-specific expression profiles

“…In contrast, B. divergens has a much more complex replicative cycle (55). Indeed, conflicting literature exists suggesting the replication cycle varies from 4-12 hr, however in most cases it appears the minimal time for division is between 4-5 hr (55,105,106). Of these, only a single study was conducted on synchronous parasites (55).…”

“…Subsequent division cycles are possible in B. divergens and the timing of these cycles can vary between 9-14 hr (55). In all studies of the dynamics of B. divergens division dynamics, there is a significant range of time for a single division, highlighting the difficulty in generating an exact measurement (55,105,106). This variability leads to difficulty in synchronizing Babesia parasites, which currently relies on mechanical release of parasites from RBCs using filtration (55, 56), and we have recently shown that parasites may be egress competent at various times in their intraerythrocytic development (37).…”