Chronic Trichuris muris infection causes neoplastic change in the intestine and exacerbates tumour formation in APC min/+ mice.

Incidences of infection-related cancers are on the rise in developing countries where the prevalence of intestinal nematode worm infections are also high. Trichuris muris (T. muris) is a murine gut-dwelling nematode that is the direct model for human T. trichiura, one of the major soil-transmitted helminth infections of humans. In order to assess whether chronic infection with T. muris does indeed influence the development of cancer hallmarks, both wild type mice and colon cancer model (APC min/+) mice were infected with this parasite. Parasite infection in wild type mice led to the development of neoplastic change similar to that seen in mice that had been treated with the carcinogen azoxymethane. Additionally, both chronic and acute infection in the APCmin/+ mice led to an enhanced tumour development that was distinct to the site of infection suggesting systemic control. By blocking the parasite induced T regulatory response in these mice, the increase in the number of tumours following infection was abrogated. Thus T. muris infection alone causes an increase in gut pathologies that are known to be markers of cancer but also increases the incidence of tumour formation in a colon cancer model. The influence of parasitic worm infection on the development of cancer may therefore be significant.

JBP1 and JBP2 proteins are Fe2+/2-oxoglutarate-dependent dioxygenases regulating hydroxylation of thymidine residues in trypanosome DNA.

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 Fe(2+)/2-oxoglutarate (2-OG)-dependent dioxygenase superfamily where hydroxylation 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 demonstrate 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 spectrum centered at 530 nm attributed to metal chelation of 2-OG bound to JBP, a spectroscopic signature of Fe(2+)/2-OG-dependent 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-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.

Two thymidine hydroxylases differentially regulate the formation of glucosylated DNA at regions flanking polymerase II polycistronic transcription units throughout the genome of Trypanosoma brucei.

Base J is a hypermodified DNA base localized primarily to telomeric regions of the genome of Trypanosoma brucei. We have previously characterized two thymidine-hydroxylases (TH), JBP1 and JBP2, which regulate J-biosynthesis. JBP2 is a chromatin re-modeling protein that induces de novo J-synthesis, allowing JBP1, a J-DNA binding protein, to stimulate additional J-synthesis. Here, we show that both JBP2 and JBP1 are capable of stimulating de novo J-synthesis. We localized the JBP1- and JBP2-stimulated J by anti-J immunoprecipitation and high-throughput sequencing. This genome-wide analysis revealed an enrichment of base J at regions flanking polymerase II polycistronic transcription units (Pol II PTUs) throughout the T. brucei genome. Chromosome-internal J deposition is primarily mediated by JBP1, whereas JBP2-stimulated J deposition at the telomeric regions. However, the maintenance of J at JBP1-specific regions is dependent on JBP2 SWI/SNF and TH activity. That similar regions of Leishmania major also contain base J highlights the functional importance of the modified base at Pol II PTUs within members of the kinetoplastid family. The regulation of J synthesis/localization by two THs and potential biological function of J in regulating kinetoplastid gene expression is discussed.

JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes.

Genomic DNA of African trypanosomes contains a hypermodified thymidine residue termed base J (beta-d-glucosyl-HOMedU). This modified base is localized primarily to repetitive DNA, namely the telomeres, and is implicated in the regulation of antigenic variation. The base is synthesized in a two-step pathway. Initially, a thymidine residue in DNA is hydroxylated by a thymidine hydroxylase (TH). This intermediate (HOMedU) is then glucosylated to form base J. Two proteins involved in J synthesis, JBP1 (J binding protein 1) and JBP2, contain a putative TH domain related to the family of Fe(2+)/2-oxoglutarate-dependent hydroxylases. We have previously shown that mutations in the TH domain of JBP1 kill its ability to stimulate J synthesis. Here we show that mutation of key residues in the TH domain of JBP2 ablate its ability to induce de novo J synthesis. While the individual JBP1 null and JBP2 null trypanosomes have reduced J levels, the deletion of both JBP1 and JBP2 generates a cell line that completely lacks base J but still contains glucosyl-transferase activity. Reintroduction of JBP2 in the J-null trypanosome stimulates HOMedU formation and site-specific synthesis of base J. We conclude that JBP2 and JBP1 are the TH enzymes involved in J biosynthesis.

An increase in epithelial cell apoptosis is associated with chronic intestinal nematode infection.

