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Male-killing symbiont damages host's dosage-compensated sex chromosome to induce embryonic apoptosis

View Article: PubMed Central - PubMed

ABSTRACT

Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner.

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The MSL complex is necessary and sufficient for Spiroplasma-induced DNA damage and abnormal apoptosis.(a–c) Apoptosis (a) and DNA damage (b,c) in Spiroplasma-infected male and female embryos of msl3 maternal–zygotic mutant (m−/z−; zygotic genotype msl31/msl31) and maternal mutant (m−/z+; zygotic genotype msl31/TM3 ActGFP). (a) Quantification of TUNEL-positive areas at stage 11–12. Different letters (a,b) indicate statistically significant differences (P<0.05; Kruskal–Wallis test followed by Mann–Whitney U-tests). (b) Quantification of focal pH2Av signals at stage 11–12. Different letters (a,b) indicate statistically significant differences (P<0.01; Kruskal–Wallis test followed by Mann–Whitney U-tests). (c) Focal pH2Av signals in msl3 mutant embryos at stage 12. (d–k) Ectopic MSL complex formation by the H83M2 transgene. (d) An uninfected H83M2 female embryo exhibiting little abnormal apoptosis. (e,f) Infected H83M2 embryos showing abnormal apoptosis (e, female; f, male). In d–f, stage 13 embryos are stained for Sxl (green) and TUNEL (magenta), whereas single-channelled TUNEL images are shown in d′–f′. (g) Quantification of TUNEL-positive areas in uninfected and infected H83M2 embryos at stage 11 (left) and 13 (right). Different letters (a–c) indicate statistically significant differences (P<0.01; Kruskal–Wallis test followed by Mann–Whitney U-tests). (h) Epidermal cells of an infected H83M2 female embryo at stage 11, stained for Sxl (green) and DNA (magenta), whereas single-channelled DNA image is shown in h′. (i,j) Enlarged images of dividing cells with a chromatin bridge (i) and an abnormally tangled DNA mass (j, arrow), representing boxed regions in h. (k) Quantification of chromatin bridges in the epidermal cells of uninfected and infected H83M2 female embryos at stage 11–12. The number of chromatin bridges per × 63 objective view are categorized into three classes: no bridge (0); 1 to 5 bridges (1–5); and 6 or more bridges (5<). In a, b and g, box plots are as in Fig. 2i,j. and sample sizes are indicated at the bottom. In b, numbers of embryos observed are shown in parentheses. Scale bars, 10 μm (c, i and j), 100 μm (d–f′) and 20 μm (h,h′).
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f5: The MSL complex is necessary and sufficient for Spiroplasma-induced DNA damage and abnormal apoptosis.(a–c) Apoptosis (a) and DNA damage (b,c) in Spiroplasma-infected male and female embryos of msl3 maternal–zygotic mutant (m−/z−; zygotic genotype msl31/msl31) and maternal mutant (m−/z+; zygotic genotype msl31/TM3 ActGFP). (a) Quantification of TUNEL-positive areas at stage 11–12. Different letters (a,b) indicate statistically significant differences (P<0.05; Kruskal–Wallis test followed by Mann–Whitney U-tests). (b) Quantification of focal pH2Av signals at stage 11–12. Different letters (a,b) indicate statistically significant differences (P<0.01; Kruskal–Wallis test followed by Mann–Whitney U-tests). (c) Focal pH2Av signals in msl3 mutant embryos at stage 12. (d–k) Ectopic MSL complex formation by the H83M2 transgene. (d) An uninfected H83M2 female embryo exhibiting little abnormal apoptosis. (e,f) Infected H83M2 embryos showing abnormal apoptosis (e, female; f, male). In d–f, stage 13 embryos are stained for Sxl (green) and TUNEL (magenta), whereas single-channelled TUNEL images are shown in d′–f′. (g) Quantification of TUNEL-positive areas in uninfected and infected H83M2 embryos at stage 11 (left) and 13 (right). Different letters (a–c) indicate statistically significant differences (P<0.01; Kruskal–Wallis test followed by Mann–Whitney U-tests). (h) Epidermal cells of an infected H83M2 female embryo at stage 11, stained for Sxl (green) and DNA (magenta), whereas single-channelled DNA image is shown in h′. (i,j) Enlarged images of dividing cells with a chromatin bridge (i) and an abnormally tangled DNA mass (j, arrow), representing boxed regions in h. (k) Quantification of chromatin bridges in the epidermal cells of uninfected and infected H83M2 female embryos at stage 11–12. The number of chromatin bridges per × 63 objective view are categorized into three classes: no bridge (0); 1 to 5 bridges (1–5); and 6 or more bridges (5<). In a, b and g, box plots are as in Fig. 2i,j. and sample sizes are indicated at the bottom. In b, numbers of embryos observed are shown in parentheses. Scale bars, 10 μm (c, i and j), 100 μm (d–f′) and 20 μm (h,h′).

