Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii

Wassim Daher,Marie-France Carlier,Dominique Soldati-Favre,Fabienne Plattner,Vern B Carruthers

doi:10.1371/journal.ppat.1001132

Abstract

The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.

Highlights

The phylum Apicomplexa encompasses pathogens of significant medical relevance including those responsible for malaria and toxoplasmosis
The presence of a canonical FH1 is not apparent but both formins possess a segment rich in proline residues upstream of the FH2 (Figure 1A). These formins lack the diaphanous autoregulatory domain (DAD) and diaphanous inhibitory domain (DID) regulatory domains, TgFRM1 possesses four tetratricopeptide repeats present on PfFRM1 (Figure 1A)
Specific antibodies were raised against bacterially produced FH2 domains of both T. gondii formins and western blot analysis confirmed that TgFRM1 and TgFRM2 are expressed in tachyzoites and migrate at their predicted sizes on SDS-PAGE (Figure 1B)

Summary

Introduction

The phylum Apicomplexa encompasses pathogens of significant medical relevance including those responsible for malaria and toxoplasmosis. These parasites cross biological barriers and enter cells by an active process that depends on a unique form of gliding motility [1]. Among the systems orchestrating actin nucleation, the Arp2/3 complex, generates a network of short, branched filaments, whereas the formin-profilin system catalyzes the processive assembly of unbranched actin filaments [9]. The Apicomplexans lack many actin-regulatory proteins including the Arp2/3 complex [10]. They contain at least two formins and a profilin that have been previously associated with parasite motility [11,12,13]

Methods

Results

Conclusion