It is well established that homeostasis of the intestinal epithelium becomes dysregulated during gastrointestinal helminth infection and is under immune control. An increase in both enterocyte proliferation and the subsequent generation of crypt hyperplasia are hallmarks of chronic infection with Trichuris muris, a large intestinal dwelling nematode. The effect of this parasitic infection on apoptosis induction in the large intestine and its regulation has been neglected. To address this, mice of resistant and susceptible phenotypes were infected with different doses of T. muris, and the levels of epithelial cell apoptosis were determined. It is clear that apoptosis is induced during chronic T. muris infection. This occurs mainly at the base of the cecal crypt, within the stem cell region. The level of apoptosis induced is independent of worm number, suggesting that it is not a consequence of worm-induced damage but rather a mechanism for controlling cell number within the crypt. Neutralization of both gamma interferon and tumor necrosis factor alpha caused a significant reduction in the levels of apoptosis, showing that proinflammatory cytokines generated in response to chronic infection play an important role in apoptosis induction in this system. It is proposed that the generation of proinflammatory cytokines during chronic T. muris infection may play a positive role, by promoting intestinal epithelial cell apoptosis, to counter infection-induced epithelial hyperplasia.

Regulation of surface coat exchange by differentiating African trypanosomes.

African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.

The characterization of intraepithelial lymphocytes, lamina propria leukocytes, and isolated lymphoid follicles in the large intestine of mice infected with the intestinal nematode parasite Trichuris muris.

Despite a growing understanding of the role of cytokines in immunity to the parasitic helminth Trichuris muris, the local effector mechanism culminating in the expulsion of worms from the large intestine is not known. We used flow cytometry and immunohistochemistry to characterize the phenotype of large intestinal intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) from resistant and susceptible strains of mouse infected with T. muris. Leukocytes accumulated in the epithelium and lamina propria after infection, revealing marked differences between the different strains of mouse. In resistant mice, which mount a Th2 response, the number of infiltrating CD4+, CD8+, B220+, and F4/80+ IEL and LPL was generally highest around the time of worm expulsion from the gut, at which point the inflammation was dominated by CD4+ IEL and F4/80+ LPL. In contrast, in susceptible mice, which mount a Th1 response, the number of IEL and LPL increased more gradually and was highest after a chronic infection had developed. At this point, CD8+ IEL and F4/80+ LPL were predominant. Therefore, this study reveals the local immune responses underlying the expulsion of worms or the persistence of a chronic infection in resistant and susceptible strains of mouse, respectively. In addition, for the first time, we illustrate isolated lymphoid follicles in the large intestine, consisting of B cells interspersed with CD4+ T cells and having a central zone of rapidly proliferating cells. Furthermore, we demonstrate the organogenesis of these structures in response to T. muris infection.

Accelerated intestinal epithelial cell turnover: a new mechanism of parasite expulsion.

The functional integrity of the intestinal epithelial barrier forms a major defense against invading pathogens, including gastrointestinal-dwelling nematodes, which are ubiquitous in their distribution worldwide. Here, we show that an increase in the rate of epithelial cell turnover in the large intestine acts like an "epithelial escalator" to expel Trichuris and that the rate of epithelial cell movement is under immune control by the cytokine interleukin-13 and the chemokine CXCL10. This host protective mechanism against intestinal pathogens has implications for our wider understanding of the multifunctional role played by intestinal epithelium in mucosal defense.

The Trichuris muris system: a paradigm of resistance and susceptibility to intestinal nematode infection.

Gastrointestinal helminths infect over 1 billion people worldwide. Although rarely causing death, such diseases are associated with high levels of morbidity and furthermore bear a large economic burden within areas where infections are endemic. Trichuris muris, a natural intestinal parasite of mice has been extensively utilised as a laboratory model for the study of human whipworm Trichuris trichiura. This has proven to be an invaluable tool in dissecting the different components involved in immunity to trichuris infection. Moreover, it has become a paradigm of cytokine mediated immunity to gastrointestinal nematodes in general. It is well established that resistance and susceptibility to T. muris infection are tightly associated with the generation of a T helper 2 (TH2) or a T helper 1 (TH1) immune response, respectively. This review gives a detailed account of the experimental work which has provided us with this knowledge, and further builds upon this, by focusing upon the most recent developments and important findings from this host-parasite relationship.