Limits...
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

renU encodes a Nudix hydrolase required for biofilm growth.(A) Relative Nudix hydrolase activity of RenU on a substrate panel (left). Nucleoside diphosphate derivatives (NDPX) are preferred substrates compared to nucleoside triphosphates (NTP). A catalytically-inactive mutant of RenU (RenUDEAD) protein, in which 3 glutamate residues (E74, E77, and E78) in the Nudix box were mutated to alanines, exhibits no phosphatase activity towards the preferred substrates (right). (B) Kinetics studies of Nudix hydrolase activity of RenU on the three NDPXs as preferred substrates ADP-ribose, FAD, and NADH. (C) Rate of RenU catalytic activity on NADH compared to its oxidative form NAD+. Fit curve is shown for NADH. (D) The Nudix hydrolase activity of RenU is required for M. smegmatis biofilm growth. Wild type M. smegmatis, MsΔrenU, and the mutant strains completed with wild type RenU or RenUDEAD were assayed for biofilm growth. Whereas renU fully restored biofilm growth to MsΔrenU, renUDEAD failed to complement the mutant. Shown images are representatives of biological triplicates. (E) The Nudix hydrolase activity of RenU is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔrenU, and the mutant strains completed with wild type RenU or RenUDEAD were was assayed for biofilm growth. Whereas renU fully restored biofilm growth to MtbΔrenU, renUDEAD failed to complement the mutant. Shown images are representatives of biological duplicates. (F) Quantitation of biofilm growth of Mtb strains. The biofilm biomass was harvested and estimated by determining total protein per plate. Error bars represent standard deviation of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401782&req=5

ppat.1004839.g003: renU encodes a Nudix hydrolase required for biofilm growth.(A) Relative Nudix hydrolase activity of RenU on a substrate panel (left). Nucleoside diphosphate derivatives (NDPX) are preferred substrates compared to nucleoside triphosphates (NTP). A catalytically-inactive mutant of RenU (RenUDEAD) protein, in which 3 glutamate residues (E74, E77, and E78) in the Nudix box were mutated to alanines, exhibits no phosphatase activity towards the preferred substrates (right). (B) Kinetics studies of Nudix hydrolase activity of RenU on the three NDPXs as preferred substrates ADP-ribose, FAD, and NADH. (C) Rate of RenU catalytic activity on NADH compared to its oxidative form NAD+. Fit curve is shown for NADH. (D) The Nudix hydrolase activity of RenU is required for M. smegmatis biofilm growth. Wild type M. smegmatis, MsΔrenU, and the mutant strains completed with wild type RenU or RenUDEAD were assayed for biofilm growth. Whereas renU fully restored biofilm growth to MsΔrenU, renUDEAD failed to complement the mutant. Shown images are representatives of biological triplicates. (E) The Nudix hydrolase activity of RenU is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔrenU, and the mutant strains completed with wild type RenU or RenUDEAD were was assayed for biofilm growth. Whereas renU fully restored biofilm growth to MtbΔrenU, renUDEAD failed to complement the mutant. Shown images are representatives of biological duplicates. (F) Quantitation of biofilm growth of Mtb strains. The biofilm biomass was harvested and estimated by determining total protein per plate. Error bars represent standard deviation of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference.

Mentions: To test whether RenU is indeed a hydrolase, and to further characterize its enzymatic activity, the recombinant M. smegmatis RenU was purified to homogeneity. Size exclusion chromatographic analysis showed that RenU was monomeric in solution (S4 Fig). Next, its enzymatic activity was determined using a coupled enzyme colorimetric assay described in the Extended Experimental Procedures. Substrate specificity was investigated with a panel of several different nucleoside diphosphate derivatives (NDPX) and nucleoside triphosphates (NTP). RenU did exhibit Nudix hydrolase activity with a substrate preference for NDPXs. Among the substrates tested, the highest activities were observed with ADP-ribose, FAD, and NADH (Figs 3A left panel, and S5). By contrast, the enzyme displayed much lower activities towards NTPs including ATP, 7,8-dihydroneopterin triphosphate (DHNTP) (Fig 3A left panel), dGTP, dCTP, dUTP, or other NDPXs such as CoA, GDP-D-mannose, NADP, ADP-ADP, and CDP-choline (S5 Fig). Importantly, mutations in glutamate residues of the Nudix box (E74, E77, and E78, see S2 Table), which are expected to coordinate the magnesium required for the activities of Nudix hydrolases, completely abolished the enzymatic activity of RenU. The mutated protein, RenUDEAD, displayed no activity towards the preferred substrates exhibited by wild type RenU (Fig 3A, right panel). Michaelis-Menten analysis revealed that, in vitro, ADP-ribose and FAD were better substrates than NADH, as evidenced by its higher kcat/Km value (Figs 3B and S6). However, previous studies with Nudix hydrolases predict that the substrate preference of RenU might be defined in vivo through its interactions with other proteins [18]. In fact, analysis of cellular levels and PknG induction experiments (see below) suggest that NADH is the physiologically relevant substrate of RenU in vivo. Interestingly, while RenU readily hydrolyzed NADH, the reduced form of nicotinamide adenine dinucleotide, it did not show significant catalytic activity towards the oxidized form, NAD+ (Fig 3C).


