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William J Sullivan

Publications and source records attributed to William J Sullivan.

17 recordsLinked to original sources
PubMed2006

Histones and histone modifications in protozoan parasites.

Protozoan parasites are early branching eukaryotes causing significant morbidity and mortality in humans and livestock. Single-celled parasites have evolved complex life cycles, which may involve multiple host organisms, and strategies to evade host immune responses. Consequently, two key aspects of virulence that underlie pathogenesis are parasite differentiation and antigenic variation, both of which require changes in the expressed genome. Complicating these requisite alterations in the parasite transcriptome is chromatin, which serves as a formidable barrier to DNA processes including transcription, repair, replication and recombination. Considerable progress has been made in the study of chromatin dynamics in other eukaryotes, and there is much to be gained in extending these analyses to protozoan parasites. Much of the work completed to date has focused on histone acetylation and methylation in the apicomplexans and trypanosomatids. As we describe in this review, such studies provide a unique vantage point of the evolutionary picture of eukaryotic cell development, and reveal unique phenomena that could be exploited pharmacologically to treat protozoal diseases.

Acetylation↗
PubMed2006

Histone mediated gene activation in Toxoplasma gondii.

DNA processes, including transcriptional activation, are largely governed by histone modifications. For most eukaryotes, chromatin-remodeling machines have been identified that move, replace, or decorate histones. Recent studies have shed some important light on similar machinery operating in apicomplexan parasites, including the opportunistic pathogen Toxoplasma gondii. Here we present a synthesis of the current knowledge of histone mediated gene expression in Toxoplasma, particularly in the context of parasite differentiation.

Animals↗
PubMed2006

Pair of unusual GCN5 histone acetyltransferases and ADA2 homologues in the protozoan parasite Toxoplasma gondii.

GCN5 is a histone acetyltransferase (HAT) essential for development in mammals and critical to stress responses in yeast. The protozoan parasite Toxoplasma gondii is a serious opportunistic pathogen. The study of epigenetics and gene expression in this ancient eukaryote has pharmacological relevance and may facilitate the understanding of these processes in higher eukaryotes. Here we show that the disruption of T. gondii GCN5 yields viable parasites, which were subsequently employed in a proteomics study to identify gene products affected by its loss. Promoter analysis of these TgGCN5-dependent genes, which were mostly parasite specific, reveals a conserved T-rich element. The loss of TgGCN5 does not attenuate virulence in an in vivo mouse model. We also discovered that T. gondii is the only invertebrate reported to date possessing a second GCN5 (TgGCN5-B). TgGCN5-B harbors a strikingly divergent N-terminal domain required for nuclear localization. Despite high homology between the HAT domains, the two TgGCN5s exhibit differing substrate specificities. In contrast to TgGCN5-A, which exclusively targets lysine 18 of H3, TgGCN5-B acetylates multiple lysines in the H3 tail. We also identify two ADA2 homologues that interact differently with the TgGCN5s. TgGCN5-B has the potential to compensate for TgGCN5-A, which probably arose from a gene duplication unique to T. gondii. Our work reveals an unexpected complexity in the GCN5 machinery of this primitive eukaryote.

Acetylation↗
PubMed2005

The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits.

Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.

Amino Acid Sequence↗
PubMed2005

Identification of proteins interacting with Toxoplasma SRCAP by yeast two-hybrid screening.

Toxoplasma gondii is an opportunistic protozoan parasite that differentiates into latent cysts (bradyzoite) that can be reactivated during immunosuppression. TgSRCAP (Toxoplasma gondii Snf2-related CBP activator protein) is a SWI2/SNF2 family chromatin remodeler whose expression increases during cyst development. Identifying the proteins associating with TgSRCAP during the pre-cyst stage (tachyzoite) will increase our understanding of how parasite differentiation is initiated. We employed the yeast two-hybrid system to identify proteins that may interact directly with TgSRCAP. A stretch of 1,060 amino acids between ATPase subdomains IV and V of TgSRCAP was chosen as "bait" since the corresponding region in human SRCAP interacts with other proteins, including CREB binding protein. We have identified several novel parasite-specific transcription factors predicted to be in the T. gondii genome. Metabolic enzymes that may participate in cyst development were also identified as interacting with TgSRCAP.

