[Show abstract][Hide abstract] ABSTRACT: We have recently demonstrated that O-linked glucosylation of thymine in trypanosome DNA (base J) regulates polymerase II transcription initiation. In vivo analysis has indicated that base J synthesis is initiated by the hydroxylation of thymidine by proteins (JBP1 and JBP2) homologous
to the Fe2+/2-oxoglutarate (2-OG)-dependent dioxygenase superfamily where hydroxylation is driven by the oxidative decarboxylation of
2-OG, forming succinate and CO2. However, no direct evidence for hydroxylase activity has been reported for the JBP proteins. We now demonstrate recombinant
JBP1 hydroxylates thymine specifically in the context of dsDNA in a Fe2+-, 2-OG-, and O2-dependent manner. Under anaerobic conditions, the addition of Fe2+ to JBP1/2-OG results in the formation of a broad absorption spectrum centered at 530 nm attributed to metal chelation of
2-OG bound to JBP, a spectroscopic signature of Fe2+/2-OG-dependent dioxygenases. The N-terminal thymidine hydroxylase domain of JBP1 is sufficient for full activity and mutation
of residues involved in coordinating Fe2+ inhibit iron binding and thymidine hydroxylation. Hydroxylation in vitro and J synthesis in vivo is inhibited by known inhibitors of Fe2+/2-OG-dependent dioxygenases. The data clearly demonstrate the JBP enzymes are dioxygenases acting directly on dsDNA, confirming
the two-step J synthesis model. Growth of trypanosomes in hypoxic conditions decreases JBP1 and -2 activity, resulting in
reduced levels of J and changes in parasite virulence previously characterized in the JBP KO. The influence of environment
upon J biosynthesis via oxygen-sensitive regulation of JBP1/2 has exciting implications for the regulation of gene expression
and parasite adaptation to different host niches.
[Show abstract][Hide abstract] ABSTRACT: Background: Base J regulates Pol II transcription. Results: JBP1 and -2 stimulate the first step of base J synthesis: hydroxylation of thymidine. Conclusion: JBP are Fe 2 /2-OG-dependent dioxygenases sensitive to physiologically relevant O 2 tensions. Significance: These results predict that JBPs can act as oxygen sensors regulating trypanosome gene expression and adaption to different host niches. We have recently demonstrated that O-linked glucosylation of thymine in trypanosome DNA (base J) regulates polymerase II transcription initiation. In vivo analysis has indicated that base J synthesis is initiated by the hydroxylation of thymidine by pro-teins (JBP1 and JBP2) homologous to the Fe 2 /2-oxoglutarate (2-OG)-dependent dioxygenase superfamily where hydroxyla-tion is driven by the oxidative decarboxylation of 2-OG, forming succinate and CO 2 . However, no direct evidence for hydroxylase activity has been reported for the JBP proteins. We now demon-strate recombinant JBP1 hydroxylates thymine specifically in the context of dsDNA in a Fe 2 -, 2-OG-, and O 2 -dependent manner. Under anaerobic conditions, the addition of Fe 2 to JBP1/2-OG results in the formation of a broad absorption spec-trum centered at 530 nm attributed to metal chelation of 2-OG bound to JBP, a spectroscopic signature of Fe 2 /2-OG-depen-dent dioxygenases. The N-terminal thymidine hydroxylase domain of JBP1 is sufficient for full activity and mutation of residues involved in coordinating Fe 2 inhibit iron binding and thymidine hydroxylation. Hydroxylation in vitro and J synthesis in vivo is inhibited by known inhibitors of Fe 2 /2-OG-depen-dent dioxygenases. The data clearly demonstrate the JBP enzymes are dioxygenases acting directly on dsDNA, confirm-ing the two-step J synthesis model. Growth of trypanosomes in hypoxic conditions decreases JBP1 and -2 activity, resulting in reduced levels of J and changes in parasite virulence previously characterized in the JBP KO. The influence of environment upon J biosynthesis via oxygen-sensitive regulation of JBP1/2 has exciting implications for the regulation of gene expression and parasite adaptation to different host niches. -D-Glucopyranosyloxymethyluracil (base J) 2 is a hyper-modified DNA base found in eukaryotes. This DNA modifica-tion is evolutionarily conserved within members of the kineto-plastid family, namely Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, where J replaces about 