المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: Phylogenetic tree of 296 M . tuberculosis and M . africanum clinical isolates representing the global diversity of the Mycobacterium tuberculosis complex (MTBC). The tree scale denotes number of mutations per site. Ancestrally derived lineage and sub-lineage defining lldD2 mutations are indicated by a bar. Triangles denote de novo or homoplastic mutations. Total number of mutations per strain is tallied to the right of the phylogenetic tree.

العلاقة: https://figshare.com/articles/figure/Stepwise_evolution_of_LldD2_/25320174Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g001Test
https://figshare.com/articles/figure/Stepwise_evolution_of_LldD2_/25320174Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: (A) Principal component analysis of the RNA-seq technical replicates based on normalized counts. (B) Volcano plot of the differential expression (DE) analysis. Grey indicates DE genes with log 2 fold change (L2FC) greater than 0.6 or less than -0.6 (P<0.01), red indicates DE genes with an L2FC greater than 1, blue indicates DE genes with an L2FC less than -1. There are 7 DE genes in 7H9 and 52 DE genes in lactate. (C) Heatmap of the 52 DE genes from the lactate condition, 7H9 shown for comparison. The genes are ordered according to chromosome position. Dots denote genes involved in the pathways shown in (D). (D) STRING protein-protein interaction network of the 52 DE genes from the lactate condition. P-value indicates the significance of Gene Ontology, KEGG, or STRING pathway enrichment after multiple test correction.

العلاقة: https://figshare.com/articles/figure/Lactate_and_i_lldD2_i_variants_remodel_the_Mtb_virulence_transcriptome_/25320186Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g005Test
https://figshare.com/articles/figure/Lactate_and_i_lldD2_i_variants_remodel_the_Mtb_virulence_transcriptome_/25320186Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: (A) lldD2 expression as measured by qPCR. Gene expression is normalized to that of -18 T>G. Each dot represents average of technical replicates from one of three independent experiments. Error bars indicate standard deviation. P-value indicates results of an ordinary one-way ANOVA test with Dunnett’s multiple comparison correction. (B) LldD2 production as measured by a Renilla luciferase assay. Luminescence normalized to that of the Msm strain expressing the L4 ancestral version of lldD2 . Each dot represents average of technical replicates from an independent experiment, error bars represent the standard deviation. P-value indicates results of an ordinary one-way ANOVA test with Dunnett’s multiple comparison correction. Nat R refers to nourseothricin resistance. (C) Alamar blue assay of clofazimine (CFZ). P-value indicates the results of two-way ANOVA with Dunnett’s multiple test correction. Triplicate replicates shown, error bars represent the standard deviation. (D) Growth curves of the indicated strains. All cultures started at OD 600 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. Representative of two independent experiments. (E) Schematic of 13 C-Lactate metabolic flux assay. Grey box indicates a proposed pathway. Dotted arrow represents the spontaneous conversion of DHAP to methylglyoxal. Dashed arrows represent abbreviated steps. (F) 13 C-Lactate metabolic flux assay results. Error bars represent the standard deviation of three technical replicates. Dark grey indicates labeled carbon ions, light grey represents total carbon ions.

العلاقة: https://figshare.com/articles/figure/_i_lldD2_i_homoplastic_mutations_modulate_gene_and_enzyme_activity_/25320183Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g004Test
https://figshare.com/articles/figure/_i_lldD2_i_homoplastic_mutations_modulate_gene_and_enzyme_activity_/25320183Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: (A) Growth curves of Mtb clinical strains from L1, L2, and L4. Strains are grouped for comparison of the lldD2 homoplastic mutation strains to closely related lineage ancestral strains. All cultures started at OD 600 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. (B) Quantification of the area under the curves (AUCs) from A. The AUC in 7H12–0.2% L-lactate is normalized to that of 7H9 for a given strain. The AUC is averaged for the V3I and V253M L4 strains and for the two L1 ancestral strains that were paired to -18 G>T. P-value indicates results of an unpaired t-test. (C) and (E) Schematic depicting the construction of recombinant lldD2 strains. (D) and (F) Growth curves of the strains indicated in (C) and (E). Hyg R , Kan R , and Zeo R refer to hygromycin, kanamycin, and zeocin resistance respectively. All cultures started at OD 600 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. Representative of two independent experiments.

العلاقة: https://figshare.com/articles/figure/Ancestral_and_homoplastic_mutations_in_i_lldD2_i_augment_Mtb_fitness_in_lactate_/25320180Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g003Test
https://figshare.com/articles/figure/Ancestral_and_homoplastic_mutations_in_i_lldD2_i_augment_Mtb_fitness_in_lactate_/25320180Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: (A) Schematic of LldD2 depicting the amino acid position of coding mutations listed in Fig 1 . The purple rectangle illustrates the FMN-dependent alpha-hydroxy acid dehydrogenase motif. (B) Percentage of lldD2 homoplastic or clade-defining mutations belonging to the indicated genotype. 17,148 mutations spanning 216 unique loci were identified across 50,270 strains. (C) Percentage of strains carrying at least one of the indicated mutations in either LldD1 or LldD2. (D) Comparison of the percentage of strains carrying at least one LldD1 or LldD2 mutation for each lineage.

