Disruption of the Toxoplasma gondii bradyzoite-specific gene BAG1 decreases in vivo cyst formation

The bradyzoite stage of the Apicomplexan protozoan parasite Toxoplasma gondii plays a critical role in maintenance of latent infection. We reported previously the cloning of a bradyzoite-specific gene BAG1/hsp30 (previously referred to as BAG5) encoding a cytoplasmic antigen related to small heat shock proteins. We have now disrupted BAG1 in the T. gondii PLK strain by homologous recombination. H7, a cloned null mutant, and Y8, a control positive for both cat and BAG1, were chosen for further characterization. Immunofluorescence and Western blot analysis of bradyzoites with BAG1 antisera demonstrated expression of BAG1 in the Y8 and the PLK strain but no expression in H7. All three strains expressed a 116 kDa bradyzoite cyst wall antigen, a 29 kDa matrix antigen and the 65 kDa matrix reactive antigen MAG1. Mice inoculated with H7 parasites formed significantly fewer cysts than those inoculated with the Y8 and the PLK strains. H7 parasites were complemented with BAG1 using phleomycin selection. Cyst formation in vivo for the BAG1-complemented H7 parasites was similar to wild-type parasites. We therefore conclude that BAG1 is not essential for cyst formation, but facilitates formation of cysts in vivo.     

A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.     

The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis -elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii , we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis -acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6–8 bp resolution and these minimal cis -elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis -elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.     

Functional Analysis of Drosophila HSP70 Promoter with Different HSE Numbers in Human Cells

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38°C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.     

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.     

A role of carboxy-terminal region of Toxoplasma gondii-heat shock protein 70 in enhancement of T. gondii infection in mice

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g.HSP70-NH2-terminal region, or rT.g.HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T. gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or r.T.g.HSP70-carboxy-terminal region increased the number of T. gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NH2-terminal region did not. These results suggest that T.g.HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.     

Isolation of a <Emphasis Type="Italic">Latimeria menadoensis</Emphasis><Emphasis Type="Italic">heat shock protein 70</Emphasis> (<Emphasis Type="Italic">Lmhsp70</Emphasis>) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system. K. W. Modisakeng and M. Jiwaji contributed equally to this study.     

Simultaneous exposure of Xenopus A6 kidney epithelial cells to concurrent mild sodium arsenite and heat stress results in enhanced hsp30 and hsp70 gene expression and the acquisition of thermotolerance

In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1–10 µM sodium arsenite at a mild heat shock temperature of 30 °C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 µM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 °C with larger responses at 28 and 30 °C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 µM) and heat shock (30 °C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 °C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.     

A consensus sequence polymer inhibits in vivo expression of heat shock genes.

Promoter function for hsp70 gene expression in Drosophila melanogaster was studied with an in vivo competition assay. A polymer of 40 tandem copies of the pair of regulatory elements of the hsp70 gene was constructed and cloned into a plasmid vector. Various marked genes were cotransfected with the polymer plasmid into Schneider line 2 cells, and their expression was determined by enzyme activity measurements. The polymer dramatically inhibited expression of cotransfected hsp70, hsp26, and hsp83 genes, but not cotransfected copia and histone genes. Our results indicate that in vivo, a trans-acting, positive regulatory factor, which can be titrated by heat shock consensus sequences, is required for activation of heat shock genes and is specific for these genes; the coordinate induction of different heat shock genes appears to be mediated by similar, but not identical, interactions of the trans-acting induction factor and the cis-acting heat shock consensus sequences; and the uninduced or basal level expression of the transfected hsp70 gene is also due to interaction of the consensus sequence with a positively acting factor.     

Heat shock gene-expression in HSP-70 and HSF1 gene-transfected human epidermoid A-431 cells

Thermotolerant cells display attenuated heat shock protein 70 kD (HSP-70) gene expression and signal transduction such as intracellular Ca2+ concentration and inositol trisphosphate in response to sublethal heat. To further investigate the regulation of heat shock gene expression, we developed constructs containing human HSP-70 and HSF1 genes and transfected human epidermoid A-431 cells. These cells were chosen because skin cells are especially vulnerable to heat shock and other environmental stressors. We report that A431 cells can be successfully transfected with HSP-70 and HSF1 genes as shown by the elevated levels of respective message and protein. Overexpression of HSP-70 in cells transfected with HSP-70 gene led to a down-regulation of the HSF1 gene expression. Interestingly, transfection of cells with the HSF1 gene was not associated with increased expression of HSP-70. Exposure of HSF1 gene-transfected cells to heat resulted in a transient but significant increase in HSP-70 gene expression as compared to that found in vector-transfected cells, which was completely inhibited by treatment with staurosporine. In conclusion, we have demonstrated successful transfection of human A-431 cells with HSF1 and HSP-70 genes, where the regulation of their expression can be studied. (Mol Cell Biochem 167: 145-152, 1997)     

Cyclic nucleotide kinases and tachyzoite-bradyzoite transition in Toxoplasma gondii

The ability of Toxoplasma gondii to cycle between the tachyzoite and bradyzoite life stages in intermediate hosts is key to parasite survival and the pathogenesis of toxoplasmosis. Studies from a number of laboratories indicate that differentiation in T. gondii is a stress-induced phenomenon. The signalling pathways or molecular mechanisms that control formation of the latent bradyzoite stage are unknown and specific effectors of differentiation have not been identified. We engineered a reporter parasite to facilitate simultaneous comparison of differentiation and replication after various treatments. Chloramphenicol acetyltransferase (CAT), expressed constitutively from the alpha-tubulin promoter (TUB1), was used to quantitate parasite number. beta-galactosidase (beta-GAL), expressed from a bradyzoite specific promoter (BAG1), was used as a measure of bradyzoite gene expression. Sodium nitroprusside, a well-known inducer of bradyzoite differentiation, reduced reporter parasite replication and caused bradyzoite differentiation. Stress-induced differentiation in many other pathogens is regulated by cyclic nucleotide kinases. Specific inhibitors of the cAMP dependent protein kinase and apicomplexan cGMP dependent protein kinase inhibited replication and induced differentiation. The beta-GAL/CAT reporter parasite provides a method to quantify and compare agents that cause differentiation in T. gondii.     

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.     

An intron-containing,heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae,Diplopoda)

The highly conserved part of the nucleotide-binding domain of the hsp70 gene family was amplified from the soil diplopod Tachypodoiulus niger (Julidae, Diplopoda). Genomic DNA yielded 701, 549 and 540 bp sequences, whereas cDNA from heat shocked animals produced only one distinct fragment of 543 bp. The sequences could be classified as a 70 kDa heat shock protein (hsp70), the corresponding 70 kDa heat shock cognate (hsc70) and a glucose-related hsp70 homologue (grp78). Comparisons of genomic and cDNA sequences of hsc70 identified two introns within the consensus sequence. Generally, stress-70 expression levels were low, which hampered successful RT-PCR and subsequent subcloning. Following experimental heat shock, however, the spliced hsc70 was amplified predominantly, instead of its inducible homologue hsp70. This finding suggests that microevolution in this soil-dwelling arthropod is directed towards low constitutive stress-70 levels and that the capacity for stress-70 induction presumably is limited. hsc70, albeit having introns, apparently is inducible and contributes to the stress-70 response.