The hows and whys of amastigote flagellum motility in Trypanosoma cruzi

ABSTRACT The protist Trypanosoma cruzi exhibits several extracellular stages characterized by the presence of a long and motile flagellum and one intracellular life cycle stage termed amastigote, which possesses a tiny flagellum barely exiting the flagellar pocket. This stage was so far described as replicative but immotile cells. Unexpectedly, the recent work of M. M. Won, T. Krüger, M. Engstler, and B. A. Burleigh (mBio 14:e03556-22, 2023, https://doi.org/10.1128/mbio.03556-22) revealed that this short flagellum actually displays beating activity. This commentary explores how such a short flagellum could be constructed and why it could affect the parasite’s survival inside the mammalian host.


THE SURPRISE
T rypanosoma cruzi, the causative agent of Chagas disease, is a flagellated parasite with a complex life cycle between two hosts. It passes through three major stages: the epimastigote, present in the insect host; the trypomastigote, found in the blood stream of the mammalian host; and the amastigote, present inside the mammalian host cells. The epimastigote and trypomastigote forms have a long motile flagellum, with the canonical 9+2 axoneme. T. cruzi and most trypanosomatid parasites have an extra-axone mal structure, the paraflagellar rod (PFR), which is present in these two life cycle stages and is required for cell motility in epimastigote (1). By contrast, the amastigote form is small and has a short flagellum, with an average length of 2.7 µm and a distal end outside the flagellar pocket. After more than a century of investigations, the recent work of Won and colleagues brings to light the unexpected motile activity of the amastigote short flagellum. They describe the flagellar beats in a quasiperiodic behavior with a variable beating frequency and a rotational movement, showing motility as an intrinsic property of the amastigote flagellum. This result opens several questions, such as (i) how is this short flagellum formed during differentiation from trypomastigote? and (ii) why does amastigote keep a motile flagellum?
How can the amastigote short flagellum have an active beating? T. cruzi amastigote maintains elements of a motile 9+2 axoneme such as dynein arms and central pair apparatus, as shown by structural ( Fig. 1A and B) and biochemical approaches (2)(3)(4), arguing in favor of a motile activity. In amastigote, it is commonly reported that the PFR is absent. Intriguingly, when observed by electron microscopy, the amastigote flagellum exhibits some electron-dense material at the same position where the PFR is observed in other stages (Fig. 1A), suggesting the existence of a reduced PFR. Moreover, proteome data reveal the presence of PFR proteins, such as PFR2 and PAR4, in amastigote forms (2,4). In epimastigote forms, where the PFR is present, the flagellar beat shows a different pattern with asymmetric base-to-tip waves (5). Won and colleagues argue that the quasiperiodic beating pattern observed in the amastigote flagellum can be given by its short length, but how much impact does a reduced or possibly absent PFR have on these differences in wave forming?

HOW IS THE SHORT MOTILE FLAGELLUM CONSTRUCTED?
How could such a short flagellum be put in place during differentiation? Does it originate from the shortening of the trypomastigote flagellum or de novo assembly, as debated for Leishmania (8)? Assembling a flagellum relies on the well-conserved transporting mechanism called intraflagellar transport (IFT), which delivers the building elements to the tip of the growing flagellum (9). A dense structure between the axoneme and the flagellar membrane that resembles an IFT particle is actually observed on the amastigote flagellar section (Fig. 1A, arrowhead). This morphological data is strengthened by the presence of some IFT proteins (IFT81, IFT88, and IFT20) identified in amastigote during the flagellum membrane protein SMP1-1 proximity proteome (4).
If the amastigote flagellum is assembled by IFT, why is it so short? Several models can be proposed. The first one is based on what was described in the T. brucei procyclic form that uses a grow-and-lock model to prevent further elongation of the existing flagellum (10). One could imagine a premature locking leading to the formation of a very short flagellum. The second is the "balance-point" model, where the axoneme length is determined by the equilibrium between flagellum assembly and disassembly (11). That would suggest that the amastigote flagellum is less stable or at least has a faster turnover when compared to other stages. A third model would be the limitation of the pool of soluble tubulin or associated material.