Mentions: While MSL1 and MSL2 act as scaffold for MSL complex formation, MSL3, MOF and MLE are required for spreading the complex across the entire X chromosome54647. Loss-of-function mutants of msl3, for example msl31, fail to form the complete MSL complex and exhibit male-specific larval lethality due to dosage compensation defects484950. On account of the maternal and zygotic sources of msl3, we investigated a maternal–zygotic mutant (m–/z–; zygotic genotype msl31/msl31) with compromised MSL complex function in comparison with a maternal mutant (m–/z+; zygotic genotype msl31/TM3 ActGFP) with the functional MSL complex. When these fly strains were infected with Spiroplasma, DNA damage and abnormal apoptosis in male embryos were attenuated under the msl3-deficient maternal–zygotic mutant genotype (Fig. 5a–c), indicating that the MSL complex is necessary for Spiroplasma-induced DNA damage and abnormal apoptosis.


Male-killing symbiont damages host's dosage-compensated sex chromosome to induce embryonic apoptosis
The MSL complex is necessary and sufficient for Spiroplasma-induced DNA damage and abnormal apoptosis.(a–c) Apoptosis (a) and DNA damage (b,c) in Spiroplasma-infected male and female embryos of msl3 maternal–zygotic mutant (m−/z−; zygotic genotype msl31/msl31) and maternal mutant (m−/z+; zygotic genotype msl31/TM3 ActGFP). (a) Quantification of TUNEL-positive areas at stage 11–12. Different letters (a,b) indicate statistically significant differences (P<0.05; Kruskal–Wallis test followed by Mann–Whitney U-tests). (b) Quantification of focal pH2Av signals at stage 11–12. Different letters (a,b) indicate statistically significant differences (P<0.01; Kruskal–Wallis test followed by Mann–Whitney U-tests). (c) Focal pH2Av signals in msl3 mutant embryos at stage 12. (d–k) Ectopic MSL complex formation by the H83M2 transgene. (d) An uninfected H83M2 female embryo exhibiting little abnormal apoptosis. (e,f) Infected H83M2 embryos showing abnormal apoptosis (e, female; f, male). In d–f, stage 13 embryos are stained for Sxl (green) and TUNEL (magenta), whereas single-channelled TUNEL images are shown in d′–f′. (g) Quantification of TUNEL-positive areas in uninfected and infected H83M2 embryos at stage 11 (left) and 13 (right). Different letters (a–c) indicate statistically significant differences (P<0.01; Kruskal–Wallis test followed by Mann–Whitney U-tests). (h) Epidermal cells of an infected H83M2 female embryo at stage 11, stained for Sxl (green) and DNA (magenta), whereas single-channelled DNA image is shown in h′. (i,j) Enlarged images of dividing cells with a chromatin bridge (i) and an abnormally tangled DNA mass (j, arrow), representing boxed regions in h. (k) Quantification of chromatin bridges in the epidermal cells of uninfected and infected H83M2 female embryos at stage 11–12. The number of chromatin bridges per × 63 objective view are categorized into three classes: no bridge (0); 1 to 5 bridges (1–5); and 6 or more bridges (5<). In a, b and g, box plots are as in Fig. 2i,j. and sample sizes are indicated at the bottom. In b, numbers of embryos observed are shown in parentheses. Scale bars, 10 μm (c, i and j), 100 μm (d–f′) and 20 μm (h,h′).
