Ira J. Blader

Microarray analysis reveals previously unknown changes in Toxoplasma gondii-infected human cells.

Cells infected with the intracellular protozoan parasite Toxoplasma gondii undergo up-regulation of pro-inflammatory cytokines, organelle redistribution, and protection from apoptosis. To examine the molecular basis of these and other changes, gene expression profiles of human foreskin fibroblasts infected with Toxoplasma were studied using human cDNA microarrays consisting of ∼22,000 known genes and uncharacterized expressed sequence tags. Early during infection (1–2 h), <1% of all genes show a significant change in the abundance of their transcripts. Of the 63 known genes in this group, 27 encode proteins associated with the immune response. These genes are also up-regulated by secreted, soluble factors from extracellular parasites indicating that the early response does not require parasite invasion. Later during infection, genes involved in numerous host cell processes, including glucose and mevalonate metabolism, are modulated. Many of these late genes are dependent on the direct presence of the parasite;i.e. secreted products from either the parasite or infected cells are insufficient to induce these changes. These results reveal several previously unknown effects on the host cell and lay the foundation for detailed analysis of their role in the host-pathogen interaction.

Toxoplasma gondii Asexual Development: Identification of Developmentally Regulated Genes and Distinct Patterns of Gene Expression

ABSTRACT Asexual development in Toxoplasma gondii is a vital aspect of the parasites life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of ∼4,400 Toxoplasma cDNAs, representing a minimum of ∼600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.

Identification and Cloning of Centaurin-α A NOVEL PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE-BINDING PROTEIN FROM RAT BRAIN

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-α. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-α displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-α is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.

The host cell transcription factor hypoxia-inducible factor 1 is required for Toxoplasma gondii growth and survival at physiological oxygen levels.

Toxoplasma gondii is an obligate intracellular protozoan pathogen. We previously found that genes mediating cellular responses to hypoxia were upregulated in Toxoplasma ‐infected cells but not in cells infected with another intracellular pathogen, Trypanosoma cruzi. The inducible expression of these genes is controlled by the hypoxia‐inducible factor 1 (HIF1) transcription factor, which is the master regulator of cells exposed to low oxygen. Because this response may be important for parasites to grow at physiological oxygen levels, we tested the hypothesis that HIF1 is important for Toxoplasma growth. Here, we demonstrate that Toxoplasma infection rapidly increased the abundance of the HIF1α subunit and activated HIF1 reporter gene expression. In addition, we found that Toxoplasma growth and survival was severely reduced in HIF1α knockout cells at 3% oxygen. While HIF1α was not required for parasite invasion, we determined that HIF1 was required for parasite cell division and organelle maintenance at 3% oxygen. These data indicate that Toxoplasma activates HIF1 and requires HIF1 for growth and survival at physiologically relevant oxygen levels.

Peculiarities of Host Cholesterol Transport to the Unique Intracellular Vacuole Containing Toxoplasma

The intracellular protozoan Toxoplasma gondii is auxotrophic for low‐density lipoprotein (LDL)‐derived cholesterol (C). We previously showed that T. gondii scavenges this essential lipid from host endolysosomal compartments and that C delivery to the parasitophorous vacuole (PV) does not require transit through host Golgi or endoplasmic reticulum. In this study, we explore the itinerary of C from the host endolysosomes to the PV. Labeled C incorporated into LDL is rapidly detected in intravacuolar parasites and partially esterified by the parasites. In contrast to diverse mammalian organelles, the post‐endolysosomal transfer of C to the PV does not involve the host plasma membrane as an intermediate. Nevertheless, the PV membrane is accessible to extracellular sterol acceptors, suggesting C trafficking from intracellular parasites to host plasma membrane. C movement to the PV requires temperatures permissive for vesicular transport, metabolic energy and functional microtubules. Host caveolae vesicles and the sterol carrier protein‐2 do not participate in this process. Proteolytic treatment of purified PV or free parasites abolishes C acquisition by the parasites. Altogether, these results support a vesicular transport system from host endolysosomes to the PV, and a requirement for PV membrane and parasite plasma membrane proteins in C delivery to T. gondii.