1% of the total T in the genome and is predominantly present in repetitive DNA sequences, such as telomeric repeats (for review, see Ref. 1). However, more recently, we localized a minor fraction of J to chromosome-internal regions coinciding with RNA polymer-ase II (Pol II) transcription initiation and termination sites (2). Loss of base J synthesis at these chromosome-internal regions in T. cruzi led to increased Pol II transcription initiation and corresponding changes in gene expression and parasite viru-lence (3, 4). Thus, base J represents a novel epigenetic modifi-cation of kinetoplastid DNA involved in regulating gene expression. Indirect evidence (for review, see Ref. 5) indicates J is synthe-sized in a two-step pathway (Fig. 1). Step one involves the hydroxylation of thymine in DNA by a thymidine hydroxylase (TH) enzyme, forming 5-hydroxymethyluracil (hmU). This intermediate is then glucosylated by a glucosyltransferase forming base J. Although the glucosyltransferase has not been identified, two proteins involved in the first step (JBP1 and JBP2) (6, 7) have been characterized. Both JBP1 and JBP2 (8, 9) contain a putative TH domain at the N terminus that has led to the designation of these enzymes belonging to the new TET/ JBP subfamily of dioxygenases that require Fe 2 and 2-oxogl-utarate (2-OG) for activity (10, 11). Family members are typi-cally identified on a structural level by the presence of a jelly roll -helix sheet that contains four key conserved residues involved in the binding of Fe 2 and 2-OG and are essential for catalytic activity (see for review, see Ref. 12). Mutation of these conserved residues within the TH domain of JBP1 and JBP2
Journal of Biological Chemistry 04/2012; 287(24):19886. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Very little is understood regarding how transcription is initiated/regulated in the early-diverging eukaryote Trypanosoma cruzi. Unusually for a eukaryote, genes transcribed by RNA polymerase (Pol) II in T. cruzi are arranged in polycistronic transcription units (PTUs). On the basis of this gene organization, it was previously thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. We recently localized a novel glucosylated thymine DNA base, called base J, to potential promoter regions of PTUs throughout the trypanosome genome. Loss of base J, following the deletion of JBP1, a thymidine hydroxylase involved with synthesis, led to a global increase in the Pol II transcription rate and gene expression. In order to determine the mechanism by which base J regulates transcription, we have characterized changes in chromatin structure and Pol II recruitment to promoter regions following the loss of base J. The loss of base J coincides with a decrease in nucleosome abundance, increased histone H3/H4 acetylation, and increased Pol II occupancy at promoter regions, including the well-characterized spliced leader RNA gene promoter. These studies present the first direct evidence for epigenetic regulation of Pol II transcription initiation via DNA modification and chromatin structure in kinetoplastids as well as provide a mechanism for regulation of trypanosome gene expression via the novel hypermodified base J.
[Show abstract][Hide abstract] ABSTRACT: Unlike other eukaryotes, the protein-coding genes of Trypanosoma cruzi are arranged in large polycistronic gene clusters transcribed by polymerase II (Pol II). Thus, it is thought that trypanosomes
rely solely on posttranscriptional processes to regulate gene expression. Here, we show that the glucosylated thymine DNA
base (β-d-glucosyl-hydroxymethyluracil or base J) is present within sequences flanking the polycistronic units (PTUs) in T. cruzi. The loss of base J at sites of transcription initiation, via deletion of the two enzymes that regulate base J synthesis (JBP1
and JBP2), correlates with an increased rate of Pol II transcription and subsequent genome-wide increase in gene expression.
The affected genes include virulence genes, and the resulting parasites are defective in host cell invasion and egress. These
studies indicate that base J is an epigenetic factor regulating Pol II transcription initiation in kinetoplastids and provides
the first biological role of the only hypermodified DNA base in eukaryotes.
