Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites

Abstract

Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity.

Plasmodium falciparumOTU-like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with noncanonical role of Atg8

“…In higher eukaryotes, it has been suggested that deconjugation of Atg8 from PE plays an important role in regulation of autophagy. Further, it is also suggested that Atg8 plays a role in microneme clearance as well as autophagosome fusion similar to mammalian systems (Voss et al, 2016). In addition, the canonical role of Plasmodium Atg8 in cellular processes such as ribophagy and piecemeal microautophagy of the nucleus has been also suggested (Cervantes et al, 2014).…”

“…In addition, the canonical role of Plasmodium Atg8 in cellular processes such as ribophagy and piecemeal microautophagy of the nucleus has been also suggested (Cervantes et al, 2014). Further, it is also suggested that Atg8 plays a role in microneme clearance as well as autophagosome fusion similar to mammalian systems (Voss et al, 2016).…”

ToxoplasmaTgATG9 is critical for autophagy and long-term persistence in tissue cysts

“…These spherical structures were reported to encapsulate a variety of cytoplasmic structures and organelles, which is similar to the apparent sequestration of ER, secretory organelles, lipid droplets, and even parts of the mitochondria itself in ∆atg9 bradyzoites. It is worth noting that previous reports have implicated ATG8 in organelle clearance in Plasmodium spp, which is essential to parasite development (34,53). We speculate that the encapsulation of organelles by multimembrane vesicles (including apparent m-vacuoles) observed here could represent an attempt at organelle clearance in the absence of ATG9-dependent canonical autophagy in the related apicomplexan T. gondii.…”

“…While a number of proteins involved in apicoplast maintenance have been studied and shown to be essential in T. gondii and Plasmodium spp. (22)(23)(24)29,(32)(33)(34)(35), little is known about the role of proteins that function earlier in the pathway. The autophagy-related protein TgATG9 has previously been shown to be non-essential for survival of T. gondii tachyzoites within the PV in vitro (31).…”

A synthetic promoter for multi-stage expression to probe complementary functions of Plasmodium adhesins

“…So far, investigation of later stages, in which the importance of the specific gene is unknown, has been attempted by using two different strategies. First, small genes can be expressed as a second copy driven by a different promoter, which leaves the main expression timing untouched (Voss et al, 2016;Sato et al, 2016).…”