Formin-2 drives intracellular polymerisation of actin filaments enabling correct segregation of apicoplasts in Plasmodium falciparum and Toxoplasma gondii

Abstract

FV, food vacuole; F-actin, filamentous actin; PfACT1, P. falciparum actin-1; PfFRM1, 22 P. falciparum Formin-1; PfFRM2, P. falciparum Formin-2; TgFRM1, Toxoplasma gondii Formin-1; 23 TgFRM2, Toxoplasma gondii Formin-2; PV, parasitophorous vacuole; RAP, rapamycin; RON4, rhoptry 24 neck protein-4; TJ, tight junction, IMC, inner membrane complex. PCR, Polymerase chain reaction; 25 IFA, (indirect) Immunofluorescence assay; HA, Haemagglutinin; YFP, yellow fluorescent protein; RT, 26 room temperature. 27 Abstract 28Pathogenic obligate-intracellular apicomplexan parasites possess an essential chloroplast-like 29 organelle called the apicoplast that undergoes division and segregation during replication. Parasite 30 actin is essential during intracellular development, implicated in vesicular transport, parasite 31 replication and apicoplast inheritance. However, the inability to visualise live actin dynamics in 32 apicomplexan parasites limited functional characterisation of both filamentous-actin (F-actin) and 33 actin regulatory factors. Apicomplexans possess at least two distinct formins, Formin-1 and Formin-34 2, predicted to serve as actin-nucleating factors, and previously implicated in regulating gliding 35 motility and host cell invasion. Here, we expressed chromobodies and validated them as F-actin-36 binding sensors in Plasmodium falciparum and characterised the in vivo dynamics of the F-actin 37 network. The F-actin network could be modulated chemically and disrupted by conditionally deleting 38 the actin-1 gene. In a comparative approach, we demonstrate that Formin-2 is closely associated 39 with apicoplasts and with the F-actin network in P. falciparum and Toxoplasma gondii. 40Consequently, disruption of Formin-2 resulted not only in an apicoplast segregation defect, but also 41 in complete abrogation of F-actin dynamics in intracellular parasites. Together, our results strongly 42 indicate that Formin-2-mediated filament formation is the common primary mechanism for F-actin 43 nucleation during apicomplexan intracellular growth effecting apicoplast segregation. 44 Plattner et al. 2010), with T. gondii Formin-2 (TgFRM2) also being implicated in apicoplast 126 maintenance (Jacot, Daher et al. 2013) -leading to inconsistencies in reports and questions whether 127 the two formins have conserved or divergent functions in both parasites. 128 Here we reanalysed the role of Formin-2 in P. falciparum and T. gondii and, in contrast to previous 129 reports, demonstrate that it localises adjacent to apicoplasts in both parasites. Conditional 130 disruption of Formin-2 not only results in a complete abrogation of actin dynamics in P. falciparum 131 and T. gondii, it also leads to loss of the apicoplast. Together our study highlights a highly conserved 132 role of Formin-2 in the intracellular development of apicomplexan parasites. Importantly, apicoplast 133 loss appears to be not the only critical phenotype caused by Formin-2 depletion, since in P. 134 falciparum the loss of fitness due to the deletion of PfFRM2 cannot ...

Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii

“…FRM1 is also confined at the front of sporozoites and at the poles of gametocytes (Douglas et al, 2018; Hliscs et al, 2015). On the other hand, FRM2 localizes diffusely throughout the merozoite cytoplasm (Baum et al, 2008) a localization compatible with a role in apicoplast inheritance (Stortz et al, 2018).…”

Molecular Docking Studies of Rifampicin – rpoB complex: Repurposing Drug Design Implications for against Plasmodium falciparum Malaria through a Computational Approach

“…The apicoplast contains a range of metabolic pathways and processes that differ radically to those of the host thereby offering opportunities and presenting ideal strategies for antimalarial drug therapy. Also, repurposing of known drug molecules for new therapeutic use for a drug in a new field is a current trend [10][11][12][13][14]. It is an effective approach in discovering drug molecules with new therapeutic indications [15][16][17].…”