Abstract
Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1–3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this “minimal Golgi” hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.
Highlights
Infectious parasite stages transmitted to a new host via the oral route require a highly resistant extracellular matrix to protect them in the environment and during passage through the stomach
Previous work shows that encystation-specific vesicles (ESVs) are most likely minimal pulsed Golgi-like compartments for exporting pre-sorted cyst wall material
By tracking immature and processed forms of the three cyst wall proteins during differentiation we discovered a novel sorting function which results in partitioning of ESV cargo and sequential secretion of the cyst wall material
Summary
Infectious parasite stages transmitted to a new host via the oral route (cysts, oocysts, eggs) require a highly resistant extracellular matrix to protect them in the environment and during passage through the stomach. Trophozoites in the small intestine or in culture undergo stage-differentiation to a cyst form in response to environmental cues, e.g. changes in pH, bile and/or cholesterol concentration [2,3]. The members of this phylum have undergone strong reductive evolution resulting in minimization or loss of cellular systems and organelles such as mitochondria, peroxisomes and the Golgi apparatus [4,5,6], but despite significant advances in phylogenetic analysis their point of divergence during evolution remains elusive [7]. Reductive evolution is the most parsimonious, albeit still unproven, explanation for the absence of a Golgi organelle and Golgi functions in Giardia trophozoites [8]
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