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)

renU encodes a Nudix hydrolase required for biofilm growth.(A) Relative Nudix hydrolase activity of RenU on a substrate panel (left). Nucleoside diphosphate derivatives (NDPX) are preferred substrates compared to nucleoside triphosphates (NTP). A catalytically-inactive mutant of RenU (RenUDEAD) protein, in which 3 glutamate residues (E74, E77, and E78) in the Nudix box were mutated to alanines, exhibits no phosphatase activity towards the preferred substrates (right). (B) Kinetics studies of Nudix hydrolase activity of RenU on the three NDPXs as preferred substrates ADP-ribose, FAD, and NADH. (C) Rate of RenU catalytic activity on NADH compared to its oxidative form NAD+. Fit curve is shown for NADH. (D) The Nudix hydrolase activity of RenU is required for M. smegmatis biofilm growth. Wild type M. smegmatis, MsΔrenU, and the mutant strains completed with wild type RenU or RenUDEAD were assayed for biofilm growth. Whereas renU fully restored biofilm growth to MsΔrenU, renUDEAD failed to complement the mutant. Shown images are representatives of biological triplicates. (E) The Nudix hydrolase activity of RenU is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔrenU, and the mutant strains completed with wild type RenU or RenUDEAD were was assayed for biofilm growth. Whereas renU fully restored biofilm growth to MtbΔrenU, renUDEAD failed to complement the mutant. Shown images are representatives of biological duplicates. (F) Quantitation of biofilm growth of Mtb strains. The biofilm biomass was harvested and estimated by determining total protein per plate. Error bars represent standard deviation of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004839.g003: renU encodes a Nudix hydrolase required for biofilm growth.(A) Relative Nudix hydrolase activity of RenU on a substrate panel (left). Nucleoside diphosphate derivatives (NDPX) are preferred substrates compared to nucleoside triphosphates (NTP). A catalytically-inactive mutant of RenU (RenUDEAD) protein, in which 3 glutamate residues (E74, E77, and E78) in the Nudix box were mutated to alanines, exhibits no phosphatase activity towards the preferred substrates (right). (B) Kinetics studies of Nudix hydrolase activity of RenU on the three NDPXs as preferred substrates ADP-ribose, FAD, and NADH. (C) Rate of RenU catalytic activity on NADH compared to its oxidative form NAD+. Fit curve is shown for NADH. (D) The Nudix hydrolase activity of RenU is required for M. smegmatis biofilm growth. Wild type M. smegmatis, MsΔrenU, and the mutant strains completed with wild type RenU or RenUDEAD were assayed for biofilm growth. Whereas renU fully restored biofilm growth to MsΔrenU, renUDEAD failed to complement the mutant. Shown images are representatives of biological triplicates. (E) The Nudix hydrolase activity of RenU is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔrenU, and the mutant strains completed with wild type RenU or RenUDEAD were was assayed for biofilm growth. Whereas renU fully restored biofilm growth to MtbΔrenU, renUDEAD failed to complement the mutant. Shown images are representatives of biological duplicates. (F) Quantitation of biofilm growth of Mtb strains. The biofilm biomass was harvested and estimated by determining total protein per plate. Error bars represent standard deviation of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference.
Mentions: To test whether RenU is indeed a hydrolase, and to further characterize its enzymatic activity, the recombinant M. smegmatis RenU was purified to homogeneity. Size exclusion chromatographic analysis showed that RenU was monomeric in solution (S4 Fig). Next, its enzymatic activity was determined using a coupled enzyme colorimetric assay described in the Extended Experimental Procedures. Substrate specificity was investigated with a panel of several different nucleoside diphosphate derivatives (NDPX) and nucleoside triphosphates (NTP). RenU did exhibit Nudix hydrolase activity with a substrate preference for NDPXs. Among the substrates tested, the highest activities were observed with ADP-ribose, FAD, and NADH (Figs 3A left panel, and S5). By contrast, the enzyme displayed much lower activities towards NTPs including ATP, 7,8-dihydroneopterin triphosphate (DHNTP) (Fig 3A left panel), dGTP, dCTP, dUTP, or other NDPXs such as CoA, GDP-D-mannose, NADP, ADP-ADP, and CDP-choline (S5 Fig). Importantly, mutations in glutamate residues of the Nudix box (E74, E77, and E78, see S2 Table), which are expected to coordinate the magnesium required for the activities of Nudix hydrolases, completely abolished the enzymatic activity of RenU. The mutated protein, RenUDEAD, displayed no activity towards the preferred substrates exhibited by wild type RenU (Fig 3A, right panel). Michaelis-Menten analysis revealed that, in vitro, ADP-ribose and FAD were better substrates than NADH, as evidenced by its higher kcat/Km value (Figs 3B and S6). However, previous studies with Nudix hydrolases predict that the substrate preference of RenU might be defined in vivo through its interactions with other proteins [18]. In fact, analysis of cellular levels and PknG induction experiments (see below) suggest that NADH is the physiologically relevant substrate of RenU in vivo. Interestingly, while RenU readily hydrolyzed NADH, the reduced form of nicotinamide adenine dinucleotide, it did not show significant catalytic activity towards the oxidized form, NAD+ (Fig 3C).

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