Distinct Phenotypes Caused by Mutation of MSH2 in Trypanosome Insect and Mammalian Life Cycle Forms Are Associated with Parasite Adaptation to Oxidative Stress

BackgroundDNA repair mechanisms are crucial for maintenance of the genome in all organisms, including parasites where successful infection is dependent both on genomic stability and sequence variation. MSH2 is an early acting, central component of the Mismatch Repair (MMR) pathway, which is responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. In addition, recent evidence suggests that MSH2 might also play an important, but poorly understood, role in responding to oxidative damage in both African and American trypanosomes.Conclusions/SignificanceTaken together, these results indicate MSH2 displays conserved, dual roles in MMR and in the response to oxidative stress. Loss of the latter function results in life cycle dependent differences in phenotypic outcomes in T. brucei MSH2 mutants, most likely because of the greater burden of oxidative stress in the insect stage of the parasite.     

Regulation and spatial organization of PCNA in Trypanosoma brucei

As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. brucei (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.     

31P NMR spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Evidence for high levels of condensed inorganic phosphates

High resolution (31)P nuclear magnetic resonance spectra at 303.6 MHz (corresponding to a (1)H resonance frequency of 750 MHz) have been obtained of perchloric acid extracts of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, the causative agents of African sleeping sickness, Chagas' disease, and leishmaniasis. Essentially complete assignments have been made based on chemical shifts and by direct addition of authentic reference compounds. The results indicate the presence of high levels of short chain condensed polyphosphates: di-, tri-, tetra-, and pentapolyphosphate. (31)P NMR spectra of purified T. brucei, T. cruzi, and L. major acidocalcisomes, calcium and phosphorus storage organelles, indicate that polyphosphates are abundant in these organelles and have an average chain length of 3.11-3.39 phosphates. In the context of the recent discovery of several pyrophosphate-utilizing enzymes in trypanosomatids, the presence of these inorganic polyphosphates implies a critical role for these molecules in these parasites and a potential new route to chemotherapy.     

What happens when Trypanosoma brucei leaves Africa

Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.     

Dinitroaniline herbicides against protozoan parasites: the case of Trypanosoma cruzi

The drugs presently in use against Chagas disease are very toxic, inducing a great number of side effects. Alternative treatments are necessary, not only for Chagas disease but also for other diseases caused by protozoan parasites where current drugs pose toxicity problems. The plant microtubule inhibitor trifluralin has previously been tested with success against Leishmania, Trypanosoma brucei and several other protozoan parasites. Trypanosoma cruzi, the causative agent of Chagas disease, is also sensitive to the drug. This sensitivity has been correlated with the deduced amino acid sequences of alpha- and beta-tubulin of T. cruzi as compared with plant, mammal and other parasite sequences.     

DNA repair BER pathway inhibition increases cell death caused by oxidative DNA damage in Trypanosoma cruzi

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas disease, an endemic and neglected pathology in Latin America. It presents a life cycle that involves a hematophagous insect and man as well as domestic and wild mammals. The parasitic infection is not eliminated by the immune system of mammals; thus, the vertebrate host serves as a parasite reservoir. Additionally, chronic processes leading to dysfunction of the cardiac and digestive systems are observed. To establish a chronic infection some parasites should resist the oxidative damage to its DNA exerted by oxygen and nitrogen free radicals (ROS/RNS) generated in host cells. Till date there are no reports directly showing oxidative DNA damage and repair in T. cruzi. We establish that ROS/RNS generate nuclear and kinetoplastid DNA damage in T. cruzi that may be partially repaired by the parasite. Furthermore, we determined that both oxidative agents diminish T. cruzi cell viability. This effect is significantly augmented in parasites subsequently incubated with methoxyamine, a DNA base excision repair (BER) pathway inhibitor, strongly suggesting that the maintenance of T. cruzi viability is a consequence of DNA repair mechanisms.     

The identification of circular extrachromosomal DNA in the nuclear genome of Trypanosoma brucei

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.     

The karyotype and ploidy of Trypanosoma cruzi.