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f5: The MSL complex is necessary and sufficient for Spiroplasma-induced DNA damage and abnormal apoptosis.(a–c) Apoptosis (a) and DNA damage (b,c) in Spiroplasma-infected male and female embryos of msl3 maternal–zygotic mutant (m−/z−; zygotic genotype msl31/msl31) and maternal mutant (m−/z+; zygotic genotype msl31/TM3 ActGFP). (a) Quantification of TUNEL-positive areas at stage 11–12. Different letters (a,b) indicate statistically significant differences (P<0.05; Kruskal–Wallis test followed by Mann–Whitney U-tests). (b) Quantification of focal pH2Av signals at stage 11–12. Different letters (a,b) indicate statistically significant differences (P<0.01; Kruskal–Wallis test followed by Mann–Whitney U-tests). (c) Focal pH2Av signals in msl3 mutant embryos at stage 12. (d–k) Ectopic MSL complex formation by the H83M2 transgene. (d) An uninfected H83M2 female embryo exhibiting little abnormal apoptosis. (e,f) Infected H83M2 embryos showing abnormal apoptosis (e, female; f, male). In d–f, stage 13 embryos are stained for Sxl (green) and TUNEL (magenta), whereas single-channelled TUNEL images are shown in d′–f′. (g) Quantification of TUNEL-positive areas in uninfected and infected H83M2 embryos at stage 11 (left) and 13 (right). Different letters (a–c) indicate statistically significant differences (P<0.01; Kruskal–Wallis test followed by Mann–Whitney U-tests). (h) Epidermal cells of an infected H83M2 female embryo at stage 11, stained for Sxl (green) and DNA (magenta), whereas single-channelled DNA image is shown in h′. (i,j) Enlarged images of dividing cells with a chromatin bridge (i) and an abnormally tangled DNA mass (j, arrow), representing boxed regions in h. (k) Quantification of chromatin bridges in the epidermal cells of uninfected and infected H83M2 female embryos at stage 11–12. The number of chromatin bridges per × 63 objective view are categorized into three classes: no bridge (0); 1 to 5 bridges (1–5); and 6 or more bridges (5<). In a, b and g, box plots are as in Fig. 2i,j. and sample sizes are indicated at the bottom. In b, numbers of embryos observed are shown in parentheses. Scale bars, 10 μm (c, i and j), 100 μm (d–f′) and 20 μm (h,h′).
Mentions: While MSL1 and MSL2 act as scaffold for MSL complex formation, MSL3, MOF and MLE are required for spreading the complex across the entire X chromosome54647. Loss-of-function mutants of msl3, for example msl31, fail to form the complete MSL complex and exhibit male-specific larval lethality due to dosage compensation defects484950. On account of the maternal and zygotic sources of msl3, we investigated a maternal–zygotic mutant (m–/z–; zygotic genotype msl31/msl31) with compromised MSL complex function in comparison with a maternal mutant (m–/z+; zygotic genotype msl31/TM3 ActGFP) with the functional MSL complex. When these fly strains were infected with Spiroplasma, DNA damage and abnormal apoptosis in male embryos were attenuated under the msl3-deficient maternal–zygotic mutant genotype (Fig. 5a–c), indicating that the MSL complex is necessary for Spiroplasma-induced DNA damage and abnormal apoptosis.

View Article: PubMed Central - PubMed

ABSTRACT

Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner.

No MeSH data available.


Related in: MedlinePlus