Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner.

SummaryInfection of bovine leucocytes by Theileria annulata results in establishment of transformed, infected cells. Infection of the host cell is known to promote constitutive activation of pro-inflammatory transcription factors that have the potential to be beneficial or detrimental. In this study we have compared the effect of LPS activation on uninfected bovine leucocytes (BL20 cells) and their Theileria-infected counterpart (TBL20). Gene expression profiles representing activated uninfected BL20 relative to TBL20 cells were also compared. The results show that while prolonged stimulation with LPS induces cell death and activation of NF-κB in BL20 cells, the viability of Theileria-infected cells was unaffected. Analysis of gene expression networks provided evidence that the parasite establishes tight control over pathways associated with cellular activation by modulating reception of extrinsic stimuli and by significantly altering the expression outcome of genes targeted by infection-activated transcription factors. Pathway analysis of the data set identified novel candidate genes involved in manipulation of cellular functions associated with the infected transformed cell. The data indicate that the T. annulata parasite can irreversibly reconfigure host cell gene expression networks associated with development of inflammatory disease and cancer to generate an outcome thatis beneficial to survival and propagation of the infected leucocyte.

SummaryApicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kB in a parasite-dependent manner. Activation of NF-kB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kB. Further characterization of these lines demonstrated that elevated levels of activated NF-kB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host-and parasitedependent NF-kB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

Noncoding RNAs can modulate gene expression by directing modifications to histones that alter chromatin structure. In fission yeast, siRNAs produced via the RNAi pathway direct modifications associated with heterochromatin formation. siRNAs associate with the RNAi effector protein Argonaute 1 (Ago1), targeting the Ago1-containing RNA-induced transcriptional silencing (RITS) complex to homologous nascent transcripts. This promotes recruitment of the Clr4 complex (CLRC), which mediates methylation of histone H3 on lysine 9 (H3K9me) in cognate chromatin. A key question is how the RNAi and chromatin modification machineries are connected. Stc1 is a small protein recently shown to associate with both Ago1 and CLRC and to play a pivotal role in mediating the RNAi-dependent recruitment of CLRC to chromatin. To understand its mode of action, we have performed a detailed structural and functional analysis of the Stc1 protein. Our analyses reveal that the conserved N-terminal region of Stc1 represents an unusual tandem zinc finger domain, with similarities to common LIM domains but distinguished by a lack of preferred relative orientation of the two zinc fingers. We demonstrate that this tandem zinc finger domain is involved in binding Ago1, whereas the nonconserved C-terminal region mediates association with CLRC. These findings elucidate the molecular basis for the coupling of RNAi to chromatin modification in fission yeast. NMR | Schizosaccharomyces pombeR NAi is a conserved gene silencing mechanism characterized by the involvement of small RNAs. These small RNAs are bound by Argonaute proteins, forming RNAi effector complexes that can mediate silencing of specific targets based on sequence homology (1). This silencing can occur either posttranscriptionally, via transcript cleavage or translational repression, or at the transcriptional level, via chromatin modification. Such RNAidependent chromatin modification has been implicated in genome regulation throughout eukaryotes; however, mechanisms underlying the coupling of RNAi to either DNA methylation and/or histone modification remain poorly understood in most systems (2, 3).One of the best-characterized examples of RNAi-directed chromatin modification occurs in the fission yeast, Schizosaccharomyces pombe, where RNAi is required to promote assembly of pericentromeric heterochromatin. Heterochromatin is a specialized form of chromatin that is generally associated with transcriptional repression, and it is important for genome stability. In fission yeast, as in higher eukaryotes, heterochromatin is characterized by high levels of methylation on lysine 9 of histone H3 (H3K9), which provides a binding site for chromodomain proteins, including the heterochromatin protein 1 (HP1) family proteins Swi6 and Chp2 (4-6). Assembly of heterochromatin in fission yeast requires the action of histone deacetylases (Sir2, Clr3, and Clr6) as well as the sole H3K9 methyltransferase, Clr4 (7-9). Clr4 resides in a multisubunit Clr4 complex (CLRC), all members of which are required to suppo...