OTHER SHORT FLAGELLA, DIFFERENT FUNCTIONS?
The presence of a life cycle stage with a short flagellum is not exclusive to T. cruzi, and it is observed in at least two other cases. The first one is the amastigote stage of L. mexicana. They are morphologically close to T. cruzi amastigotes and are also found inside the host cell; however, their short flagellum ( Fig. 1C and D) lacks the dynein arms and the central pair (7) and presumably is immotile. Therefore, although L. mexicana and T. cruzi share a morphologically similar amastigote life cycle stage, their functions are clearly different. This opens the discussion of how these differences can be related to specific interactions between these parasites and their host cells.
The second case is the T. brucei short epimastigote found in the cardia of the insect host, the tsetse fly. The short epimastigote keeps a short motile flagellum that has the canonical axoneme and the PFR. Moreover, it has been proposed that this form is Commentary mBio responsible for the invasion of the salivary glands and that the short flagellum could be responsible for anchoring to the epithelium, possibly via a sensory mechanism (12,13). This form also keeps an active IFT system that could be responsible for the flagellum elongation during differentiation to the attached epimastigote stage (14). This situation is therefore more akin to that of the T. cruzi amastigote.

WHY WOULD THE AMASTIGOTE STAGE NEED MOTILITY?
Is the amastigote parasite able to move in the cytosol of host cells? It was observed in the alga Chlamydomonas reinhardtii that a flagellum longer than 2 µm can generate a rotation force in the cell body (15). Given that, the amastigote flagellum could, in theory, power cell movement (Fig. 1E-a), although that has not been reported yet. In this context, the T. cruzi flagellum could be guided by sensing means, as this organelle is considered a sensory platform since it contains different signaling proteins localised to its membrane like the putative calcium-sensor FCaBP (16) and the cation channel TcCat (17). The close contact of amastigote flagellum with host cell mitochondria was already reported, and a sensory role was speculated at the time (18). Motility could contribute to this interaction, with cell movement possibly helping the search for host organelle targets through signaling molecules ( Fig. 1E-b). Flagellar motility could also guide amastigote parasites when searching for and uptaking nutrients within the host cell ( Fig. 1E-c). In that matter, it is noteworthy that T. cruzi intracellular amastigote has an active endocytic system, in contrast to the extracellular amastigote stage or during differentiation to trypomastigotes (19). Flagellar movement could also confer advantages for the infectivity of extracellular amastigote. These forms represent 10% of circulating parasites in the blood during acute infection (20), and their capability to infect host cells is well known (21,22). Could flagellum beating play an active role in generating host cell response to the parasite? Trypomastigote forms can invade host cells by recognition and attachment throughout their flagellum, producing membrane damage that triggers the repairing mechanism by lysosome fusion (23). Even more, the cell body distortions conferred by flagellar movement can also affect infectivity. Trypomastigote shows an active gliding movement while attached to the host cell surface through its posterior region, leading to membrane deformations that help parasite invasion (24). Although extracellular amastigote invades the host cell through a phagocytosis-like pathway, the flagellar beat could confer motility to the cell, helping with the search and recognition of the entry site by a sensory mechanism or even triggering the host cell response by generating movements of the whole cell ( Fig. 1E-d).
The amazing discovery of motility in this short flagellum reminds us that, after more than a century of observing T. cruzi, we can still have surprises. The finding changes the way we look at the working mechanisms of a well-known structure as the flagellum and turns the amastigote short and active flagellum into an intriguing model to explore flagellum construction and motility. This discovery opens the field to other possible roles the short beating flagellum could play on parasite survival inside and outside the host cell.
We thank Parul Sharma and Jean Marc Tsagmo Ngoune for critical reading of the manuscript.