Transient inhibition of poly(ADP-ribose) polymerase expression and activity by Toxoplasma gondii is dispensable for parasite-mediated blockade of host cell apoptosis and intracellular parasite replication.

Poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) is an abundant nuclear protein that is involved in DNA repair, cell cycle control, programmed cell death and transcriptional regulation. It also plays critical roles in the pathogenesis of inflammatory disorders. Here we have performed a detailed analysis of the interplay between the apicomplexan parasite Toxoplasma gondii and host cell PARP and its consequences for the host-parasite relationship. Our results have shown that T. gondii significantly decreased PARP expression in its host cells within 10min of infection but that the amount of PARP normalized during prolonged infection. Importantly, down-regulation of PARP expression after infection abrogated the ADP-ribosylation of acceptor proteins in response to oxidative stress. Overexpression of PARP in RAW264.7 cells revealed that elevated amounts of PARP neither affected host cell invasion nor intracellular development of T. gondii in non-stimulated or IFN-gamma/LPS-stimulated monocytes/macrophages. Furthermore, measurements of the activities of effector caspases 3 and 7 indicated that the blockade of host cell apoptosis by T. gondii occurs independently of the inhibition of PARP after infection. These findings suggest that the prominent decrease of host cell PARP and poly(ADP-ribos)ylation after parasitic infection do not affect the intracellular development of T. gondii in vitro.

Diverse mechanisms employed by Toxoplasma gondii to inhibit IFN-gamma-induced major histocompatibility complex class II gene expression.

The intracellular parasite Toxoplasma gondii is able to establish persistent infections in immunocompetent hosts and this may be facilitated by different immune evasion mechanisms. In the present study, we describe that infection of murine monocyte/macrophage RAW 264.7 cells with T. gondii blocks the IFN-gamma-induced upregulation of major histocompatibility complex (MHC) class II mRNAs and proteins. Heat inactivation of the parasites prior to host cell invasion, but not inhibition of the intracellular replication of T. gondii abolished the inhibition of MHC class II upregulation. Interestingly, a T. gondii lysate (TL) mimicked the inhibitory effect of viable parasites on MHC class II expression. Nuclear translocation of the signal transducer and activator of transcription in response to IFN-gamma were normal both in cells incubated with TL or infected with viable parasites. Transcript levels of the class II transactivator and consequently H2-Ab were nevertheless diminished by both viable parasites and TL. In contrast, interferon regulatory factor-1 mRNA was only decreased in response to viable T. gondii. Luciferase reporter assays confirmed differential effects of viable parasites and TL on minimal or complex IFN-gamma-responsive promoters. Furthermore, only TL, and not viable parasites, strongly induced the secretion of IL-10 by murine macrophages. Whereas TL also inhibited MHC class II expression in macrophages from IL-10-deficient mice, increased IL-10 secretion by wild type macrophages did not mediate the block in MHC class II upregulation. In conclusion, T. gondii employs different mechanisms to inhibit MHC class II expression, suggesting a complex regulation of this immune evasion strategy.

Systematic gene targeting on the X chromosome of Drosophila melanogaster.

The genome of the model organism Drosophila melanogaster has been sequenced and annotated. Based on this groundwork, we performed a systematic genetic screen of the D. melanogaster X chromosome, which carries about one sixth of the genes of the organism. We generated a collection of single P-element insertions to provide genetic and molecular access to virtually all X-chromosomal genes. The study complements earlier work designed to systematically identify vital genes on the X chromosome by targeting transcription units which are phenotypically silent. We describe single UAS sequence-bearing P-element insertions throughout the X chromosome, which allows one to express the tagged genes under control of tissue/organ-directed GAL4 activity. In addition, the present collection of single insertion lines provides a tool to generate chromosomal deletions which are on average less than 33 kb in size.

Mapping and identification of essential gene functions on the X chromosome of Drosophila.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.