Toxoplasma gondii Activates Hypoxia-inducible Factor (HIF) by Stabilizing the HIF-1α Subunit via Type I Activin-like Receptor Kinase Receptor Signaling

Toxoplasma gondii is an intracellular protozoan parasite that can cause devastating disease in fetuses and immune-compromised individuals. We previously reported that the α subunit of the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), is up-regulated by infection and necessary for Toxoplasma growth. Under basal conditions, HIF-1α is constitutively expressed but rapidly targeted for proteasomal degradation after two proline residues are hydroxylated by a family of prolyl hydroxylases (PHDs). The PHDs are α-ketoglutarate-dependent dioxygenases that have low Km values for oxygen, making them important cellular oxygen sensors. Thus, when oxygen levels decrease, HIF-1α is not hydroxylated, and HIF-1 is activated. How Toxoplasma activates HIF-1 under normoxic conditions remains unknown. Here, we report that Toxoplasma infection increases HIF-1α stability by preventing HIF-1α prolyl hydroxylation. Infection significantly decreases PHD2 abundance, which is the key prolyl hydroxylase for regulating HIF-1α. The effects of Toxoplasma on HIF-1α abundance and prolyl hydroxylase activity require activin-like receptor kinase signaling. Finally, parasite growth is severely diminished when signaling from this family of receptors is inhibited. Together, these data indicate that PHD2 is a key host cell factor for T. gondii growth and represent a novel mechanism by which a microbial pathogen subverts host cell signaling and transcription to establish its replicative niche.

Lytic Cycle of Toxoplasma gondii: 15 Years Later

Toxoplasmosis is the clinical and pathological consequence of acute infection with the obligate intracellular apicomplexan parasite Toxoplasma gondii. Symptoms result from tissue destruction that accompanies lytic parasite growth. This review updates current understanding of the host cell invasion, parasite replication, and eventual egress that constitute the lytic cycle, as well as the ways T. gondii manipulates host cells to ensure its survival. Since the publication of a previous iteration of this review 15 years ago, important advances have been made in our molecular understanding of parasite growth and mechanisms of host cell egress, and knowledge of the parasites manipulation of the host has rapidly progressed. Here we cover molecular advances and current conceptual frameworks that include each of these topics, with an eye to what may be known 15 years from now.

Host cell invasion by Toxoplasma gondii is temporally regulated by the host microtubule cytoskeleton.

ABSTRACT 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 cells 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 Rhoptry Discharge Correlates with Activation of the Early Growth Response 2 Host Cell Transcription Factor

ABSTRACT Toxoplasma gondii is a ubiquitous apicomplexan parasite that can cause severe disease in fetuses and immune-compromised patients. Rhoptries, micronemes, and dense granules, which are secretory organelles unique to Toxoplasma and other apicomplexan parasites, play critical roles in parasite growth and virulence. To understand how these organelles modulate infected host cells, we sought to identify host cell transcription factors triggered by their release. Early growth response 2 (EGR2) is a host cell transcription factor that is rapidly upregulated and activated in Toxoplasma-infected cells but not in cells infected with the closely related apicomplexan parasite Neospora caninum. EGR2 upregulation occurred only when live parasites were in direct contact with the host cell and not from exposure to cell extracts that contain dense granule or micronemal proteins. When microneme-mediated attachment was blocked by pretreating parasites with a calcium chelator, EGR2 expression was significantly reduced. In contrast, when host cells were infected with parasites in the presence of cytochalasin D, which allows rhoptry secretion but prevents parasite invasion, EGR2 was activated. Finally, we demonstrate that Toxoplasma activation of host p38 mitogen-activated protein kinase is necessary but not sufficient for EGR2 activation. Collectively, these data indicate that EGR2 is specifically upregulated by a parasite-derived secreted factor that is most likely a resident rhoptry protein.

Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1–30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F’ pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions. DOI: http://dx.doi.org/10.7554/eLife.12556.001