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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

Susceptibility of T. brucei and T. cruzi MMR knockout mutants to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).(A) T. brucei wild type (WT) and procyclic form mutants (Tbmsh2+/-, Tbmsh2-/-, Tbmlh1+/- and Tbmlh1-/-) 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 cultures. (B) WT T. cruzi epimastigotes and MSH2 mutants (Tcmsh2+/- and msh2-/-) were grown in culture medium with 0 μM or 5 μM MNNG. Cell viability was measured after 72 hours and is plotted as the percentage survival of the MNNG treated cells relative to untreated cultures. Vertical lines indicate standard deviation. ***p<0.001, **p<0.01, *p<0.05: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type cells.
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pntd.0003870.g002: Susceptibility of T. brucei and T. cruzi MMR knockout mutants to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).(A) T. brucei wild type (WT) and procyclic form mutants (Tbmsh2+/-, Tbmsh2-/-, Tbmlh1+/- and Tbmlh1-/-) 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 cultures. (B) WT T. cruzi epimastigotes and MSH2 mutants (Tcmsh2+/- and msh2-/-) were grown in culture medium with 0 μM or 5 μM MNNG. Cell viability was measured after 72 hours and is plotted as the percentage survival of the MNNG treated cells relative to untreated cultures. Vertical lines indicate standard deviation. ***p<0.001, **p<0.01, *p<0.05: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type cells.

Mentions: To ask if the mutations described above result in detectable loss of MMR, we measured the sensitivity of the parasites to MNNG [26]. T. brucei and T. cruzi mutants and WT cells were grown for 72 hours with increasing concentrations of MMNG. Survival of the cells was determined by measuring the cell density of the WT or mutant cells after MNNG treatment relative to untreated cells. In T. brucei, all cells showed increasing growth impairment as MNNG was increased from 2.5 to 5 μM. However, similar to what has been described in BSF cells [21], and consistent with the proposed futile cycle of alkylation repair in MMR-proficient cells [26, 27], deletion of one allele of Tbmsh2 or Tbmlh1 in the PCF+/- mutants caused increased tolerance to MNNG, and this tolerance increased yet further when both alleles were deleted in the-/- mutants (Fig 2A). Deletion of TbMSH2 or TbMLH1 in PCF cells also resulted in the-/- parasites displaying increased microsatellite instability, as was observed in T. brucei BSF mutants, indicating decreased replication fidelity (S4 Fig). In T. cruzi increased tolerance to 5 μM MNNG was seen in the Tcmsh2+/- cells and was not detectably increased in the-/- mutants (Fig 2B). Though this somewhat contrasts with the response of T. brucei MMR mutants to MNNG, these data nonetheless show that mutation of MMR genes in either parasite resulted in the expected enhanced survival in the presence of this alkylating drug.


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)

Susceptibility of T. brucei and T. cruzi MMR knockout mutants to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).(A) T. brucei wild type (WT) and procyclic form mutants (Tbmsh2+/-, Tbmsh2-/-, Tbmlh1+/- and Tbmlh1-/-) 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 cultures. (B) WT T. cruzi epimastigotes and MSH2 mutants (Tcmsh2+/- and msh2-/-) were grown in culture medium with 0 μM or 5 μM MNNG. Cell viability was measured after 72 hours and is plotted as the percentage survival of the MNNG treated cells relative to untreated cultures. Vertical lines indicate standard deviation. ***p<0.001, **p<0.01, *p<0.05: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type cells.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003870.g002: Susceptibility of T. brucei and T. cruzi MMR knockout mutants to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).(A) T. brucei wild type (WT) and procyclic form mutants (Tbmsh2+/-, Tbmsh2-/-, Tbmlh1+/- and Tbmlh1-/-) 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 cultures. (B) WT T. cruzi epimastigotes and MSH2 mutants (Tcmsh2+/- and msh2-/-) were grown in culture medium with 0 μM or 5 μM MNNG. Cell viability was measured after 72 hours and is plotted as the percentage survival of the MNNG treated cells relative to untreated cultures. Vertical lines indicate standard deviation. ***p<0.001, **p<0.01, *p<0.05: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type cells.
Mentions: To ask if the mutations described above result in detectable loss of MMR, we measured the sensitivity of the parasites to MNNG [26]. T. brucei and T. cruzi mutants and WT cells were grown for 72 hours with increasing concentrations of MMNG. Survival of the cells was determined by measuring the cell density of the WT or mutant cells after MNNG treatment relative to untreated cells. In T. brucei, all cells showed increasing growth impairment as MNNG was increased from 2.5 to 5 μM. However, similar to what has been described in BSF cells [21], and consistent with the proposed futile cycle of alkylation repair in MMR-proficient cells [26, 27], deletion of one allele of Tbmsh2 or Tbmlh1 in the PCF+/- mutants caused increased tolerance to MNNG, and this tolerance increased yet further when both alleles were deleted in the-/- mutants (Fig 2A). Deletion of TbMSH2 or TbMLH1 in PCF cells also resulted in the-/- parasites displaying increased microsatellite instability, as was observed in T. brucei BSF mutants, indicating decreased replication fidelity (S4 Fig). In T. cruzi increased tolerance to 5 μM MNNG was seen in the Tcmsh2+/- cells and was not detectably increased in the-/- mutants (Fig 2B). Though this somewhat contrasts with the response of T. brucei MMR mutants to MNNG, these data nonetheless show that mutation of MMR genes in either parasite resulted in the expected enhanced survival in the presence of this alkylating drug.

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