siRNA Screening Identifies the Host Hexokinase 2 (HK2) Gene as an Important Hypoxia-Inducible Transcription Factor 1 (HIF-1) Target Gene in Toxoplasma gondii-Infected Cells.

UNLABELLED: Although it is established that oxygen availability regulates cellular metabolism and growth, little is known regarding how intracellular pathogens use host factors to grow at physiological oxygen levels. Therefore, large-scale human small interfering RNA screening was performed to identify host genes important for growth of the intracellular protozoan parasite Toxoplasma gondii at tissue oxygen tensions. Among the genes identified by this screen, we focused on the hexokinase 2 (HK2) gene because its expression is regulated by hypoxia-inducible transcription factor 1 (HIF-1), which is important for Toxoplasma growth. Toxoplasma increases host HK2 transcript and protein levels in a HIF-1-dependent manner. In addition, parasite growth at 3% oxygen is restored in HIF-1-deficient cells transfected with HK2 expression plasmids. Both HIF-1 activation and HK2 expression were accompanied by increases in host glycolytic flux, suggesting that enhanced HK2 expression in parasite-infected cells is functionally significant. Parasite dependence on host HK2 and HIF-1 expression is not restricted to transformed cell lines, as both are required for parasite growth in nontransformed C2C12 myoblasts and HK2 is upregulated in vivo following infection. While HK2 is normally associated with the cytoplasmic face of the outer mitochondrial membrane at physiological O2 levels, HK2 relocalizes to the host cytoplasm following infection, a process that is required for parasite growth at 3% oxygen. Taken together, our findings show that HIF-1-dependent expression and relocalization of HK2 represent a novel mechanism by which Toxoplasma establishes its replicative niche at tissue oxygen tensions. IMPORTANCE: Little is known regarding how the host cell contributes to the survival of the intracellular parasite Toxoplasma gondii at oxygen levels that mimic those found in tissues. Our previous work showed that Toxoplasma activates the expression of an oxygen-regulated transcription factor that is required for growth. Here, we report that Toxoplasma regulates the abundance and activity of a key host metabolic enzyme, hexokinase 2, by activating HIF-1 and by promoting dissociation of hexokinase 2 from the mitochondrial membrane. Collectively, our data reveal HIF-1/hexokinase 2 as a novel target for an intracellular pathogen that acts by reprograming the host cell's metabolism to create an environment conducive for parasite replication at physiological oxygen levels.

Host microtubule plus-end binding protein CLASP1 influences sequential steps in the Trypanosoma cruzi infection process.

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin-dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus-end tracking proteins (+TIPs) in the co-ordination of T. cruzi trypomastigote internalization and post-entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium-regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1-depleted cells. These post-entry phenotypes correlate with a generalized impairment of minus-end directed transport of lysosomes in CLASP1 knock-down cells and mimic the effects of dynactin disruption. Consistent with GSK3ß acting as a negative regulator of CLASP function, inhibition of GSK3ß activity enhances T. cruzi entry in a CLASP1-dependent manner and expression of constitutively active GSK3ß dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite-elicited signalling, host microtubule plus-end tracking proteins and dynein-based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells.

The Skp1 protein from Toxoplasma is modified by a cytoplasmic prolyl 4-hydroxylase associated with oxygen sensing in the social amoeba Dictyostelium.

In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O(2) and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O(2) regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3(SCF) ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O(2) sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O(2) levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O(2) requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O(2). The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O(2) by detecting the levels of O(2)-regulated metabolites such as α-ketoglutarate.

Serum response factor regulates immediate early host gene expression in Toxoplasma gondii-infected host cells.

Toxoplasma gondii is a wide spread pathogen that can cause severe and even fatal disease in fetuses and immune-compromised hosts. As an obligate intracellular parasite, Toxoplasma must alter the environment of its host cell in order to establish its replicative niche. This is accomplished, in part, by secretion of factors into the host cell that act to modulate processes such as transcription. Previous studies demonstrated that genes encoding transcription factors such as c-jun, junB, EGR1, and EGR2 were amongst the host genes that were the most rapidly upregulated following infection. In cells stimulated with growth factors, these genes are regulated by a transcription factor named Serum Response Factor. Serum Response Factor is a ubiquitously expressed DNA binding protein that regulates growth and actin cytoskeleton genes via MAP kinase or actin cytoskeletal signaling, respectively. Here, we report that Toxoplasma infection leads to the rapid activation of Serum Response Factor. Serum Response Factor activation is a Toxoplasma-specific event since the transcription factor is not activated by the closely related protozoan parasite, Neospora caninum. We further demonstrate that Serum Response Factor activation requires a parasite-derived secreted factor that signals via host MAP kinases but independently of the host actin cytoskeleton. Together, these data define Serum Response Factor as a host cell transcription factor that regulates immediate early gene expression in Toxoplasma-infected cells.

Identification of the RLBP1 gene promoter.

PURPOSE: Cellular retinaldehyde-binding protein (CRALBP), transcribed from the RLBP1 gene, is a 36-kDa water-soluble protein with 316 amino acids found in the retinal pigment epithelium (RPE) and in retinal Müller cells. It is thought to play a critical role in the visual cycle by functioning as an acceptor of 11-cis-retinol from the isomerohydrolase reaction. The goal here was to evaluate the functional promoter of this gene. METHODS: 5' RACE analysis, promoter-reporter assays, and semiquantitative PCR with exon-specific primers were performed using human-derived RPE cells (ARPE-19 and D407) in culture to evaluate the 5' sequence flanking the RLBP1 gene. In addition, the murine, bovine, and porcine RLBP1 genes were evaluated in silico to identify likely proximal promoter/exon 1 sequences similar to the human gene. RESULTS: 5' RACE analysis revealed the presence of a previously undescribed exon in the RLBP1 gene. This was confirmed by analysis of the GenBank Human EST database, which revealed the presence of 18 sequences matching exon 1. Exon-specific PCR revealed that most CRALBP transcripts expressed in ARPE-19 cells contain both exon 1 and the final exon, suggesting that the primary promoter of CRALBP exists 5' of the newly identified exon 1. Highly homologous sequences in the murine, bovine, and porcine genes were also identified. Finally, promoter-reporter constructs revealed a minimal sequence necessary for promoter function and indicated significantly greater promoter activity compared with previously described RLBP1 promoters. CONCLUSIONS: The findings presented here suggest that CRALBP transcripts in RPE cells contain a noncoding exon in addition to a newly described promoter and, by definition, an additional intron. This finding sets the stage for a mechanistic understanding of the high degree of cell type-specific expression of RLBP1.