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. 2020 Oct 14;5(5):e00928-20.
doi: 10.1128/mSphere.00928-20.

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

José Javier Conesa #  1   2 , Elena Sevilla #  3 , María Carmen Terrón  4 , Luis Miguel González  3 , Jeremy Gray  5 , Ana J Pérez-Berná  2 , José L Carrascosa  1 , Eva Pereiro  2 , Francisco Javier Chichón  1 , Daniel Luque  6 , Estrella Montero  7
Affiliations

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

José Javier Conesa et al. mSphere. .

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.

Keywords: Babesia divergens; cryo-soft X-ray tomography; intraerythrocytic asexual cycle; pathogen-host cell interactions; time-lapse video microscopy.

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Figures

FIG 1
FIG 1
Correlative cryo-epifluorescence and cryo-soft X-ray tomography imaging of B. divergens human iRBCs. (a) This correlative workflow, used at MISTRAL beamline at ALBA synchrotron, provides biological images and structural information of whole B. divergens human iRBCs close to their native state at a spatial resolution of around 50 nm. B. divergens iRBCs are tilted to different angles, and an image is acquired at each angle. The tilt series of images are reconstructed into a 3D tomogram providing structural information of the whole cells. (b) B. divergens asynchronous in vitro cultures labeled with MitoTracker Red (red fluorescence) are deposited on to holey carbon EM grids in an optimal cell confluence (105 cells per grid) and plunge-frozen in liquid ethane. The vitrified grids are screened with an online epifluorescence microscope to generate a fluorescence map and select the most relevant cells (yellow square). (c) Grids are loaded into the MISTRAL transmission X-ray microscope at the ALBA synchrotron light source for screening. An X-ray mosaic of projection images is generated and the previous fluorescence map helps in locating again the same cells in the yellow square. (d) Cryo-SXT tomogram sections of a B. divergens multiparasite stage acquired in the yellow squared area in panel c. (e) 3D rendering of the acquired cryo-SXT tomogram shown in panel d. The scale bars in panels b, c, and d represent 100, 20, and 2 μm, respectively.
FIG 2
FIG 2
3D architecture of intact B. divergens parasites. Panels show the cryo-SXT tomogram sections of the B. divergens free merozoite and six main stages within human RBCs. The corresponding 3D tomogram renderings show the architecture of intact parasites and the organelle distribution in color. Insets show the equivalent parasite stages stained with Giemsa and observed by light microscopy (top inset) or stained with MitoTracker (green fluorescence) and observed using a confocal laser microscope (lower inset). (a and b) Free merozoite. (c and d) Single round trophozoite stage. (e and f) Double round trophozoite stage. (g and h) Quadruple round trophozoite stage. (i and j) Paired-pyriform stage. (k to l) Tetrad (Maltese Cross). (m, n, and o) Tomogram sections of a double paired pyriform stage at different depths over the same 3D reconstruction. (p) 3D tomogram rendering of the corresponding double paired pyriform stage. The scale bars in panels a to p represent 2 μm. The scale bars in the insets in panels a to p represent 5 μm.
FIG 3
FIG 3
Dynamic development of single trophozoite and paired pyriform stages. The figure shows the development of the single trophozoite and paired pyriform stages and the transient forms that intersperse in a chronological order between both main stages within the human RBC. (a to f) Time-lapse image sequences, captured by video microscopy, of B. divergens parasites stained with MitoTracker (green fluorescence) within the human RBC. Equivalent IE parasite forms are identified in in vitro cultures by cryo-SXT and Giemsa stain and light microscopy. (g to k) Cryo-SXT tomogram sections of the B. divergens iRBCs. (l to p) The corresponding 3D tomogram renderings show the architecture of intact parasites and the organelle distribution in color. (q to u) B. divergens iRBCs stained with Giemsa. Panels are organized sequentially according to the video microscopy data. (a and b) A single trophozoite adopts amoeboid shapes until it reaches the budding form at 4 h and 15 min. (c) The budding form develops into an early paired pyriform at 4 h and 40 min. (d) The early paired pyriform ultimately develops into a paired pyriform stage at 6 h and 10 min. (e and f) The paired pyriform stage develops into the double trophozoite stage. (g, l, and q) Single trophozoite stage. (h, m, and r) Budding form. (i, n, and s) Early paired pyriform under development. (j, o, and t) Paired-pyriform stage. (k, p, and u) Double trophozoite stage. (h to m) Budding form showing the initial segregation of cell material that appears concentrate in both buds. (n) Detail of the transverse constriction of the main cellular body and the presence of some organelles across the constriction zone. (o) Each daughter pear-shaped cell inherits a complete set of organelles at the end of the binary fission. The scale bars in panels a to k and q to u represent 5 μm. The scale bars in panels l to p represent 2 μm. Time-lapse imaging was captured every 5 min. The time lapse between each frame is indicated in hours and minutes (see also Movie S1 at https://figshare.com/s/8ba6afd9e161899d682c).
FIG 4
FIG 4
4D reconstruction of the main and intermediate IE stages that encompass the B. divergens asexual cycle. The cycle model shows a detailed chronological development that main IE stages undergo within the human RBC after the free merozoite invasion. (a to p) 3D rendering of cryo-SXT tomograms organized according to the time-lapse images generated for parasites at different stages of development (green fluorescence). Cellular compartments and the distribution of the parasite organelles are indicated in color. (a) A free merozoite about to invade a new human RBC. (b) Single trophozoite (main stage). (c) Budding form. (d) Early paired pyriform under development. (e) Paired-pyriform (main stage). (f) Initial development process of a paired pyriform into a tetrad. (g) Tetrad (main stage). (h) Two unattached pear-shaped parasites and a paired pyriform. (i) Quadruple trophozoites (main stage). (j) Paired pyriform (main stage). (k) Double trophozoites (main stage). (l) Two trophozoites adopting amoeboid forms during their development into double paired pyriforms. (m) Double budding form. (n and o) Double paired pyriforms (main stage). (p) Multiparasite stages (main stage). IE, intraerythrocytic.
FIG 5
FIG 5
Simplified model of the B. divergens asexual cycle: from the single trophozoite to the multiparasite stage. The virtual model shows the transformation that a single trophozoite undergoes to become a multiparasite stage within one human RBC (red dotted line). This is possible through several rounds of multiplication by binary fission involving budding within the same host cell. This process starts with the invasion by a free merozoite (surrounded by a white dotted line) and its transformation into a single trophozoite inside the RBC. The single trophozoite develops into a paired pyriform. This new stage develops into double trophozoites or tetrads that precede double paired pyriforms. This last one may develop into quadruple trophozoites and/or multiparasite stages. Quadruple trophozoites can also develop into multiparasite stages. Importantly, paired pyriforms, double paired pyriforms, tetrads, and multiparasite stages, instead of developing within the RBC, can exit the host cell as free merozoites capable of invading new RBCs resulting in a rise in parasitemia.
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