Figure 7.
Ak4 regulates mtDNA synthesis through its kinase activity to enhance host defense against bacterial infection. WT and Ak4 KO TG-pMacs were pretreated with DMSO, 10 μM dNs, 5 μM Gem, or 10 μM ddC for 1 h, followed by infection with Listeria at an MOI of 5 or Salmonella at an MOI of 10 for 1 h. Cells were then treated with 250 μg/ml gentamicin, washed with PBS, and maintained in 50 μg/ml gentamicin for 6 h prior to analysis. (A–E) Intracellular bacterial loads of Listeria or Salmonella in dNs- (A and B), Gem- (C and D), or ddC-treated (E) WT and Ak4 KO TG-pMacs were assessed by plating cell lysates onto TSA plates and quantified by CFU assay at 24 h after plating (n = 4). (F) Mock, Ak4 WT, or kinase-dead Ak4 mutants were transduced into Ak4 KO TG-pMacs using lentiviral vectors. Mock-transduced WT TG-pMacs served as controls. Intracellular bacterial load was assessed by plating cell lysates onto TSA plates and quantified by CFU assay at 24 h after plating (n = 4). (G and H) Female mice were i.p. infected with 5 × 104Listeria or 1 × 104Salmonella. Survival was monitored daily for 14 days. On day 3 after infection, spleens and livers were harvested, homogenized, and plated on TSA plates to assess bacterial load. Bacterial burden (G) and survival rate (H) in WT (n = 12), Ak4 KO (n = 14), Ak4K18 (n = 15), and Ak4K18A (n = 16) mice after i.p. infection with 5 × 104Listeria. (I) Survival rate in WT (n = 7), Ak4 KO (n = 8), Ak4K18 (n = 8), and Ak4K18A (n = 6) mice after i.p. infection with 1 × 104Salmonella. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (A–G) or log-rank test (H–I). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats. Panels A to E show intracellular bacterial loads of Listeria or Salmonella in wild-type and A k 4 knockout thioglycolate-elicited peritoneal macrophages treated with DMSO, deoxynucleosides (d N s), gemcitabine (Gem), or 2 prime, 3 prime-dideoxycytidine (d d C). The y-axis represents colony-forming units (C F U times 10 to the power 5). Panel F presents a bacterial killing assay in A k 4 knockout macrophages reconstituted with wild-type or mutant A K 4 constructs, with C F U times 10 to the power 5 on the y-axis. Panel G displays bacterial burden in spleen and liver across different mouse genotypes, expressed as log 10 (C F U per gram). Panels H and I show survival curves after Listeria or Salmonella infection, with percentage survival plotted against days post-infection.

Ak4 regulates mtDNA synthesis through its kinase activity to enhance host defense against bacterial infection. WT and Ak4 KO TG-pMacs were pretreated with DMSO, 10 μM dNs, 5 μM Gem, or 10 μM ddC for 1 h, followed by infection with Listeria at an MOI of 5 or Salmonella at an MOI of 10 for 1 h. Cells were then treated with 250 μg/ml gentamicin, washed with PBS, and maintained in 50 μg/ml gentamicin for 6 h prior to analysis. (A–E) Intracellular bacterial loads of Listeria or Salmonella in dNs- (A and B), Gem- (C and D), or ddC-treated (E) WT and Ak4 KO TG-pMacs were assessed by plating cell lysates onto TSA plates and quantified by CFU assay at 24 h after plating (n = 4). (F) Mock, Ak4 WT, or kinase-dead Ak4 mutants were transduced into Ak4 KO TG-pMacs using lentiviral vectors. Mock-transduced WT TG-pMacs served as controls. Intracellular bacterial load was assessed by plating cell lysates onto TSA plates and quantified by CFU assay at 24 h after plating (n = 4). (G and H) Female mice were i.p. infected with 5 × 104Listeria or 1 × 104Salmonella. Survival was monitored daily for 14 days. On day 3 after infection, spleens and livers were harvested, homogenized, and plated on TSA plates to assess bacterial load. Bacterial burden (G) and survival rate (H) in WT (n = 12), Ak4 KO (n = 14), Ak4K18 (n = 15), and Ak4K18A (n = 16) mice after i.p. infection with 5 × 104Listeria. (I) Survival rate in WT (n = 7), Ak4 KO (n = 8), Ak4K18 (n = 8), and Ak4K18A (n = 6) mice after i.p. infection with 1 × 104Salmonella. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (A–G) or log-rank test (H–I). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats.

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