[Show abstract][Hide abstract] ABSTRACT: Infections from Cryptosporidium parvum are of interest not only to public health, but also to wildlife conservation, particularly when humans and livestock encroach on nature and thereby increase the risk of cross-species transmissions. To clarify this risk, we used polymerase chain reaction to examine the hypervariable region of the C. parvum 18S rRNA gene in feces from three monkey species. Samples were isolated from regions where disease transmission between monkeys, livestock, and humans was likely (soiled habitat) or unlikely (clean habitat). Monkey individuals, their social groups, and different species shared multiple genotypes/isolates of C. parvum. Ecological and molecular analyses suggested that Cryptosporidium infection among Toque macaques in soiled habitats was mainly the bovine genotype C. parvum. Monkeys inhabiting clean habitat, particularly gray and purple-faced langurs, lacked Cryptosporidium species/types associated with bovines. Livestock apparently was a main source of infection for wild primates.
The American journal of tropical medicine and hygiene 12/2007; 77(5):818-22. · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Synthesis of the modified thymine base, beta-d-glucosyl-hydroxymethyluracil or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream form specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. In order to analyze the function of base J in the regulation of antigenic variation, we are characterizing the regulatory mechanism of J biosynthesis. We have recently proposed a model in which chromatin remodeling by a SWI2/SNF2-like protein (JBP2) regulates the developmental and de novo site-specific localization of J synthesis within bloodstream form trypanosome DNA. Consistent with this model, we now show that JBP2 (-/-) bloodstream form trypanosomes contain five-fold less base J and are unable to stimulate de novo J synthesis in newly generated telomeric arrays.
[Show abstract][Hide abstract] ABSTRACT: Base J or beta-d-glucosylhydroxymethyluracil is a modification of thymine residues within the genome of kinetoplastid parasites. In organisms known to contain the modified base, J is located mainly within the telomeric repeats. However, in Trypanosoma brucei, a small fraction of J is also located within the silent subtelomeric variant surface glycoprotein (VSG) gene expression sites, but not in the active expression site, suggesting a role for J in regulating telomeric genes involved in pathogenesis. With the identification of surface glycoprotein genes adjacent to telomeres in the South American Trypanosome, Trypanosoma cruzi, we became interested in the telomeric distribution of base J. Analysis of J and telomeric repeat sequences by J immunoblots and Southern blots following DNA digestion, reveals approximately 25% of J outside the telomeric repeat sequences. Moreover, the analysis of DNA sequences immunoprecipitated with J antiserum, localized J within subtelomeric regions rich in life-stage-specific surface glycoprotein genes involved in pathogenesis. Interestingly, the pattern of J within these regions is developmentally regulated. These studies provide a framework to characterize the role of base J in the regulation of telomeric gene expression/diversity in T. cruzi.
Nucleic Acids Research 02/2007; 35(19):6367-77. DOI:10.1093/nar/gkm693 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Serum samples collected during August 2003-June 2004 from 45 privately owned captive and 8 elephants from the Pinnawala Elephant Orphanage were tested for the presence of antibodies against Toxoplasma gondii using the direct modified agglutination test (MAT). Antibodies were found in sera of 14 of 45 (32%) privately owned elephants with titers of 1:25 in three, 1:50 in three, 1:100 in three, 1:200 in three, and 1:400 in three elephants. The elephants from Pinnawala Elephant Orphanage were seronegative. This is the first report of T. gondii seroprevalence in elephants in Sri Lanka.
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidiosis is a rapidly emerging disease in the tropics. This is the first report of Cryptosporidium and other protozoan infections (Entamoeba spp., Iodamoeba, Chilomastix, and Balantidium spp.) in wild primates that inhabit the natural forest of Sri Lanka. It is unclear if non-human primates serve as a reservoir for these parasites under certain conditions. A cross-sectional coprologic survey among 125 monkeys (89 toque macaques, 21 gray langurs, and 15 purple-faced langurs) indicated that Cryptosporidium was detected in all three primate species and was most common among monkeys using areas and water that had been heavily soiled by human feces and livestock. Most macaques (96%) shedding Cryptosporidium oocysts were co-infected with other protozoans and important anthropozoonotic gastrointestinal parasites (e.g., Enterobius and Strongyloides). The transmission of these parasites among primates in the wild may have important implications for public health as well as wildlife conservation management.