Little is known of the number or organization of chromosomes in Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease in man in the New World. Straightforward cytogenetic analysis is precluded because trypanosome chromosomes fail to condense during the cell cycle. We have size-fractionated the chromosome-sized DNA molecules of representative T. cruzi strains by pulsed field gradient (PFG) gel electrophoresis and located several housekeeping genes by Southern blotting using cDNA probes from the related trypanosome T. brucei. We show that DNA molecules from homologous chromosomes of T. cruzi migrate differently in the PFG system and infer that T. cruzi epimastigotes are at minimum diploid. In contrast to T. brucei, mini-chromosomes are absent in T. cruzi. All the housekeeping genes studied hybridize to DNA molecules which can be resolved in the PFG system, suggesting that T. cruzi may have no chromosomes larger than a few megabase pairs.     

Validation of Trypanosoma brucei trypanothione synthetase as drug target

In trypanosomes, the parasite-specific thiol trypanothione [T(SH)2] fulfills various functions, the best established being detoxification of H2O2 and organic hydroperoxides and ribonucleotide reduction. Recently, a trypanothione synthetase (Tb-TryS) gene from Trypanosoma brucei was isolated and the heterologously expressed Tb-TryS catalyzed the entire synthesis of T(SH)2 from glutathione (GSH) and spermidine in vitro. To confirm the in situ function of the complex Tb-TryS activities and to evaluate the importance of T(SH)2 metabolism in T. brucei, TryS suppression by double-stranded RNA interference was performed. Knockdown of TryS led to depletion of both T(SH)2 and glutathionylspermidine (Gsp) and accumulation of GSH, while concomitantly impairment of viability and arrest of proliferation were observed. TryS-downregulated cells displayed a significantly increased sensitivity to H2O2 and tert.-butyl hydroperoxide. These data verify the hypothesis that in T. brucei, a single enzyme synthesizes the spermidine-conjugated thiols (Gsp and T(SH)2) and further confirms the significance of trypanothione in the defense against oxidative stress and the maintenance of viability and proliferation in unstressed parasites.     

Trypanosoma cruzi: Oxidative stress induces arginine kinase expression

Trypanosoma cruzi arginine kinase is a key enzyme in cell energy management and is also involved in pH and nutritional stress response mechanisms. T. cruzi epimastigotes treated with hydrogen peroxide presented a time-dependent increase in arginine kinase expression, up to 10-fold, when compared with untreated parasites. Among other oxidative stress-generating compounds tested, only nifurtimox produced more than 2-fold increase in arginine kinase expression. Moreover, parasites overexpressing arginine kinase showed significantly increased survival capability during hydrogen peroxide exposure. These findings suggest the participation of arginine kinase in oxidative stress response systems.     

A Trypanosoma cruzi monoclonal antibody that recognizes a superficial tubulin-like antigen

A monoclonal antibody (MAB 10), obtained from mice infected with Trypanosoma cruzi, was found to recognize a superficial antigen in living or fixed parasites. It reacted more strongly with T. cruzi than with related parasites such as T. brucei and Leishmania. In immunoblots it recognized a single trypanosoma polypeptide and also brain tubulin, both of which had the same electrophoretic mobility. Further analysis suggested that the alpha-tubulin subunit contained the epitope recognized by MAB 10. These results suggest that a surface tubulin-like protein is present is T. cruzi.     

Telomeric co-localization of the modified base J and contingency genes in the protozoan parasite Trypanosoma cruzi

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.     

Mechanisms of Trypanosoma cruzi persistence in Chagas disease

Trypanosoma cruzi infection leads to development of chronic Chagas disease. In this article, we provide an update on the current knowledge of the mechanisms employed by the parasite to gain entry into the host cells and establish persistent infection despite activation of a potent immune response by the host. Recent studies point to a number of T. cruzi molecules that interact with host cell receptors to promote parasite invasion of the diverse host cells. T. cruzi expresses an antioxidant system and thromboxane A(2) to evade phagosomal oxidative assault and suppress the host's ability to clear parasites. Additional studies suggest that besides cardiac and smooth muscle cells that are the major target of T. cruzi infection, adipocytes and adipose tissue serve as reservoirs from where T. cruzi can recrudesce and cause disease decades later. Further, T. cruzi employs at least four strategies to maintain a symbiotic-like relationship with the host, and ensure consistent supply of nutrients for its own survival and long-term persistence. Ongoing and future research will continue to help refining the models of T. cruzi invasion and persistence in diverse tissues and organs in the host.     

Functional Characterization of 8-Oxoguanine DNA Glycosylase of Trypanosoma cruzi

The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-)/(-) (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2)O(2)). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2)O(2) of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.     

