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A redox regulatory system critical for mycobacterial survival in macrophages and biofilm development.

Wolff KA, de la Peña AH, Nguyen HT, Pham TH, Amzel LM, Gabelli SB, Nguyen L - PLoS Pathog. (2015)

Bottom Line: Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress.We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis.Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT
Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostatic system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

No MeSH data available.


Related in: MedlinePlus

Intracellular trafficking and survival of M. tuberculosis RHOCS mutants.(A) Intracellular survival of Mtb strains. Macrophages were infected with Mtb strains for 3 hours, followed by 0 or 72-hour chase. CFUs were counted after 4–5 weeks of growth at 37°C. Bars represent percentages of CFUs remaining at 72-hour compared to 0-hour time point. Error bars represent standard deviations from 3–6 repeats. *, p < 0.001; ns, not significant relative to Mtb H37Rv; #ns, not significant between the two indicated groups. Order of strains is as in 7B. (B) Quantitative analysis of lysosomal delivery following phagocytosis of Mtb strains by macrophages. Macrophages were infected with FLUOS-stained Mtb strains for 1 hour, followed by 16-hour chase. Infected macrophages (see C below) were used for quantitation. Biological triplicates of 50 events were counted for each Mtb strain. Error bars represent standard deviations. Error bars represent standard deviations from 3–6 repeats. *, p < 0.0001; ns, not significant relative to Mtb H37Rv; ** ns, not significant between the two indicated groups. (C) A model depicting activity and function of RHOCS in mycobacteria. PknG was previously shown to de-repress the TCA cycle through its phosphorylation of GarA, an inhibitor of α-ketoglutarate decarboxylase and glutamate dehydrogenase. Increased TCA cycle activities, hypoxia, or impaired oxidative phosphorylation (OXPHOS), lead to elevated NADH levels. To protect mycobacterial cells against the change in redox status, PknG expression is up-regulated, leading to the signaling cascade including L13 and RenU, which degrades NADH and FAD and restores their optimal level. AMP, adenosine monophosphate, FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NMNH, nicotinamide mononucleotide.
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ppat.1004839.g007: Intracellular trafficking and survival of M. tuberculosis RHOCS mutants.(A) Intracellular survival of Mtb strains. Macrophages were infected with Mtb strains for 3 hours, followed by 0 or 72-hour chase. CFUs were counted after 4–5 weeks of growth at 37°C. Bars represent percentages of CFUs remaining at 72-hour compared to 0-hour time point. Error bars represent standard deviations from 3–6 repeats. *, p < 0.001; ns, not significant relative to Mtb H37Rv; #ns, not significant between the two indicated groups. Order of strains is as in 7B. (B) Quantitative analysis of lysosomal delivery following phagocytosis of Mtb strains by macrophages. Macrophages were infected with FLUOS-stained Mtb strains for 1 hour, followed by 16-hour chase. Infected macrophages (see C below) were used for quantitation. Biological triplicates of 50 events were counted for each Mtb strain. Error bars represent standard deviations. Error bars represent standard deviations from 3–6 repeats. *, p < 0.0001; ns, not significant relative to Mtb H37Rv; ** ns, not significant between the two indicated groups. (C) A model depicting activity and function of RHOCS in mycobacteria. PknG was previously shown to de-repress the TCA cycle through its phosphorylation of GarA, an inhibitor of α-ketoglutarate decarboxylase and glutamate dehydrogenase. Increased TCA cycle activities, hypoxia, or impaired oxidative phosphorylation (OXPHOS), lead to elevated NADH levels. To protect mycobacterial cells against the change in redox status, PknG expression is up-regulated, leading to the signaling cascade including L13 and RenU, which degrades NADH and FAD and restores their optimal level. AMP, adenosine monophosphate, FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NMNH, nicotinamide mononucleotide.

Mentions: To investigate if RHOCS is related to the role of PknG in the survival and lysosomal delivery of pathogenic mycobacteria in infected macrophages [3], Mtb RHOCS mutants and the parental strain H37Rv were used to infect murine bone-marrow derived macrophages. Survival was determined by measuring colony forming units (CFUs) of internalized Mtb cells at day 0 and day 4 following the infection. As shown in Fig 7A, deletion of pknG or renU, or the single mutation L13(T11A), significantly reduced the percentages of Mtb survival at day 4 relative to day 0. By contrast, the complemented strains, namely MtbΔpknG/pknG, MtbΔrenU/renU, and L13(T11A)/(T11E), showed survival similar to wild type Mtb (Fig 7A). As shown with biofilm growth, the mutant allele renUDEAD failed to restore the survival of the MtbΔrenU mutant (Fig 7A). In addition, in trans expression of the phosphorylation-mimic form of L13, L13(T11E), also made the L13(T11A)/(T11E) strain resistant to AX20017, the PknG specific inhibitor. These results, all together, indicated that the kinase activity of PknG, the Nudix hydrolase activity of RenU, as well as the phosphorylation of L13, each is required for the survival of Mtb in host macrophages.


