Trypanosoma brucei and Trypanosoma cruzi DNA Mismatch Repair Proteins Act Differently in the Response to DNA Damage Caused by Oxidative Stress

Abstract

MSH2, associated with MSH3 or MSH6, is a central component of the eukaryotic DNA Mismatch Repair (MMR) pathway responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. Previous studies have shown that MSH2 plays an additional DNA repair role in response to oxidative damage in Trypanosoma cruzi and Trypanosoma brucei. By performing co-immunoprecipitation followed by mass spectrometry with parasites expressing tagged proteins, we confirmed that the parasites' MSH2 forms complexes with MSH3 and MSH6. To investigate the involvement of these two other MMR components in the oxidative stress response, we generated knockout mutants of MSH6 and MSH3 in T. brucei bloodstream forms and MSH6 mutants in T. cruzi epimastigotes. Differently from the phenotype observed with T. cruzi MSH2 knockout epimastigotes, loss of one or two alleles of T. cruzi msh6 resulted in increased susceptibility to H₂O₂ exposure, besides impaired MMR. In contrast, T. brucei msh6 or msh3 null mutants displayed increased tolerance to MNNG treatment, indicating that MMR is affected, but no difference in the response to H₂O₂ treatment when compared to wild type cells. Taken together, our results suggest that, while T. cruzi MSH6 and MSH2 are involved with the oxidative stress response in addition to their role as components of the MMR, the DNA repair pathway that deals with oxidative stress damage operates differently in T. brucei.

Keywords: DNA Mismatch Repair; MSH2; MSH6; Trypanosoma brucei; Trypanosoma cruzi; oxidative stress.

Figure 1

Interaction of MSH2 with MSH6 and MSH3 in T. brucei and T. cruzi. (A) Cell extract of T. brucei BSF wild type (WT), BSF expressing MSH2 with a myc tag (MSH2::myc) and BSFs co-expressing MSH2::myc and MSH6 with an HA tag (MSH6::HA) were used for co-immunoprecipitation with α-myc antibody bound to magnetic beads. Western blot of input and precipitated (IP) fractions were incubated with α-myc (1:7,000) or α-HA (1:5,000) antibodies. (B) Polyacrylamide gel electrophoresis stained with Brilliant Blue G showing protein bands present on the IP fraction described in Figure 1A. The bands identified in MSH2:myc/MSH6::HA expressing parasites but not on WT BSFs were cropped and sent for mass spectrometry. Black arrows show identified MSH2 and MSH3 proteins. (C) Cell extract of T. brucei PCF WT and PCF expressing MSH2::myc were used to immunoprecipitate MSH2 with α-myc. The IP fraction as well as the unbound and input fractions were analyzed on western blot with anti-myc antibodies. In addition to MSH2, a 50 KDa band corresponding to immunoglobulin heavy chain is identified in the IP fraction. (D) IP fractions from WT and PCF expressing MSH2::myc were separated on polyacrylamide gel electrophoresis stained with Brilliant Blue G. The two bands present in the IP fraction of MSH2::myc expressing cells but not on WT PCFs were cropped and sent to mass spectrometry. Arrows show identified MSH2 and MSH3 proteins. (E) Cell extract of T. cruzi epimastigote WT and epimastigotes transfected to co-express MSH2::myc and MSH6 with an HA tag (MSH6::HA) were used for co-immunoprecipitation with α-HA antibody. Western blot of input and precipitated (IP) fractions were incubated with α-HA (1:3,000) or α-myc (1:3,000) antibodies.

Figure 2

RT-PCR analysis of BSF msh3 and msh6 mutants. Agarose gel electrophoresis of PCR products generated after reverse transcriptase (RT) reactions of RNA isolated from T. brucei BSFs msh3 and msh6 knockout mutants (-/-), WT cells and heterozygous (+/-) mutants. (A) PCR products generated with cDNA from WT and Tbmsh3 mutants and primers that amplify part of msh3 CDS with an expected fragment of 437 bp. (B) PCR with cDNA from Tbmsh6 mutants and primers that amplify part of msh6 CDS with an expected fragment of 502 bp. (C) PCR with RNA samples in the absence of RT isolated from wild type and mutants and TbRAD51 primers; gDNA from WT T. brucei was used as a positive control for the reaction, and blank is a control PCR with water instead of a DNA template. PCR-amplification has the expected size 1,122 bp. (D) PCR products from cDNA samples derived from RNA isolated from samples as shown in (C) and amplified with Tbrad51 primers. PCR-amplification has the expected size 1,122 bp.

Figure 3

Evaluation of MMR deficiency in T. brucei msh3 and msh6 knockouts. (A) The conversion of Alamar blue to fluorescent Resazurin was used to determine the sensitivity of T. brucei WT cells and Tbmsh3 and Tbmsh6 mutants toward MNNG and shown by IC50 values. IC50 values are the mean of three experiments. (B) Susceptibility of T. brucei MMR knockout mutants to the oxidative stress caused by H₂O₂. WT, Tbmsh3+/-, Tbmsh3-/-, Tbmsh6+/-, and Tbmsh6-/- were grown in culture media containing 0, 50, or 100 μM H₂O₂. Cell densities were counted 72 h after incubation beginning. Values are shown as percentage survival, which was calculated from the cell density of each cell type in the presence of H₂O₂ as a percentage of the same cells grown in the absence of damage (which was taken as 100%). Vertical lines show standard deviation. **p < 0.01: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to WT cells. Ns indicates no significant difference.