Properties of phosphoenolpyruvate mutase,the first enzyme in the aminoethylphosphonate biosynthetic pathway in Trypanosoma cruzi

Phosphoenolpyruvate (PEP) mutase catalyzes the conversion of phosphoenolpyruvate to phosphonopyruvate, the initial step in the formation of many naturally occurring phosphonate compounds. The phosphonate compound 2-aminoethylphosphonate is present as a component of complex carbohydrates on the surface membrane of many trypanosomatids including glycosylinositolphospholipids of Trypanosoma cruzi. Using partial sequence information from the T. cruzi genome project we have isolated a full-length gene with significant homology to PEP mutase from the free-living protozoan Tetrahymena pyriformis and the edible mussel Mytilus edulis. Recombinant expression in Escherichia coli confirms that it encodes a functional PEP mutase with a Km apparent of 8 microM for phosphonopyruvate and a kcat of 12 s-1. The native enzyme is a homotetramer with an absolute requirement for divalent metal ions and displays negative cooperativity for Mg2+ (S0.5 0.4 microM; n = 0.46). Immunofluorescence and sub-cellular fractionation indicates that PEP mutase has a dual localization in the cell. Further evidence to support this was obtained by Western analysis of a partial sub-cellular fractionation of T. cruzi cells. Southern and Western analysis suggests that PEP mutase is unique to T. cruzi and is not present in the other medically important parasites, Trypanosoma brucei and Leishmania spp.     

Molecular cloning and characterization of the DNA mismatch repair gene class 2 from the Trypanosoma cruzi

Genes with homology to the bacterial mutS gene, which encodes a protein involved in post-replication DNA mismatch repair, are known in several organisms. Using a degenerate PCR strategy, we cloned a Trypanosoma cruzi genomic DNA fragment homologous to the mutS gene class two (MSH2). This fragment was used as a probe to select the corresponding cDNAs from a T. cruzi cDNA library. The complete sequence of the gene (3304 bp), denominated TcMSH2, was obtained. The sequence contained an open reading frame of 2889 bp coding for a putative protein of 962 amino acids. Computational analyses of the amino acid sequence showed 36% identity with MSH2 proteins from other eukaryotes and revealed the presence of all functional domains of MutS proteins. Hybridization analyses indicated that the TcMSH2 gene is present as a single copy gene that is expressed in all forms of the T. cruzi life cycle. The role of the product of the TcMSH2 gene in mismatch repair was investigated by negative dominance phenotype analyses in Escherichia coli. When eukaryotic muts genes are expressed in a prokaryotic system, they increase the bacterial mutation rate. The same phenomenon was observed with the TcMSH2 cDNA, indicating that T. cruzi MSH2 interferes with the bacterial mismatch system. Phylogenetic analyses showed that the T. cruzi gene grouped with the MSH2 clade confirming the nature of the gene isolated in this work.     

DNA metabolism and genetic diversity in Trypanosomes

Trypanosomes are protozoan parasites that cause major diseases in humans and other animals. Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of African and American Trypanosomiasis, respectively. In spite of large amounts of information regarding various aspects of their biology, including the essentially complete sequences of their genomes, studies directed towards an understanding of mechanisms related to DNA metabolism have been very limited. Recent reports, however, describing genes involved with DNA recombination and repair in T. brucei and T. cruzi, indicated the importance of these processes in the generation of genetic variability, which is crucial to the success of these parasites. Here, we review these data and discuss how the DNA repair and recombination machineries may contribute to strikingly different strategies evolved by the two Trypanosomes to create genetic variability that is needed for survival in their hosts. In T. brucei, two genetic components are critical to the success of antigenic variation, a strategy that allows the parasite to evade the host immune system by periodically changing the expression of a group of variant surface glycoproteins (VSGs). One component is a mechanism that provides for the exclusive expression of a single VSG at any one time, and the second is a large repository of antigenically distinct VSGs. Work from various groups showing the importance of recombination reactions in T. brucei, primarily to move a silent VSG into an active VSG expression site, is discussed. T. cruzi does not use the strategy of antigenic variation for host immune evasion but counts on the extreme heterogeneity of their population for parasite adaptation to different hosts. We discuss recent evidence indicating the existence of major differences in the levels of genomic heterogeneity among T. cruzi strains, and suggest that metabolic changes in the mismatch repair pathway could be an important source of antigenic diversity found within the T. cruzi population.     

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.