A redox regulatory system critical for mycobacterial survival in macrophages and biofilm development.

Wolff KA, de la Peña AH, Nguyen HT, Pham TH, Amzel LM, Gabelli SB, Nguyen L - PLoS Pathog. (2015)

Intracellular trafficking and survival of M. tuberculosis RHOCS mutants.(A) Intracellular survival of Mtb strains. Macrophages were infected with Mtb strains for 3 hours, followed by 0 or 72-hour chase. CFUs were counted after 4–5 weeks of growth at 37°C. Bars represent percentages of CFUs remaining at 72-hour compared to 0-hour time point. Error bars represent standard deviations from 3–6 repeats. *, p < 0.001; ns, not significant relative to Mtb H37Rv; #ns, not significant between the two indicated groups. Order of strains is as in 7B. (B) Quantitative analysis of lysosomal delivery following phagocytosis of Mtb strains by macrophages. Macrophages were infected with FLUOS-stained Mtb strains for 1 hour, followed by 16-hour chase. Infected macrophages (see C below) were used for quantitation. Biological triplicates of 50 events were counted for each Mtb strain. Error bars represent standard deviations. Error bars represent standard deviations from 3–6 repeats. *, p < 0.0001; ns, not significant relative to Mtb H37Rv; ** ns, not significant between the two indicated groups. (C) A model depicting activity and function of RHOCS in mycobacteria. PknG was previously shown to de-repress the TCA cycle through its phosphorylation of GarA, an inhibitor of α-ketoglutarate decarboxylase and glutamate dehydrogenase. Increased TCA cycle activities, hypoxia, or impaired oxidative phosphorylation (OXPHOS), lead to elevated NADH levels. To protect mycobacterial cells against the change in redox status, PknG expression is up-regulated, leading to the signaling cascade including L13 and RenU, which degrades NADH and FAD and restores their optimal level. AMP, adenosine monophosphate, FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NMNH, nicotinamide mononucleotide.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401782&req=5

ppat.1004839.g007: Intracellular trafficking and survival of M. tuberculosis RHOCS mutants.(A) Intracellular survival of Mtb strains. Macrophages were infected with Mtb strains for 3 hours, followed by 0 or 72-hour chase. CFUs were counted after 4–5 weeks of growth at 37°C. Bars represent percentages of CFUs remaining at 72-hour compared to 0-hour time point. Error bars represent standard deviations from 3–6 repeats. *, p < 0.001; ns, not significant relative to Mtb H37Rv; #ns, not significant between the two indicated groups. Order of strains is as in 7B. (B) Quantitative analysis of lysosomal delivery following phagocytosis of Mtb strains by macrophages. Macrophages were infected with FLUOS-stained Mtb strains for 1 hour, followed by 16-hour chase. Infected macrophages (see C below) were used for quantitation. Biological triplicates of 50 events were counted for each Mtb strain. Error bars represent standard deviations. Error bars represent standard deviations from 3–6 repeats. *, p < 0.0001; ns, not significant relative to Mtb H37Rv; ** ns, not significant between the two indicated groups. (C) A model depicting activity and function of RHOCS in mycobacteria. PknG was previously shown to de-repress the TCA cycle through its phosphorylation of GarA, an inhibitor of α-ketoglutarate decarboxylase and glutamate dehydrogenase. Increased TCA cycle activities, hypoxia, or impaired oxidative phosphorylation (OXPHOS), lead to elevated NADH levels. To protect mycobacterial cells against the change in redox status, PknG expression is up-regulated, leading to the signaling cascade including L13 and RenU, which degrades NADH and FAD and restores their optimal level. AMP, adenosine monophosphate, FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NMNH, nicotinamide mononucleotide.
Mentions: To investigate if RHOCS is related to the role of PknG in the survival and lysosomal delivery of pathogenic mycobacteria in infected macrophages [3], Mtb RHOCS mutants and the parental strain H37Rv were used to infect murine bone-marrow derived macrophages. Survival was determined by measuring colony forming units (CFUs) of internalized Mtb cells at day 0 and day 4 following the infection. As shown in Fig 7A, deletion of pknG or renU, or the single mutation L13(T11A), significantly reduced the percentages of Mtb survival at day 4 relative to day 0. By contrast, the complemented strains, namely MtbΔpknG/pknG, MtbΔrenU/renU, and L13(T11A)/(T11E), showed survival similar to wild type Mtb (Fig 7A). As shown with biofilm growth, the mutant allele renUDEAD failed to restore the survival of the MtbΔrenU mutant (Fig 7A). In addition, in trans expression of the phosphorylation-mimic form of L13, L13(T11E), also made the L13(T11A)/(T11E) strain resistant to AX20017, the PknG specific inhibitor. These results, all together, indicated that the kinase activity of PknG, the Nudix hydrolase activity of RenU, as well as the phosphorylation of L13, each is required for the survival of Mtb in host macrophages.

Bottom Line: Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress.We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis.Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.

ABSTRACT
Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostatic system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

No MeSH data available.


Related in: MedlinePlus