Date Published: June 17, 2015
Publisher: Public Library of Science
Author(s): Viviane Grazielle-Silva, Tehseen Fatima Zeb, Jason Bolderson, Priscila C. Campos, Julia B. Miranda, Ceres L. Alves, Carlos R. Machado, Richard McCulloch, Santuza M. R. Teixeira, Alvaro Acosta-Serrano. http://doi.org/10.1371/journal.pntd.0003870
Abstract: 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.Methodology/Principal FindingsTo investigate the involvement of MMR in the oxidative stress response, null mutants of MSH2 were generated in Trypanosoma brucei procyclic forms and in Trypanosoma cruzi epimastigote forms. Unexpectedly, the MSH2 null mutants showed increased resistance to H2O2 exposure when compared with wild type cells, a phenotype distinct from the previously observed increased sensitivity of T. brucei bloodstream forms MSH2 mutants. Complementation studies indicated that the increased oxidative resistance of procyclic T. brucei was due to adaptation to MSH2 loss. In both parasites, loss of MSH2 was shown to result in increased tolerance to alkylation by MNNG and increased accumulation of 8-oxo-guanine in the nuclear and mitochondrial genomes, indicating impaired MMR. In T. cruzi, loss of MSH2 also increases the parasite capacity to survive within host macrophages.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.
Partial Text: Two members of the trypanosomatidae family, Trypanosoma cruzi and Trypanosoma brucei, are important human pathogens, since they cause, respectively, Chagas disease, or American trypanosomiasis, and African Sleeping Sickness, or Human African trypanosomiasis. Together, T. cruzi and T. brucei infections affect almost 20 million people [1, 2]. The life cycles of both these parasites involve two hosts: an invertebrate vector and a mammalian host. In the digestive tract of the insect vector T. cruzi multiplies as epimastigotes and differentiates into metacyclic trypomastigotes, which are expelled with the vector’s faeces. After a blood meal, trypomastigotes injected in the host bloodstream can invade different cell types, where they replicate as intracellular amastigotes that, after a number of replication cycles in the host cell cytoplasm, differentiate into trypomastigotes and lyse the host cell membrane. Despite being similar in general strategy, the life cycle of T. brucei is different to that of T. cruzi in several key details. Notably, T. brucei does not display any intracellular replicative stages. In the mammal, T. brucei is exclusively extracellular, replicating in the bloodstream and tissue fluids as bloodstream form (BSF) cells, which can be taken up by the tsetse fly vector during a bloodmeal. In the insect vector BSF cells differentiate into replicative procyclic forms (PCF), which then undergo several further differentiation events associated with migration to the fly salivary glands, where non-replicative metacyclic trypomastigotes are formed and can be passed into a new mammalian host through the proboscis when the infected fly is feeding . Irrespective of the detailed differences in the life cycles, differentiation between the mammal-infective and vector-infective forms of both T. cruzi and T. brucei is accompanied by dramatic metabolic changes and morphological alterations .
In previous studies we have described the phenotypic effects of deleting both alleles of MSH2 or MLH1 in T. brucei BSF cells, and the effects of loss of a single allele of MSH2 in T. cruzi epimastigotes [21, 37]. Here, we provide several new insights into the function of MMR in African and American trypanosomes. First, we show that genetic ablation of MSH2 or MLH1 is possible in T. brucei PCF cells, as is MSH2 ablation in T. cruzi epimastigotes. In all cases, loss of MSH2 results in detectable impairment in MMR, as demonstrated by the increased tolerance to the alkylator MNNG, a phenotype specifically seen in MMR mutants in other eukaryotes  and in T. brucei BSF cells . Second, we reveal a striking life cycle dependent difference in the effect of MSH2 ablation in T. brucei: in PCF cells loss of MSH2 results in increased tolerance to H2O2, whereas MSH2 loss results in increased sensitivity in BSF cells . The same increased tolerance is also seen in T. cruzi epimastigote MSH2 mutants, where it impacts on the capacity of the parasite to grow in ROS-producing host macrophages. Importantly, altered H2O2 resistance was not observed when T. brucei BSF or PCF cells lacking MLH1 were examined. Finally, we provide evidence that the increased resistance to H2O2 in T. brucei after loss of MSH2 is due to a life cycle dependent adaptation in a facet of the cell that is distinct from MSH2, since re-expression of the protein restores MMR activity in PCF cells but does not alter the response to H2O2. In contrast, re-expression of MSH2 in T. brucei BSF msh2-/- mutants reverts both MMR impairment and H2O2 sensitivity.