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

Grazielle-Silva V, Zeb TF, Bolderson J, Campos PC, Miranda JB, Alves CL, Machado CR, McCulloch R, Teixeira SM - PLoS Negl Trop Dis (2015)

Bottom Line: 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.Taken 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; The Wellcome Trust Center for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, Scotland, United Kingdom.

ABSTRACT

Background: DNA 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 findings: To investigate the involvement of MMR in the oxidative stress response, mutants of MSH2 were generated in Trypanosoma brucei procyclic forms and in Trypanosoma cruzi epimastigote forms. Unexpectedly, the MSH2 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/significance: Taken 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.

No MeSH data available.


Related in: MedlinePlus

8-oxoguanine (8-oxoG) accumulation in MSH2 knockout cells.FITC-avidin was used to estimate 8-oxoG levels based on the fluorescence intensity in the nuclear DNA (nDNA) and kinetoplast DNA (kDNA) of T. brucei procyclic forms and T. cruzi epimastigotes. (A) Representative images of FITC-avidin or DAPI stained T. brucei WT cells and Tbmsh2+/- or Tbmsh2-/- mutants are shown. (Bar = 1.9 μM) (B) Fluorescence intensity of FITC-avidin signals were quantified in the nDNA and kDNA using ImageJ software and plotted as arbitrary units; values shown are the average signal from 100 WT, Tbmsh2+/-, Tbmsh2-/-, Tbmlh1+/- or Tbmlh1-/- PCF cells; vertical lines show standard error (SEM). (C) FITC-avidin signal evaluated by the same process in T. cruzi epimastigote WT cells and in msh2+/- and msh2-/- knockout mutants. ***p<0.001: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type; ns indicates no signifcant difference.
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pntd.0003870.g005: 8-oxoguanine (8-oxoG) accumulation in MSH2 knockout cells.FITC-avidin was used to estimate 8-oxoG levels based on the fluorescence intensity in the nuclear DNA (nDNA) and kinetoplast DNA (kDNA) of T. brucei procyclic forms and T. cruzi epimastigotes. (A) Representative images of FITC-avidin or DAPI stained T. brucei WT cells and Tbmsh2+/- or Tbmsh2-/- mutants are shown. (Bar = 1.9 μM) (B) Fluorescence intensity of FITC-avidin signals were quantified in the nDNA and kDNA using ImageJ software and plotted as arbitrary units; values shown are the average signal from 100 WT, Tbmsh2+/-, Tbmsh2-/-, Tbmlh1+/- or Tbmlh1-/- PCF cells; vertical lines show standard error (SEM). (C) FITC-avidin signal evaluated by the same process in T. cruzi epimastigote WT cells and in msh2+/- and msh2-/- knockout mutants. ***p<0.001: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type; ns indicates no signifcant difference.

Mentions: The increased resistance to oxidative stress observed in T. brucei PCF cells and T. cruzi epimastigotes after MSH2 mutation could be a direct consequence of the loss of this MMR component, or could be due to indirect effects, such as a metabolic adaptation. To begin to address this, we determined the capacity of mutant cells to limit the accumulation of oxidized bases in their genome, which can arise from exposure to ROS generated by endogenous parasite metabolism or from the host. The primary DNA lesion generated by oxidative damage is 8-oxoG, which, if not removed by base-excision repair, can be recognized by MMR [26]. We therefore measured the levels of this oxidized base in the WT and msh2 mutant parasite genomes using avidin-conjugated FITC and measuring fluorescence [47]. As shown in Fig 5A and 5B, PCF T. brucei msh2+/- and msh2-/- mutants each displayed ~2 fold greater fluorescence in their nuclear DNA (nDNA) and kinetoplast DNA (kDNA) compared with WT cells. These fluorescence data indicate that the levels of 8oxoG were no greater in the T. brucei msh2-/- mutants than in the Tbmsh2+/- mutants, consistent with the observation that the two mutants display equivalent levels of resistance to H2O2 (Fig 3). In contrast, no difference was observed in the levels of fluorescence in the nDNA or kDNA of mlh1 mutants related to WT (Fig 5B), a distinction from T. brucei msh2 mutants again consistent with the separation of MMR functions observed when examining levels of resistance to H2O2. Increased levels of avidin-FITC fluorescence were also seen in the nDNA and kDNA of T. cruzi epimastigote msh2 mutants: 1.7 to 1.9 fold increase in fluorescence was seen in the nDNA and kDNA of both Tcmsh2+/- clones and both Tcmsh2-/- clones examined relative to WT (Fig 5C). These data suggest that loss or reduction of MSH2 expression (but not MLH1) in PCF T. brucei and in epimastigote T. cruzi results in the impairment of a pathway involved in the repair of DNA-directed oxidative damage, such as 8-oxoG. Thus, the increased resistance to H2O2 in the mutants is best explained by an indirect adaptation to cope with oxidative stress following MSH2 mutation.


