Fig 1.
Generation of mismatch repair null mutants in T. cruzi and T. brucei.
(A) Agarose gel electrophoresis of reverse transcriptase (RT) PCR products to verify the absence of MSH2 or MLH1 mRNA in T. brucei procyclic form mutants. cDNA derived from wild type (WT) cells, blasticidin (BSD) or puromycin (PUR) resistant clones of Tbmsh2 single allele knockouts (+/-) and Tbmsh2 double allele knockouts (-/-) were PCR-amplified with MSH2 or MLH1- specific primers; the size of the PCR product is indicated and a control reaction without cDNA is indicated by C. The bottom panel shows, as positive controls, cDNAs from the same cells PCR-amplified (RT+) with primers specific for the RAD51 gene; a control for genomic DNA contamination, in which reverse transcriptase was excluded from the cDNA synthesis reaction (indicated by RT-) is also shown. (B) Total RNA extracted from T. cruzi epimastigote form wild type (WT) cells, a Tcmsh2 single allele knockout (+/-) mutant and three independent clones of Tcmsh2 double allele knockouts (-/-) were transferred to a nylon membrane and hybridized with [α-32P]-labeled probe specific for the T. cruzi MSH2 gene. The bottom panel shows hybridization with a probe for 24Sα rRNA, used as loading control.
Fig 2.
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.
Fig 3.
MSH2 knockout mutants are more resistant to oxidative stress generated by H2O2.
(A) T. brucei wild type (WT), msh2+/-, msh2-/-, mlh1+/- and mlh1-/- procyclic form cells were grown in culture medium with 0 μM, 10 μM or 20 μM H2O2. Cell density was measured after 48 hours and plotted as the percentage survival of the H2O2 treated cells relative to untreated. (B) T. cruzi epimastigote WT, msh2+/- and msh2-/- cells were incubated with or without 75 μM H2O2 for 20 minutes in PBS 1x and then allowed to grow in LIT medium for 48 hours, after which cell viability was determined and plotted as percentage survival of the treated cells relative to untreated. Vertical lines show standard deviation. ***p<0.001, **p<0.01, *p<0.05: determined by one-way ANOVA with Bonferroni post-test of mutants relative to wild type cells; ns indicates no signifcant difference.
Fig 4.
Assessment of T. cruzi msh2 knockout mutant infectivity in vitro.
(A) T. cruzi trypomastigote cells released by Vero cells infected with either WT or with three cloned cell lines of Tcmsh2-/- mutants were counted and equal numbers were used to infect Vero cells attached to glass coverslips or (B) cultured intraperitoneal macrophages extracted from BALB/c mice. Graphs show the average number of intracellular parasites counted per 100 cells; vertical lines denote standard deviation. **p<0.01, *p<0.05: one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type.
Fig 5.
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.
Fig 6.
Re-expression of MSH2 in T. brucei msh2 null 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.
Fig 7.
MSH2 displays nuclear localisation in T. brucei and T. cruzi.
Parasites expressing MSH2 C-terminally fused to 12 copies of the MYC epitope were used for immunolocalization of MSH2 using anti-MYC antiserum. (A) Transfected T. brucei bloodstream forms, (B) T. brucei procyclic forms, (C) T. cruzi epimastigotes, (D) T. cruzi intracellular amastigote cells inside infected Vero cels and (E) T. cruzi epimastigotes after 20 min treatment with 70μM H2O2 are shown. Representive images of DAPI staining of DNA, immunoflourescence with anti-MYC antiserum and phase contrast images of parasite cells are shown together with merged images. (Bars = 2.3 μm).