Animals↗
PubMed2005

The downed runner.

Race coverage can be a rewarding experience for the sports medicine clinician. Several conditions are likely to present to the medical tent, and accurate diagnosis is critical to proper treatment. An algorithm approach as outlined in this article can provide a starting point for the assessment of the downed runner. Recognition of the primary causes for collapse can help to instigate the correct treatment approach. A proper history and physical examination often can help to differentiate significant cardiac events from the more innocuous EAC. Furthermore, avoiding immediate i.v. fluids in the downed runner is prudent, at least until an appropriate diagnosis is made. This will help to prevent iatrogenic hyponatremia. In sum, proper preparation and knowledge of the ailments that affect long distance runners will help to maintain an effective medical tent on race day.

Algorithms↗
PubMed2005

IMP dehydrogenase from the protozoan parasite Toxoplasma gondii.

The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03+/-0.004 microM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable beta-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.

Amino Acid Sequence↗
PubMed2005

MYST family histone acetyltransferases in the protozoan parasite Toxoplasma gondii.

The restructuring of chromatin precedes tightly regulated events such as DNA transcription, replication, and repair. One type of chromatin remodeling involves the covalent modification of nucleosomes by histone acetyltransferase (HAT) complexes. The observation that apicidin exerts antiprotozoal activity by targeting a histone deacetyltransferase has prompted our search for more components of the histone modifying machinery in parasitic protozoa. We have previously identified GNAT family HATs in the opportunistic pathogen Toxoplasma gondii and now describe the first MYST (named for members MOZ, Ybf2/Sas3, Sas2, and Tip60) family HATs in apicomplexa (TgMYST-A and -B). The TgMYST-A genomic locus is singular and generates a approximately 3.5-kb transcript that can encode two proteins of 411 or 471 amino acids. TgMYST-B mRNA is approximately 7.0 kb and encodes a second MYST homologue. In addition to the canonical MYST HAT catalytic domain, both TgMYST-A and -B possess an atypical C2HC zinc finger and a chromodomain. Recombinant TgMYST-A exhibits a predilection to acetylate histone H4 in vitro at lysines 5, 8, 12, and 16. Antibody generated to TgMYST-A reveals that both the long and short (predominant) versions are present in the nucleus and are also plentiful in the cytoplasm. Moreover, both TgMYST-A forms are far more abundant in rapidly replicating parasites (tachyzoites) than encysted parasites (bradyzoites). A bioinformatics survey of the Toxoplasma genome reveals numerous homologues known to operate in native MYST complexes. The characterization of TgMYST HATs represents another important step toward understanding the regulation of gene expression in pathogenic protozoa and provides evolutionary insight into how these processes operate in eukaryotic cells in general.

5' Untranslated Regions↗
PubMed2005

Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii.

Pathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.

Acetylation↗
PubMed2004

Histone acetylase GCN5 enters the nucleus via importin-alpha in protozoan parasite Toxoplasma gondii.

The histone acetyltransferase GCN5 acetylates nucleosomal histones to alter gene expression. How GCN5 gains entry into the nucleus of the cell has not been determined. We have mapped a six-amino acid motif (RKRVKR) that serves as a necessary and sufficient nuclear localization signal (NLS) for GCN5 in the protozoan pathogen Toxoplasma gondii (TgGCN5). Virtually nothing is known about nucleocytoplasmic transport in these parasites (phylum Apicomplexa), and this study marks the first demonstrated NLS delineated for members of the phylum. The TgGCN5 NLS has predictive value because it successfully identifies other nuclear proteins in three different apicomplexan genomic databases. Given the basic composition of the T. gondii NLS, we hypothesized that TgGCN5 physically interacts with importin-alpha, the main transport receptor in the importin/karyopherin nuclear import pathway. We cloned the importin-alpha gene from T. gondii (TgIMPalpha), which encodes a protein of 545 amino acids that possesses an importin-beta-binding domain and armadillo/beta-catenin-like repeats. In vitro co-immunoprecipitation experiments confirm that TgIMPalpha directly interacts with TgGCN5, but this interaction is abolished if the TgGCN5 NLS is deleted. Taken together, these data argue that TgGCN5 gains access to the parasite nucleus by interacting with TgIMPalpha. Bioinformatics analysis of the T. gondii genome reveals that other components of the importin pathway are present in the organism. This study demonstrates the utility of T. gondii as a model for the study of nucleocytoplasmic trafficking in early eukaryotic cells.

