Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii.

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.

Hijacking of host cellular functions by the Apicomplexa.

Intracellular pathogens such as viruses and bacteria subvert all the major cellular functions of their hosts. Targeted host processes include protein synthesis, membrane trafficking, modulation of gene expression, antigen presentation, and apoptosis. In recent years, it has become evident that protozoan pathogens, including members of the phylum Apicomplexa, also hijack their host cell's functions to access nutrients and to escape cellular defenses and immune responses. These obligate intracellular parasites provide superb illustrations of the subversion of host cell processes such as the recruitment and reorganization of host cell compartments without fusion around the parasitophorous vacuole of Toxoplasma gondii; the export of Plasmodium falciparum proteins on the surface of infected erythrocytes; and the induced transformation of the lymphocytes infected by Theileria parva, which leads to clonal extension.

Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response.

Apicomplexan parasites exhibit actin-dependent gliding motility that is essential for migration across biological barriers and host cell invasion. Profilins are key contributors to actin polymerization, and the parasite Toxoplasma gondii possesses a profilin-like protein that is recognized by Toll-like receptor TLR11 in the host innate immune system. Here, we show by conditional disruption of the corresponding gene that T.gondii profilin, while not required for intracellular growth, is indispensable for gliding motility, host cell invasion, active egress from host cells, and virulence in mice. Furthermore, parasites lacking profilin are unable to induce TLR11-dependent production in vitro and in vivo of the defensive host cytokine interleukin-12. Thus, profilin is an essential element of two aspects of T. gondii infection. Like bacterial flagellin, profilin plays a role in motility while serving as a microbial ligand recognized by the host innate immune system.

Yeast Mon2p is a highly conserved protein that functions in the cytoplasm-to-vacuole transport pathway and is required for Golgi homeostasis.

Although the small Arf-like GTPases Arl1-3 are highly conserved eukaryotic proteins, they remain relatively poorly characterized. The yeast and mammalian Arl1 proteins bind to the Golgi complex, where they recruit specific structural proteins such as Golgins. Yeast Arl1p directly interacts with Mon2p/Ysl2p, a protein that displays some sequence homology to the large Sec7 guanine exchange factors (GEFs) of Arf1. Mon2p also binds the putative aminophospholipid translocase (APT) Neo1p, which performs essential function(s) in membrane trafficking. Our detailed analysis reveals that Mon2p contains six distinct amino acid regions (A to F) that are conserved in several other uncharacterized homologs in higher eukaryotes. As the conserved A, E and F domains are unique to these homologues, they represent the signature of a new protein family. To investigate the role of these domains, we made a series of N- and C-terminal deletions of Mon2p. Although fluorescence and biochemical studies showed that the B and C domains (also present in the large Sec7 GEFs) predominantly mediate interaction with Golgi/endosomal membranes, growth complementation studies revealed that the C-terminal F domain is essential for the activity of Mon2p, indicating that Mon2p might also function independently of Arl1p. We provide evidence that Mon2p is required for efficient recycling from endosomes to the late Golgi. Intriguingly, although transport of CPY to the vacuole was nearly normal in the Deltamon2 strain, we found the constitutive delivery of Aminopeptidase 1 from the cytosol to the vacuole to be almost completely blocked. Finally, we show that Mon2p exhibits genetic and physical interactions with Dop1p, a protein with a putative function in cell polarity. We propose that Mon2p is a scaffold protein with novel conserved domains, and is involved in multiple aspects of endomembrane trafficking.