The American journal of tropical medicine and hygiene 03/2006; 74(2):322-9. · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 100 free-range chickens (Gallus domesticus) from Sri Lanka was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 39 chickens with titers of 1:5 in 8, 1:10 in 8, 1:20 in 4, 1:40 in 5, 1:80 in 5, 1:160 in 5, 1:320 in 2, 1:640 or more in 2. Hearts and brains of 36 chickens with MAT titers of 1:5 or more were bioassayed in mice. Tissues of 3 chickens with doubtful titers of 1:5 were pooled and fed to a cat; the cat shed T. gondii oocysts in its feces. Tissues from 61 chickens with titers of less than 1:5 were pooled and fed to 2 T. gondii-free cats; the cats did not shed oocysts. Toxoplasma gondii was isolated from 11 of 36 seropositive chickens by bioassay in mice. All 12 T. gondii isolates were avirulent for mice. Genotyping of 12 isolates using the SAG2 locus indicated that 6 were type III, and 6 were type II. This is the first report of genetic characterization of T. gondii from any host in Sri Lanka.
Journal of Parasitology 01/2006; 91(6):1480-2. DOI:10.1645/GE-479R.1 · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: From a natural population that inhabits the dry evergreen forest at Polonnaruwa, serum samples of 170 toque macaques were examined for antibodies to Toxoplasma gondii by the modified agglutination test. Of these, 21 (12%) were found with titers of 1:16 in 9, 1:32 in 9, 1:256 in 1, 1:1,024 in 1, and 1:4,096 in 1. There was no evidence of maternal transmission of antibodies or congenital toxoplasmosis. None of the infected macaques died within 1 yr after sampling. Toxoplasma gondii infection was closely linked to human environments where domestic cats were common. Macaques having frequent contact with human settlements showed a significantly greater (P < 0.0001) prevalence (19% infected) than macaques restricted to forest habitat, none of which was infected. Although infection with T. gondii has been noted in several species of Asian primates, this is the first report of T. gondii antibodies in toque macaques (Macaca sinica) that are endemic to the island of Sri Lanka.
Journal of Parasitology 09/2004; 90(4):870-1. DOI:10.1645/GE-291R · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hematological studies were conducted in three wild groups of toque macaques (Macaca sinica) inhabiting the Polonnaruwa Sanctuary in northeastern Sri Lanka. The macaques were temporarily trapped and anesthetized, and femoral blood was drawn from 35 males and 37 females (age range: 0.33-24.5 yr). Statistically significant (P<0.05) differences were observed by sex for total plasma proteins (PP), and by age for red blood cell (RBC) counts, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), PP, erythrocyte sedimentation rate (ESR), differential and absolute neutrophil counts, differential lymphocyte counts, and absolute eosinophil counts. In general, the results were similar to those reported for other species of colony-bred and free-ranging macaques. However, there also were differences. First, in contrast to earlier studies of nonhuman primates, we examined the hematology of infants. Compared to other age classes, infants (<1 yr old) had lower RBC, Hb, MCHC, and ESR values, and a higher MCV. These findings were similar to those obtained in human infants. Second, we observed variations in hematology among social groups in relation to their ecology. Two groups (IH3 and M3) had ready access to water throughout the dry season (the period of sampling), whereas the third group (J) did not. The Hb, RBC, and PP values obtained in groups IH3 and M3 were similar to those reported in other macaque species. However, these parameters in group J were significantly (P<0.01) higher, which suggests that this group (representing about 26% of the sample) had been dehydrated during the dry season. Finally, two indices indicative of injury and infection--the ESR and leukocyte counts--were higher in the wild toque macaques than has been reported for other species of macaques held in captivity, and about 15% of the toque macaques sampled had extreme outlier values for these parameters; however, none were visibly ill or died. These results suggest that wild toque macaques are subject to a wide array of physical and biological insults that are unique to natural populations.
American Journal of Primatology 09/2003; 61(1):13-28. DOI:10.1002/ajp.10105 · 2.44 Impact Factor