Figure 4

Characterization of T. cruzi MSH6. (A) A recognition site for the restriction enzyme NarI (highlighted in blue) is present in the DNA sequence for the Esmeraldo-like allele but is absent in the Non-Esmeraldo-like allele of the msh6 gene in the CL Brener clone. Non-conserved nucleotides are shown in pink. (B) Left panel shows that PCR amplified CDS of the Esmeraldo-like allele after digestion with NarI, resulting in DNA fragments of 1,318 bp and 1,691 bp, while the non-digested fragment corresponding to Tcmsh6 CDS has 3,009 bp. Black arrows denote primer binding site. Agarose gel electrophoresis of the PCR product from amplified Tcmsh6 CDS not digested (ND) and digested (D) with NarI. (C) Epimastigotes transfected with the pTREX_MSH6::mRFP were analyzed under fluorescence microscopy. Parasite nucleus and kinetoplast DNA are stained with DAPI (upper panel). Parasites transfected with a plasmid that inserts an HA tag in the msh6 locus were analyzed by immunofluoresce after stained with anti-HA antibody (1:50) and anti-rat conjugated with Alexa 488 (1:100). Parasite nucleus and kinetoplast DNA are stained with DAPI (lower panel). (Bar = 5 μM).

Figure 5

Generation of T. cruzi msh6 null mutants. (A) Schematic representation of the strategy to generate Tcmsh6 knockouts. T. cruzi CL Brener WT parasites were transfected with buffer, recombinant rSaCas9 (in pink), two different sgRNA sequences and a donor construction named msh6_HX1_Neo_Gapdh_msh6 or donor sequence only. (B) After transfection, genomic DNA was extracted and PCR-amplified using primers to amplify the entire CDS (black arrows mark primer binding site—left panel). Agarose gel electrophoresis of PCR fragments to evaluate knockout efficiency. Non-interrupted Tcmsh6 CDS results in a PCR fragment of 3,009 bp. CDS interrupted by donor construction results in a PCR fragment of 2,673 bp. (C) Assessing the integration of DNA constructions to delete Tcmsh6. The panel above each agarose gel represents only one allele of WT or knockout (KO) culture. Black arrows denote regions of the WT or mutated loci complementary to the different primers. PCR amplifications were carried out to verify the knockout of Tcmsh6 using specific primers annealing in the target gene (P1F and P2R) or in the Neo (P3R) or Hygro (P4R) resistance genes. PCR products generated using genomic DNA from T. cruzi WT or four KO clones in which Neomycin resistance gene and Hygromycin resistance have integrated are shown after separation on 1% agarose gels.

Figure 6

Evaluation of susceptibility of T. cruzi msh6 mutants to N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) and H₂O₂. (A) T. cruzi WT and MSH6 mutants (Tcmsh6+/- and Tcmsh6-/-) were grown in culture medium with 0 μM or 5 μM MNNG. Cell viability was measured after 72 h and is plotted as the percentage survival of the MNNG treated cells relative to untreated cultures. Vertical lines indicate standard deviation. The graph represents the average of two independent experiments performed in duplicate. (B) T. cruzi epimastigote cells wild type (WT), Tcmsh6+/- (clones 1 and 2) and Tcmsh6-/- (clones 1 and 2) mutants were incubated in the presence or absence of H₂O₂ 100 μM for 30 min in PBS 1x and then allowed to grow in LIT medium for 72 h, after which cell viability was determined and plotted as percentage survival of the treated cells relative to untreated. (C) T. cruzi WT, Tcmsh6-/- and Tcmsh6-/- transfected to express MSH6::HA were grown in culture medium with 0 μM or 5 μM MNNG. Cell viability was measured after 72 h and is plotted as the percentage survival of the MNNG treated cells relative to untreated cultures. Vertical lines indicate standard deviation. The graph represents the average of two independent experiments, each performed in duplicate. (D) T. cruzi WT, Tcmsh6-/- and Tcmsh6-/- transfected to express MSH6::HA mutants were incubated in the presence or absence of 100 μM H₂O₂ for 30 min in PBS 1x and then allowed to grow in LIT medium for 72 h, after which cell viability was determined and plotted as percentage survival of the treated cells relative to untreated. Data represent the average of three independent experiments, each performed in duplicate. Vertical lines show standard deviation. ^****p < 0.0001, ^***p < 0.001, ^*p < 0.1: determined by one-way ANOVA with Bonferroni post-test of mutants relative to wild type cells.

Figure 7

Assessment of T. cruzi msh6 null mutants infectivity in vitro. (A) T. cruzi trypomastigote cells released by Vero cells infected with either WT or with two cloned cell lines of Tcmsh6-/- mutants were counted and equal numbers were used to infect Vero cells attached to glass coverslips. (B) Infection of intraperitoneal macrophages extracted from Balb/C mice with trypomastigotes released from infected Vero cells. Twenty-four and forty-eight hours post infection the infection index was estimated. Infection index = percentage of infected cells × mean number of parasites per infected cells. Values are expressed as means ± SD of one representative experiment performed in triplicate. Ns indicates no significant difference: two-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type.