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

Grazielle-Silva V, Zeb TF, Bolderson J, Campos PC, Miranda JB, Alves CL, Machado CR, McCulloch R, Teixeira SM - PLoS Negl Trop Dis (2015)

8-oxoguanine (8-oxoG) accumulation in MSH2 knockout cells.FITC-avidin was used to estimate 8-oxoG levels based on the fluorescence intensity in the nuclear DNA (nDNA) and kinetoplast DNA (kDNA) of T. brucei procyclic forms and T. cruzi epimastigotes. (A) Representative images of FITC-avidin or DAPI stained T. brucei WT cells and Tbmsh2+/- or Tbmsh2-/- mutants are shown. (Bar = 1.9 μM) (B) Fluorescence intensity of FITC-avidin signals were quantified in the nDNA and kDNA using ImageJ software and plotted as arbitrary units; values shown are the average signal from 100 WT, Tbmsh2+/-, Tbmsh2-/-, Tbmlh1+/- or Tbmlh1-/- PCF cells; vertical lines show standard error (SEM). (C) FITC-avidin signal evaluated by the same process in T. cruzi epimastigote WT cells and in msh2+/- and msh2-/- knockout mutants. ***p<0.001: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type; ns indicates no signifcant difference.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4470938&req=5

pntd.0003870.g005: 8-oxoguanine (8-oxoG) accumulation in MSH2 knockout cells.FITC-avidin was used to estimate 8-oxoG levels based on the fluorescence intensity in the nuclear DNA (nDNA) and kinetoplast DNA (kDNA) of T. brucei procyclic forms and T. cruzi epimastigotes. (A) Representative images of FITC-avidin or DAPI stained T. brucei WT cells and Tbmsh2+/- or Tbmsh2-/- mutants are shown. (Bar = 1.9 μM) (B) Fluorescence intensity of FITC-avidin signals were quantified in the nDNA and kDNA using ImageJ software and plotted as arbitrary units; values shown are the average signal from 100 WT, Tbmsh2+/-, Tbmsh2-/-, Tbmlh1+/- or Tbmlh1-/- PCF cells; vertical lines show standard error (SEM). (C) FITC-avidin signal evaluated by the same process in T. cruzi epimastigote WT cells and in msh2+/- and msh2-/- knockout mutants. ***p<0.001: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type; ns indicates no signifcant difference.
Mentions: The increased resistance to oxidative stress observed in T. brucei PCF cells and T. cruzi epimastigotes after MSH2 mutation could be a direct consequence of the loss of this MMR component, or could be due to indirect effects, such as a metabolic adaptation. To begin to address this, we determined the capacity of mutant cells to limit the accumulation of oxidized bases in their genome, which can arise from exposure to ROS generated by endogenous parasite metabolism or from the host. The primary DNA lesion generated by oxidative damage is 8-oxoG, which, if not removed by base-excision repair, can be recognized by MMR [26]. We therefore measured the levels of this oxidized base in the WT and msh2 mutant parasite genomes using avidin-conjugated FITC and measuring fluorescence [47]. As shown in Fig 5A and 5B, PCF T. brucei msh2+/- and msh2-/- mutants each displayed ~2 fold greater fluorescence in their nuclear DNA (nDNA) and kinetoplast DNA (kDNA) compared with WT cells. These fluorescence data indicate that the levels of 8oxoG were no greater in the T. brucei msh2-/- mutants than in the Tbmsh2+/- mutants, consistent with the observation that the two mutants display equivalent levels of resistance to H2O2 (Fig 3). In contrast, no difference was observed in the levels of fluorescence in the nDNA or kDNA of mlh1 mutants related to WT (Fig 5B), a distinction from T. brucei msh2 mutants again consistent with the separation of MMR functions observed when examining levels of resistance to H2O2. Increased levels of avidin-FITC fluorescence were also seen in the nDNA and kDNA of T. cruzi epimastigote msh2 mutants: 1.7 to 1.9 fold increase in fluorescence was seen in the nDNA and kDNA of both Tcmsh2+/- clones and both Tcmsh2-/- clones examined relative to WT (Fig 5C). These data suggest that loss or reduction of MSH2 expression (but not MLH1) in PCF T. brucei and in epimastigote T. cruzi results in the impairment of a pathway involved in the repair of DNA-directed oxidative damage, such as 8-oxoG. Thus, the increased resistance to H2O2 in the mutants is best explained by an indirect adaptation to cope with oxidative stress following MSH2 mutation.

Bottom Line: 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.Taken 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; The Wellcome Trust Center for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, Scotland, United Kingdom.

ABSTRACT

Background: DNA 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 findings: To investigate the involvement of MMR in the oxidative stress response, mutants of MSH2 were generated in Trypanosoma brucei procyclic forms and in Trypanosoma cruzi epimastigote forms. Unexpectedly, the MSH2 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/significance: Taken 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.

No MeSH data available.


Related in: MedlinePlus