L. David Sibley

Toxoplasma Invasion of Mammalian Cells Is Powered by the Actin Cytoskeleton of the Parasite

Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. We demonstrate that invasion is critically dependent on actin filaments in the parasite, but not the host cell. Invasion into cytochalasin D (CD)-resistant host cells was blocked by CD, while parasite mutants invaded wild-type host cells in the presence of drug. CD resistance in Toxoplasma was mediated by a point mutation in the single-copy actin gene ACT1. Transfection of the mutant act1 allele into wild-type Toxoplasma conferred motility and invasion in the presence of CD. We conclude that host cell invasion by Toxoplasma, and likely by related Apicomplexans, is actively powered by an actin-based contractile system in the parasite.

Cytoskeleton of Apicomplexan Parasites

SUMMARY The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; Toxoplasma gondii and Cryptosporidium parvum, opportunistic pathogens of immunocompromised individuals; and Eimeria spp. and Theileria spp., parasites of considerable agricultural importance. These protozoan parasites share distinctive morphological features, cytoskeletal organization, and modes of replication, motility, and invasion. This review summarizes our current understanding of the cytoskeletal elements, the properties of cytoskeletal proteins, and the role of the cytoskeleton in polarity, motility, invasion, and replication. We discuss the unusual properties of actin and myosin in the Apicomplexa, the highly stereotyped microtubule populations in apicomplexans, and a network of recently discovered novel intermediate filament-like elements in these parasites.

Gr1+ Inflammatory Monocytes Are Required for Mucosal Resistance to the Pathogen Toxoplasma gondii

The enteric pathogen Toxoplasma gondii is controlled by a vigorous innate T helper 1 (Th1) cell response in the murine model. We demonstrated that after oral infection, the parasite rapidly recruited inflammatory monocytes [Gr1(+) (Ly6C(+), Ly6G(-)) F4/80(+)CD11b(+)CD11c(-)], which established a vital defensive perimeter within the villi of the ileum in the small intestine. Mice deficient of the chemokine receptor CCR2 or the ligand CCL2 failed to recruit Gr1(+) inflammatory monocytes, whereas dendritic cells and resident tissue macrophages remained unaltered. The selective lack of Gr1(+) inflammatory monocytes resulted in an inability of mice to control replication of the parasite, high influx of neutrophils, extensive intestinal necrosis, and rapid death. Adoptive transfer of sorted Gr1(+) inflammatory monocytes demonstrated their ability to home to the ileum in infected animals and protect Ccr2(-/-) mice, which were otherwise highly susceptible to oral toxoplasmosis. Collectively, these findings illustrate the critical importance of inflammatory monocytes as a first line of defense in controlling intestinal pathogens.

Aldolase Forms a Bridge between Cell Surface Adhesins and the Actin Cytoskeleton in Apicomplexan Parasites

Host cell invasion by apicomplexan parasites requires coordinated interactions between cell surface adhesins and the parasite cytoskeleton. We have identified a complex of parasite proteins, including the actin binding protein aldolase, which specifically interacts with the C-terminal domains of several parasite adhesins belonging to the thrombospondin-related anonymous protein (TRAP) family. Binding of aldolase to the adhesin was disrupted by mutation of a critical tryptophan in the C domain, a residue that was previously shown to be essential for parasite motility. Our findings reveal a potential role for aldolase in connecting TRAP family adhesins with the cytoskeleton, and provide a model linking adhesion with motility in apicomplexan parasites.

Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii

Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of external Ca2+, while chelation of intracellular Ca2+ prevented release. Mobilization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca2+ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore‐stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin–myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus‐coupled secretion system responsible for releasing adhesins involved in cell attachment.

Secretion of micronemal proteins is associated with toxoplasma invasion of host cells

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here, we demonstrate, using several independent assays, that Toxoplasma invasion of host cells is tightly coupled to the release of proteins stored within apical secretory granules called micronemes. Both microneme secretion and cell invasion were highly temperature dependent, and partial depletion of microneme resulted in a transient loss of infectivity. Chelation of parasite intracellular calcium strongly inhibited both microneme release and invasion of host cells, and this effect was partially reversed by raising intracellular calcium using the ionophore A23187. We also provide evidence that a staurosporine‐sensitive kinase activity regulates microneme discharge and is required for parasite invasion of host cells. Additionally, we demonstrate that, during apical attachment to the host cell, the micronemal protein MIC2 is released at the junction between the parasite and the host cell. During invasion, MIC2 is successively translocated towards the posterior end of the parasite and is shed before entry of the parasite into the vacuole. Furthermore, we show that the full‐length cellular form of MIC2, but not the proteolytically modified secreted form of MIC2, binds specifically to host cells. Collectively, these observations strongly imply that micronemal proteins play a role in Toxoplasma invasion of host cells.

Acute Toxoplasmosis Leads to Lethal Overproduction of Th1 Cytokines

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-γ, TNF-α, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-α or IFN-γ. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.

Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis.

Circulating murine monocytes comprise two largely exclusive subpopulations that are responsible for seeding normal tissues (Gr-1−/CCR2−/CX3CR1high) or responding to sites of inflammation (Gr-1+/CCR2+/CX3CR1lo). Gr-1+ monocytes are recruited to the site of infection during the early stages of immune response to the intracellular pathogen Toxoplasma gondii. A murine model of toxoplasmosis was thus used to examine the importance of Gr-1+ monocytes in the control of disseminated parasitic infection in vivo. The recruitment of Gr-1+ monocytes was intimately associated with the ability to suppress early parasite replication at the site of inoculation. Infection of CCR2−/− and MCP-1−/− mice with typically nonlethal, low doses of T. gondii resulted in the abrogated recruitment of Gr-1+ monocytes. The failure to recruit Gr-1+ monocytes resulted in greatly enhanced mortality despite the induction of normal Th1 cell responses leading to high levels of IL-12, TNF-α, and IFN-γ. The profound susceptibility of CCR2−/− mice establishes Gr-1+ monocytes as necessary effector cells in the resistance to acute toxoplasmosis and suggests that the CCR2-dependent recruitment of Gr-1+ monocytes may be an important general mechanism for resistance to intracellular pathogens.

Genetic Divergence of Toxoplasma gondii Strains Associated with Ocular Toxoplasmosis, Brazil

Brazilian strains of T. gondii differ from lineages in North America and Europe; these differences may underlie severe ocular disease.

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry‐derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein‐rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite‐containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre‐existing, parasite‐containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two‐step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by Toxoplasma. The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.