Delayed cytokinesis generates multinuclearity and potential advantages in the amoeba Acanthamoeba castellanii Neff strain

We showed that agitating conditions promoted multinuclearity in A. castellanii and V. vermiformis by generating a cytokinesis delay. Interestingly, multinuclearity induced by agitation has been shown for other aquatic protists as well, such as Naegleria gruberi⁴ and Hartmannella rhysodes⁵. Tetranucleated cysts are part of the infection cycle of E. histolytica¹. Based on the data presented in our study, we can suggest that this specific multinucleate stage in E. histolytica could provide a growth advantage during infection that allows this pathogenic amoeba to efficiently produce numerous descendants when colonizing a new host. This hypothesis remains to be tested. In this light, the involvement of multinucleate cells in A. castellanii pathogenicity deserves to be investigated, and direct single-cell level analyses on human samples, instead of classical amoeba isolation protocols¹⁷, could render these observations possible.

The strain used in our study is the Acanthamoeba castellanii (Douglas) ATCC30010 (strain Designations: Neff, Depositor RJ Neff, biosafety level 1). This A. castellanii isolate has kept its cell cycle related ability to encyst, giving rise to non-adherent mature cysts under starvation conditions^18,19. In addition, this isolate retains its capacity to efficiently feed on bacterial preys. Taken together, these observations show that the validated amoebal isolate used in our study is not axenically adapted, at least to the extents regarding its cell cycle and phagocytosis processes.

At least two different, yet non-exclusive mechanisms can generate multinuclearity (Fig. S3). Firstly, uninucleate amoebae could fuse together leading to the formation of a multinucleate cell, therefore forming a syncytium. This hypothesis is interesting knowing that A. castellanii are asexual organisms replicating through mitosis¹⁵, while possessing meiotic genes^20,21,22. Reuniting multiple nuclei, originating from different cells, in a single amoeba could promote genetic material exchange and create diversity, essential for evolution. In this light, the amoeba Cochliopodium spp. was previously reported to undergo cellular followed by nuclear fusions²³. In case of cellular fusions of uni- and multinucleate amoebae occurring randomly in A. castellanii, we would expect another distribution of the number of nuclei per amoeba including even and odd numbers, which is not what we report in our study (Figs. 3 and S3). A potential mechanism that regulates particular A. castellanii cell types to fuse together, such as the mating types designated MATa and MATα in yeast²⁴, cannot be excluded at this stage. Also, if binucleated cells are more prone to fuse with binucleated cells, this would generate tetranucleated cells. Using live cell imaging and time lapse microscopy, we never observed such fusion events. However, at this stage, we cannot exclude that the frequency of cellular fusion is too low to be detected in our conditions or that the tested conditions in our laboratory did not promote fusion. A second hypothesis proposes that multinuclearity originates from endoreplication, consisting of synchronous nuclei replication without cytokinesis, successively generating from an uninucleate cell (1n) polyploid cells such as 2n, 4n, 8n, … (Fig. S3). The fact that under non-adherent conditions bi- and tetra- nucleated amoebae represent the vast majority of the population (and to a minor extent 8 nuclei) provides support for this second hypothesis (Figs. 2, 3). In our study, no case of uneven nuclei per amoeba were observed (except for single nucleated amoebae). The endoreplication hypothesis is supported by the fact that amoebae must be adherent to their substrate to divide, because the locomotion system is important to tear apart the daughter cells⁵. Agitation would impair such adhesion, and multinuclearity would be a consequence of amoebae trying to divide but ultimately failing at the cytokinesis stage, resulting in 2ⁿ number of nuclei. This endoreplication hypothesis does not exclude the fusion hypothesis and both phenomena could occur concomitantly at different rates, depending on the tested conditions.

Producing a higher number of clonal progenies for the multinucleate cells (Fig. 5) and a better substratum colonization (Fig. 6) is equivalent to a greater individual reproductive success, which is one of the aspects of a fitness advantage^25,26. Upon cellular attachment to a new solid surface-to-be-colonized and based on cytokinesis events only (without the need to replicate its genome at that moment), multinucleate amoebae can generate sibling cells faster than uninucleate amoebae, all else being equal (Figs. 3, 5, 7). The observed generation advantage takes place specifically upon the process of new substrate colonization. Indeed, multinucleate amoebae had to replicate their DNA content before, as for uninucleate ones, and if we take this into account, it might be possible that no fitness advantage could be highlighted. Nevertheless, when focusing on the colonization timing, when two amoeba cells arrive together (uni-versus multinucleate) and compete for resources, multinucleate cells do have a colonization advantage as they do not need to replicate their DNA content, resulting in a faster division accompanied by an increased population growth (Fig. 7). In addition, the observation that multinucleates are giant cells might help amoebae to sediment and adhere to their new substrate faster than uninucleate ones. This observed advantage relies on the quantification of the number of single celled amoebae and might differ at the level of biomass or total nuclei number.

Concerning the non-adherent conditions, as A. castellanii amoebae are frequently found in aquatic environments, it is possible that in their natural niche, A. castellanii cells regularly detach from a biotic or abiotic surface. For example, an early step during encystation is cell detachment. Before this differentiation process being irreversible^9,11, detached amoebae could have the time to be transported in the water column to colonize a new environment. In addition, any flux perturbations, water currents or waves might detach amoebae from their substrate and suspend them in water, in a non-adherent state. This process might help amoebae to colonize new environmental niches that are difficult to reach using an adherent state. This cellular detachment is seen in our control conditions when adherent amoebae are agitated, which leads to a small percentage of detached multinucleate amoebae even if the majority of the population remain adherent and uninucleate (Fig. 3). Detached amoebae would therefore get disseminated by water currents, replicating their DNA content without cytokinesis, preparing themselves to efficiently colonize a new habitat. To test this hypothesis, such colonizers could be collected directly from natural environmental water columns. However, the current isolation methods do not allow any direct and high-throughput single cell based-observations of collected amoebae, together with their precise identification, as they are rather based on isolation of amoebae through enrichment on bacterial lawns¹⁷. As shown in our study, even if any multinucleate amoebae are collected and inoculated on solid media from natural environment, the multinuclearity could be lost after a few stretchy cytokineses.

Excessive cell growth leads to cytoplasm dilution that impairs cellular processes such as cell signalling and translation²⁷. There is an optimal DNA/cytoplasm ratio, and values outside the tolerance zone impair cellular fitness as RNA and protein synthesis become limiting, leading to cellular senescence²⁷. Giant multinucleate cells could therefore face cytoplasm dilution. Hence, as a physiological response, amoebae may increase the number of nuclei to counteract the cytoplasm dilution phenomenon.

Source: Ecology - nature.com