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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

Re-expression of MSH2 in T. brucei msh2  mutants.(A) Procyclic form (PCF) T. brucei (Tb) wild type (WT), Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/- cells in which MSH2 is re-expressed (Tbmsh2-/-/+) were grown in culture medium with 0 μM, 2.5 μM or 5 μM MNNG. Cell density was measured after 72 hours growth and is plotted as the percentage survival of the MNNG treated cells relative to untreated. (B) PCF WT, Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/-/+ cells were grown in culture medium with 0 μM, 10 μM or 20 μM H2O2 and cell density was determined 48 and 72 hours later; growth is shown percentage survival of the treated cells relative to untreated (C) Growth of wild type bloodstream form (BSF) T. brucei cells was compared to Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/-/+ BSF mutants in the presence of 100 μM or 200 μM H2O2 as described above; graph shows survival of the mutants after 48 hours growth plotting the density of the treated cells as a percentage of the untreated; vertical lines show standard deviation. ***p<0.001, **p<0.001: determined by one-way ANOVA with Bonferroni post-test of mutants relative to wild type; ns indicates no significant difference.
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pntd.0003870.g006: Re-expression of MSH2 in T. brucei msh2 mutants.(A) Procyclic form (PCF) T. brucei (Tb) wild type (WT), Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/- cells in which MSH2 is re-expressed (Tbmsh2-/-/+) were grown in culture medium with 0 μM, 2.5 μM or 5 μM MNNG. Cell density was measured after 72 hours growth and is plotted as the percentage survival of the MNNG treated cells relative to untreated. (B) PCF WT, Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/-/+ cells were grown in culture medium with 0 μM, 10 μM or 20 μM H2O2 and cell density was determined 48 and 72 hours later; growth is shown percentage survival of the treated cells relative to untreated (C) Growth of wild type bloodstream form (BSF) T. brucei cells was compared to Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/-/+ BSF mutants in the presence of 100 μM or 200 μM H2O2 as described above; graph shows survival of the mutants after 48 hours growth plotting the density of the treated cells as a percentage of the untreated; vertical lines show standard deviation. ***p<0.001, **p<0.001: determined by one-way ANOVA with Bonferroni post-test of mutants relative to wild type; ns indicates no significant difference.

Mentions: In order to test further if H2O2 resistance in msh2 mutants results from an adaptation process that occurred in insect life stages of T. brucei, we re-expressed MSH2 in both the BSF and PCF msh2-/- cells, using a previously described construct and conditions [21]. Integration of the MSH2 re-expression construct was confirmed by PCR (S6 Fig), and southern blot in BSF [21]. Both BSF and PCF msh2 mutants display increased tolerance to MNNG (Fig 2A), and re-expression of MSH2 in BSF msh2-/- mutants reverts this tolerance to the levels of msh2+/- mutants [21, 38]. As shown in Fig 6A, re-expressing MSH2 (Tbmsh2-/-/+) in the PCF msh2-/- mutants also resulted in levels of MNNG survival similar to msh2+/- cells. In addition, and as seen in T. brucei BSF msh2-/-/+ cells [21], PCF msh2-/-/+ cells no longer showed detectable microsatellite variation in clonal growth assays (S7 Fig). Taken together, these assays indicate that MMR function can be restored in T. brucei msh2 mutants in both life cycle stages after re-introduction of MSH2 into the genome. In contrast, MSH2 re-expression had a different outcome for H2O2 sensitivity in the two life cycle stages. When T. brucei PCF msh2-/-/+ cells were grown in the presence of 10 μM or 20 μM H2O2 for 48 or 72 hrs, there was no significant difference in survival relative to the msh2-/- mutants, and survival was significantly different from the msh2+/- mutants (Fig 6B). However, in BSF T. brucei, the survival of the msh2-/-/+ cells (in this case after 48 hrs growth in either 100 or 200 μM H2O2) was indistinguishable from the msh2+/- cells and significantly greater than the msh2-/- mutants (Fig 6C). Thus, while re-expression of MSH2 in T. brucei msh2-/- mutants was able to restore MMR function in both life cycle stages, the same re-expression was able to revert the increased sensitivity to H2O2 only in BSF msh2-/- mutants. In contrast, the increased tolerance to H2O2 observed in PCF after loss of MSH2 could not be reverted by re-expressing this gene in this life cycle form of T. brucei. This lack of MSH2 complementation is most simply explained by changes in expression or function of another factor(s) that allowed PCF MSH2 mutants to cope specifically with oxidative stress, though we cannot rule out the possibility of this specific phenotypic difference between BSF and PCF cells arising due to differing levels of MSH2 in the two life cycle stages and in the msh2-/-/+ re-expressers. As suggested by RNAi data (S8 Fig), the adaptation process that may have occurred during the cloning period needed to generate the msh2-/- mutants requires several generations. When MSH2 expression is abruptly inhibited by tetracycline induction of siRNA in T. brucei PCF cells, a discernible slowing of growth is observed as a consequence of the reduced msh2 mRNA expression.


