Toxoplasmaaldolase is required for metabolism but dispensable for host-cell invasion

Abstract

Gliding motility and host-cell invasion by apicomplexan parasites depend on cell-surface adhesins that are translocated via an actinmyosin motor beneath the membrane. The current model posits that fructose-1,6-bisphosphate aldolase (ALD) provides a critical link between the cytoplasmic tails of transmembrane adhesins and the actin-myosin motor. Here we tested this model using the Toxoplasma gondii apical membrane protein 1 (TgAMA1), which binds to aldolase in vitro. TgAMA1 cytoplasmic tail mutations that disrupt ALD binding in vitro showed no correlation with host-cell invasion, indicating this interaction is not essential. Furthermore, ALD-depleted parasites were impaired when grown in glucose, yet they showed normal gliding and invasion in glucose-free medium. Depletion of ALD in the presence of glucose led to accumulation of fructose-1,6-bisphosphate, which has been associated with toxicity in other systems. Finally, TgALD knockout parasites and an ALD mutant that specifically disrupts adhesin binding in vitro also supported normal invasion when cultured in glucosefree medium. Taken together, these results suggest that ALD is primarily important for energy metabolism rather than interacting with microneme adhesins, challenging the current model for apicomplexan motility and invasion.micronemal adhesin | motor complex | glycolysis T he phylum Apicomplexa is a group of mostly intracellular parasites that contains a number of human pathogens, including Toxoplasma gondii and Plasmodium spp., the causative agent of malaria. As intracellular pathogens, efficient host-cell invasion is critical for survival, dissemination, and transmission of these parasites. Although they infect different types of host cells, apicomplexan parasites share a conserved mode of host-cell invasion that relies on regulated secretion of adhesive proteins and active motility that is powered by an actin-myosin motor complex (1, 2). According to the current model, motility and host-cell invasion depend on transmembrane adhesins that are secreted apically from the micronemes and translocated along the cell surface in a conveyor belt fashion, using the force generated by the motor complex beneath the parasite membrane (1, 2).Micronemal adhesins contain a variety of extracellular adhesive domains, a transmembrane domain, and a short cytoplasmic tail that is rich in acidic residues and contains a tryptophan residue (Trp) at or near the extreme carboxyl terminus (3). These conserved features of the cytoplasmic tails are critical to their function, as shown by mutational studies and functional replacement of the thrombospondin-related adhesive protein (TRAP) tail (TRAPt) in P. berghei with the T. gondii Microneme Protein 2 (MIC2) tail (TgMIC2t) (4). The cytoplasmic tails of several micronemal adhesins are thought to be anchored to the actin-myosin motor through a bridging molecule, the glycolytic enzyme fructose-1,6-bisphosphate aldolase (ALD) (5, 6). Mutational analysis has shown that in vitro binding of TgMIC2t and PbTRAPt to ALD is mediated by t...

Plasticity and redundancy among AMA–RON pairs ensure host cell entry of Toxoplasma parasites

“…An association between the cytoplasmic tail of AMA1 and aldolase 17,21,26 was previously reported, and proposed to act as a bridge with F-actin and the gliding machinery necessary for internalization. However, a recent report revisiting the implication of aldolase in gliding and invasion 27 showed that aldolase does not have an essential role in coupling AMA1 or other MIC proteins to the actomyosin system during invasion. How the AMA1 tail functions during internalization remains an important question for the future.…”

Gliding motility powers invasion and egress in Apicomplexa

“…In addition, TgALD-depleted T. gondii parasites showed a defect in motility and invasion, and a substantial loss of fitness 89 . However, another report showed that this fitness loss could be due to an accumulation of toxic metabolites caused by incomplete glycolysis, which cast some doubt on the role of TgALD as a connector 90 . Finally, TgALD was proven dispensable for gliding in parasites that are grown in the absence of glucose 90 .…”

“…However, another report showed that this fitness loss could be due to an accumulation of toxic metabolites caused by incomplete glycolysis, which cast some doubt on the role of TgALD as a connector 90 . Finally, TgALD was proven dispensable for gliding in parasites that are grown in the absence of glucose 90 . The physiologically relevant glideosome-associated connector (GAC) was discovered following the functional characterization of a large armadillo repeats-containing protein that accumulates at the apical tip of T. gondii tachyzoites and in the invasive stages of malaria parasites 40 .…”

Structural Basis of Toxoplasma gondii MIC2-associated Protein Interaction with MIC2

“…MIC2 is the transmembrane (TM) member of the complex, with an extracellular region composed of an integrin-like I-domain followed by five thrombospondinlike repeats (TSRs) and a degenerate sixth TSR near the TM domain. The C-terminal cytosolic tail is thought to interact with the actin-myosin motility system, but the molecular basis of this connection remains to be determined (9,10).…”