Cysteine Proteases and Cell Differentiation: Excystment of the Ciliated Protist Sterkiella histriomuscorum

Abstract

The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.The exit from dormancy, of which excystment is a particular case, is a widespread process in many eukaryotic and prokaryotic organisms. It is generally accompanied by cellular differentiation, during which numerous intra-and extracellular structures are highly modified, as observed during the germination of fungal conidia and plant seeds (reviewed in references 5, 31, and 39), among others. Cysteine proteases, which are known to be involved in intracellular protein turnover and extracellular matrix remodeling (37), constitute good candidates to take part in this general process. In this paper, we describe the involvement of such proteins in the process of excystment of the ciliate Sterkiella histriomuscorum.S. histriomuscorum (previously called Oxytricha fallax and O. trifallax) (3) is a free-living ciliated protist from the hypotrich subgroup that is characterized by a highly differentiated pattern of the vegetative cell. This pattern includes a complex oral apparatus and a clustered body ciliature which are duplicated in an orderly process during cell division (22). The S. histriomuscorum life cycle is interesting in that the vegetative cell undergoes a complete dedifferentiation process when it is deprived of food, leading in about 3 days to a resting stage which is called a cyst. The mature cyst has lost much water, the whole ciliature and basal bodies have disappeared, numerous autophagic vacuoles (lysosomes) that were particularly activ...

Phylogenetic and Primary Sequence Characterization of Cathepsin B Cysteine Proteases from the Oxymonad Flagellate Monocercomonoides

“…1, 2). Importantly, despite all of the sequences retrieved being verified as cathepsin B homologues, only some of them possessed the occluding loop motif (Sajid and McKerrow 2002;Villalobo et al 2003) (Fig. 6).…”

“…Cathepsin B has been implicated in excystation in both free-living (Villalobo et al 2003) and parasitic protists (DuBois et al 2006). In both cases, high expression of cathepsin B was observed in the vegetative life-stage of the cells post-excystation.…”

“…Cathepsin B homologues are widely distributed in eukaryotes but have been best characterized in metazoans, where they are involved in lysosomal degradation, and in kinetoplastid parasites, where they are involved in immune system evasion and pathogenesis (Mottram, Brooks, and Coombs 1998;Somanna, Mundodi, and Gedamu 2002). A role for cathepsin B in excystation has also been documented in both free-living (Villalobo et al 2003) and parasitic protists (DuBois et al 2006).…”

“…This motif has been considered a defining feature of cathepsin B and is well conserved in many of the characterized homologues to date. However, the occluding loop is absent from some cathepsin B sequences, including homologues from plants, chromalveolates, Giardia, and animals (Sajid and McKerrow 2002;Villalobo et al 2003).…”

Microfossils from the lower Mesoproterozoic Kaltasy Formation, East European Platform

“…Leiosphaerids generally lack ultrastructural features known to be associated with prasinophytes, but a distinctive TLS (trilaminar sheath structure) ultrastructure has been recognized in TEM images of Cambrian and Neoproterozoic specimens, supporting their interpretation as chlorophytes (Talyzina and Moczydłowska, 2000;and, with less certainty, Moczydłowska et al, 2010). This, however, does not mean that all spheroidal acritarchs were sourced by green algae, as potentially preservable spheroidal envelopes are made by organisms ranging from cyanobacteria (e.g., Fairchild, 1985;Sun, 1987;Sergeev, 1992) to ciliates (e.g., Villalobo et al, 2003). Questions of systematic affinity become more challenging in older successions, where the probability of encountering extinct stem group lineages increases substantially.…”