العلاقة: https://figshare.com/articles/figure/Prevalence_of_various_LldD2_mutations_/25320177Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g002Test
https://figshare.com/articles/figure/Prevalence_of_various_LldD2_mutations_/25320177Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: (A) Growth curves of Mtb clinical strains from L1, L2, and L4. Strains are grouped for comparison of the lldD2 homoplastic mutation strains to closely related lineage ancestral strains. All cultures started at OD 600 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. (B) Quantification of the area under the curves (AUCs) from A. The AUC in 7H12–0.2% L-lactate is normalized to that of 7H9 for a given strain. The AUC is averaged for the V3I and V253M L4 strains and for the two L1 ancestral strains that were paired to -18 G>T. P-value indicates results of an unpaired t-test. (C) and (E) Schematic depicting the construction of recombinant lldD2 strains. (D) and (F) Growth curves of the strains indicated in (C) and (E). Hyg R , Kan R , and Zeo R refer to hygromycin, kanamycin, and zeocin resistance respectively. All cultures started at OD 600 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. Representative of two independent experiments.

العلاقة: https://figshare.com/articles/figure/Ancestral_and_homoplastic_mutations_in_i_lldD2_i_augment_Mtb_fitness_in_lactate_/25320180Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g003Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: Phylogenetic tree of 296 M . tuberculosis and M . africanum clinical isolates representing the global diversity of the Mycobacterium tuberculosis complex (MTBC). The tree scale denotes number of mutations per site. Ancestrally derived lineage and sub-lineage defining lldD2 mutations are indicated by a bar. Triangles denote de novo or homoplastic mutations. Total number of mutations per strain is tallied to the right of the phylogenetic tree.

العلاقة: https://figshare.com/articles/figure/Stepwise_evolution_of_LldD2_/25320174Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g001Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: (A) Schematic of LldD2 depicting the amino acid position of coding mutations listed in Fig 1 . The purple rectangle illustrates the FMN-dependent alpha-hydroxy acid dehydrogenase motif. (B) Percentage of lldD2 homoplastic or clade-defining mutations belonging to the indicated genotype. 17,148 mutations spanning 216 unique loci were identified across 50,270 strains. (C) Percentage of strains carrying at least one of the indicated mutations in either LldD1 or LldD2. (D) Comparison of the percentage of strains carrying at least one LldD1 or LldD2 mutation for each lineage.

العلاقة: https://figshare.com/articles/figure/Prevalence_of_various_LldD2_mutations_/25320177Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g002Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: (A) Principal component analysis of the RNA-seq technical replicates based on normalized counts. (B) Volcano plot of the differential expression (DE) analysis. Grey indicates DE genes with log 2 fold change (L2FC) greater than 0.6 or less than -0.6 (P<0.01), red indicates DE genes with an L2FC greater than 1, blue indicates DE genes with an L2FC less than -1. There are 7 DE genes in 7H9 and 52 DE genes in lactate. (C) Heatmap of the 52 DE genes from the lactate condition, 7H9 shown for comparison. The genes are ordered according to chromosome position. Dots denote genes involved in the pathways shown in (D). (D) STRING protein-protein interaction network of the 52 DE genes from the lactate condition. P-value indicates the significance of Gene Ontology, KEGG, or STRING pathway enrichment after multiple test correction.

العلاقة: https://figshare.com/articles/figure/Lactate_and_i_lldD2_i_variants_remodel_the_Mtb_virulence_transcriptome_/25320186Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g005Test

المؤلفون: Sydney Stanley, Xin Wang, Qingyun Liu, Young Yon Kwon, Abigail M. Frey, Nathan D. Hicks, Andrew J. Vickers, Sheng Hui, Sarah M. Fortune

مصطلحات موضوعية: Biochemistry, Microbiology, Genetics, Molecular Biology, Evolutionary Biology, Ecology, Cancer, Infectious Diseases, Computational Biology, Environmental Sciences not elsewhere classified, providing experimental evidence, div >< p, central carbon metabolism, 1 virulence system, mycobacterium tuberculosis <, discernible growth defect, dependent antibiotic susceptibility, mutations functionally increase, human host environment, global clinical isolates, lactate dehydrogenase reveals, assessing mtb genes, 13 , clinical isolates, vivo <, mtb growth, lactate dehydrogenase, intracellular environment, host pressure, genes involved

الوصف: (A) lldD2 expression as measured by qPCR. Gene expression is normalized to that of -18 T>G. Each dot represents average of technical replicates from one of three independent experiments. Error bars indicate standard deviation. P-value indicates results of an ordinary one-way ANOVA test with Dunnett’s multiple comparison correction. (B) LldD2 production as measured by a Renilla luciferase assay. Luminescence normalized to that of the Msm strain expressing the L4 ancestral version of lldD2 . Each dot represents average of technical replicates from an independent experiment, error bars represent the standard deviation. P-value indicates results of an ordinary one-way ANOVA test with Dunnett’s multiple comparison correction. Nat R refers to nourseothricin resistance. (C) Alamar blue assay of clofazimine (CFZ). P-value indicates the results of two-way ANOVA with Dunnett’s multiple test correction. Triplicate replicates shown, error bars represent the standard deviation. (D) Growth curves of the indicated strains. All cultures started at OD 600 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. Representative of two independent experiments. (E) Schematic of 13 C-Lactate metabolic flux assay. Grey box indicates a proposed pathway. Dotted arrow represents the spontaneous conversion of DHAP to methylglyoxal. Dashed arrows represent abbreviated steps. (F) 13 C-Lactate metabolic flux assay results. Error bars represent the standard deviation of three technical replicates. Dark grey indicates labeled carbon ions, light grey represents total carbon ions.

العلاقة: https://figshare.com/articles/figure/_i_lldD2_i_homoplastic_mutations_modulate_gene_and_enzyme_activity_/25320183Test

الإتاحة: https://doi.org/10.1371/journal.ppat.1012050.g004Test