Acetyltransferases↗
PubMed2004

Parasite-specific eIF2 (eukaryotic initiation factor-2) kinase required for stress-induced translation control.

The ubiquitous intracellular parasite Toxoplasma gondii (phylum Apicomplexa) differentiates into an encysted form (bradyzoite) that can repeatedly re-emerge as a life-threatening acute infection (tachyzoite) upon impairment of immunity. Since the switch from tachyzoite to bradyzoite is a stress-induced response, we sought to identify components related to the phosphorylation of the alpha subunit of eIF2 (eukaryotic initiation factor-2), a well-characterized event associated with stress remediation in other eukaryotic systems. In addition to characterizing Toxoplasma eIF2alpha (TgIF2alpha), we have discovered a novel eIF2 protein kinase, designated TgIF2K-A (Toxoplasma gondii initiation factor-2kinase). Although the catalytic domain of TgIF2K-A contains sequence and structural features that are conserved among members of the eIF2 kinase family, TgIF2K-A has an extended N-terminal region that is highly divergent from other eIF2 kinases. TgIF2K-A specifically phosphorylates the regulatory serine residue of yeast eIF2alpha in vitro and in vivo, and can modulate translation when expressed in the yeast model system. We also demonstrate that TgIF2K-A phosphorylates the analogous regulatory serine residue of recombinant TgIF2alpha in vitro. Finally, we demonstrate that TgIF2alpha phosphorylation in tachyzoites is enhanced in response to heat shock or alkaline stress, conditions known to induce parasite differentiation in vitro. Collectively, this study suggests that eIF2 kinase-mediated stress responses are conserved in Apicomplexa, and a novel family member exists that may control parasite-specific events, including the clinically relevant conversion into bradyzoite cysts.

Amino Acid Sequence↗
PubMed2004

Purine salvage pathways in the apicomplexan parasite Toxoplasma gondii.

We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.

Adenine Phosphoribosyltransferase↗
PubMed2003

Molecular cloning and characterization of an SRCAP chromatin remodeling homologue in Toxoplasma gondii.

We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.

Adenosine Triphosphatases↗
PubMed2003

Histone H3 and H3.3 variants in the protozoan pathogens Plasmodium falciparum and Toxoplasma gondii.

Histones constitute the fundamental component of chromatin and participate in the regulation of gene expression by virtue of covalent modifications to their N-terminal domains. The discovery that histone-modifying enzymes are targeted by the antiprotozoal agent apicidin has prompted further investigation of gene expression regulation in protozoan parasites; consequently, several chromatin remodeling homologues with unusual features have been isolated. To facilitate investigation of these chromatin remodeling homologues using parasite-specific substrates, we sought to clone and characterize histone H3 from two medically significant pathogens in the phylum Apicomplexa: Plasmodium falciparum (malaria) and Toxoplasma gondii (opportunistic pathogen of immunocompromised individuals). Like most eukaryotic organisms, these parasites each contain at least two histone H3 variants, termed H3 and H3.3. Sequence analysis reveals the Apicomplexan H3 proteins harbor novel and rare features. Expression and purification of recombinant H3 variants will provide species-specific substrate for the analysis of the histone-modifying machinery of these parasites.

Amino Acid Sequence↗