Host Cell Invasion by Toxoplasma gondii Is Temporally Regulated by the Host Microtubule Cytoskeleton

Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogen-derived material from the host cell''s periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing disease in fetuses and immunocompromised individuals (23). The parasite infects a wide range of nucleated cells of most warm-blooded animals. The mechanisms underlying this wide tropism are not known but could be due to either the parasite infecting cells using a ubiquitously expressed host receptor and associated machinery, inserting its own receptor into the host cell''s plasma membrane, or using multiple host cell receptors/machinery (5).Toxoplasma invasion is a multistep, complex process consisting of parasite contact to host cells, intimate attachment, parasite motility, and then penetration (5). Host cell contact is a loose, low-affinity interaction that is mediated by parasite surface antigens. An unknown signal then triggers the release of proteins from a specialized secretory organelle called micronemes whose contents include proteins that function as adhesins. This is then followed by parasite gliding motility on the host cell surface. At some point, proteins from a second secretory organelle, named rhoptries, are exocytosed. Among these rhoptry proteins, several (RON2, RON4, RON5, and RON8) are part of a preformed complex that binds the previously secreted AMA1 microneme protein (1, 2, 20, 33). Together, these proteins form the moving junction complex, which defines the parasite entry site on the host cell plasma membrane. Parasite penetration occurs by the parasite propelling itself forward, via acto-myosin-dependent motility, into the host plasma membrane (35). This causes an invagination of the plasma membrane resulting in the formation of the parasitophorous vacuole (PV), which is the compartment that the parasite resides in throughout its time in the host cell. However, host plasma membrane-associated proteins are selectively incorporated into the developing PV such that glycosylphosphatidylinositol (GPI)-linked proteins are included, while single-pass transmembrane proteins are excluded (7, 24).In contrast to parasite molecules that function during invasion, few host cell components involved in this process are known. A notable exception is the finding that host Arp2/3-dependent actin polymerization promotes Toxoplasma invasion (11). Nevertheless, how actin or other host molecules function during invasion remains to be determined. The host microtubule cytoskeleton has been widely studied for its role during receptor-mediated endocytosis, as well as in bacterial and viral infections, where microtubules act to facilitate cargo transport from the host cell periphery to the interior (8, 15, 27, 29, 40). Consistent with this role in cargo transport, host microtubules also promote trafficking of rhoptry proteins secreted into the host cell (12). However, whether this host cell structure functions during parasite invasion per se is unknown.Here, we tested the hypothesis that host microtubules are used by Toxoplasma tachyzoites to penetrate into its host cell. Using synchronized parasite invasion assays, we find that disruption of host microtubules significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are localized to the moving junction but, unlike their previously described role in pathogen invasion, host microtubules promote tachyzoite invasion by hastening the time that parasites initiate invasion.