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)

Re-expression of MSH2 in T. brucei msh2  mutants.(A) Procyclic form (PCF) T. brucei (Tb) wild type (WT), Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/- cells in which MSH2 is re-expressed (Tbmsh2-/-/+) were grown in culture medium with 0 μM, 2.5 μM or 5 μM MNNG. Cell density was measured after 72 hours growth and is plotted as the percentage survival of the MNNG treated cells relative to untreated. (B) PCF WT, Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/-/+ cells were grown in culture medium with 0 μM, 10 μM or 20 μM H2O2 and cell density was determined 48 and 72 hours later; growth is shown percentage survival of the treated cells relative to untreated (C) Growth of wild type bloodstream form (BSF) T. brucei cells was compared to Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/-/+ BSF mutants in the presence of 100 μM or 200 μM H2O2 as described above; graph shows survival of the mutants after 48 hours growth plotting the density of the treated cells as a percentage of the untreated; vertical lines show standard deviation. ***p<0.001, **p<0.001: determined by one-way ANOVA with Bonferroni post-test of mutants relative to wild type; ns indicates no significant difference.
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Show All Figures
getmorefigures.php?uid=PMC4470938&req=5

pntd.0003870.g006: Re-expression of MSH2 in T. brucei msh2 mutants.(A) Procyclic form (PCF) T. brucei (Tb) wild type (WT), Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/- cells in which MSH2 is re-expressed (Tbmsh2-/-/+) were grown in culture medium with 0 μM, 2.5 μM or 5 μM MNNG. Cell density was measured after 72 hours growth and is plotted as the percentage survival of the MNNG treated cells relative to untreated. (B) PCF WT, Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/-/+ cells were grown in culture medium with 0 μM, 10 μM or 20 μM H2O2 and cell density was determined 48 and 72 hours later; growth is shown percentage survival of the treated cells relative to untreated (C) Growth of wild type bloodstream form (BSF) T. brucei cells was compared to Tbmsh2+/-, Tbmsh2-/- and Tbmsh2-/-/+ BSF mutants in the presence of 100 μM or 200 μM H2O2 as described above; graph shows survival of the mutants after 48 hours growth plotting the density of the treated cells as a percentage of the untreated; vertical lines show standard deviation. ***p<0.001, **p<0.001: determined by one-way ANOVA with Bonferroni post-test of mutants relative to wild type; ns indicates no significant difference.
Mentions: In order to test further if H2O2 resistance in msh2 mutants results from an adaptation process that occurred in insect life stages of T. brucei, we re-expressed MSH2 in both the BSF and PCF msh2-/- cells, using a previously described construct and conditions [21]. Integration of the MSH2 re-expression construct was confirmed by PCR (S6 Fig), and southern blot in BSF [21]. Both BSF and PCF msh2 mutants display increased tolerance to MNNG (Fig 2A), and re-expression of MSH2 in BSF msh2-/- mutants reverts this tolerance to the levels of msh2+/- mutants [21, 38]. As shown in Fig 6A, re-expressing MSH2 (Tbmsh2-/-/+) in the PCF msh2-/- mutants also resulted in levels of MNNG survival similar to msh2+/- cells. In addition, and as seen in T. brucei BSF msh2-/-/+ cells [21], PCF msh2-/-/+ cells no longer showed detectable microsatellite variation in clonal growth assays (S7 Fig). Taken together, these assays indicate that MMR function can be restored in T. brucei msh2 mutants in both life cycle stages after re-introduction of MSH2 into the genome. In contrast, MSH2 re-expression had a different outcome for H2O2 sensitivity in the two life cycle stages. When T. brucei PCF msh2-/-/+ cells were grown in the presence of 10 μM or 20 μM H2O2 for 48 or 72 hrs, there was no significant difference in survival relative to the msh2-/- mutants, and survival was significantly different from the msh2+/- mutants (Fig 6B). However, in BSF T. brucei, the survival of the msh2-/-/+ cells (in this case after 48 hrs growth in either 100 or 200 μM H2O2) was indistinguishable from the msh2+/- cells and significantly greater than the msh2-/- mutants (Fig 6C). Thus, while re-expression of MSH2 in T. brucei msh2-/- mutants was able to restore MMR function in both life cycle stages, the same re-expression was able to revert the increased sensitivity to H2O2 only in BSF msh2-/- mutants. In contrast, the increased tolerance to H2O2 observed in PCF after loss of MSH2 could not be reverted by re-expressing this gene in this life cycle form of T. brucei. This lack of MSH2 complementation is most simply explained by changes in expression or function of another factor(s) that allowed PCF MSH2 mutants to cope specifically with oxidative stress, though we cannot rule out the possibility of this specific phenotypic difference between BSF and PCF cells arising due to differing levels of MSH2 in the two life cycle stages and in the msh2-/-/+ re-expressers. As suggested by RNAi data (S8 Fig), the adaptation process that may have occurred during the cloning period needed to generate the msh2-/- mutants requires several generations. When MSH2 expression is abruptly inhibited by tetracycline induction of siRNA in T. brucei PCF cells, a discernible slowing of growth is observed as a consequence of the reduced msh2 mRNA expression.

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