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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

Correlation of NADH and RHOCS, and role of L13 phosphorylation by PknG.(A) Induction of PknG expression in M. smegmatis. Western analysis was used to detect PknG expression following the exposure of wild type M. smegmatis cultures (OD600 of 2) to various oxidative stimuli including NADH (upper) and FAD (lower) for 30 minutes. All chemicals were used at 10 mM except for bleomycin, which was used at 10 μg/ml. Samples were separated on SDS-PAGE, followed by immunodetection using an anti-PknG antibody or an anti-DivIVA antibody, as a control. Non-induced lysates from wild type M. smegmatis and MsΔpknG were used as controls. NADH uniquely induced expression of PknG. (B) Titration of the induced PknG expression by increasing NADH concentrations (0–30 mM) for 30 minutes, followed by Western analysis using anti-PknG antibody. (C) Time course of PknG expression (0–60 minutes) following cell exposure to 10 mM NADH. Detection of PknG was similar to (A) and (B). (D) Quantitation of cellular NADH (top), NAD+ (middle), and FAD (bottom) levels following oxidative stress induced by H2O2. M. smegmatis cells were exposed to 1 mM H2O2 for 1 hour. Bars show means with standard deviations from 3–6 biological repeats. *, p < 0.0001; ns, not significant relative to wild type M. smegmatis). (E) Effect of PknG-catalyzed phosphorylation of L13 on its association with RenU in the cytoplasm. Expression of PknG in M. smegmatis strains was induced by NADH. Cells were disintegrated by French Press, followed by ultracentrifugation to remove ribosomes. RenU.6H was added to the non-ribosomal fraction, followed by pull-down using Cobalt-agarose beads. The presence of L13 in the pulled down materials was detected by Western analysis using anti-L13 antibody. (F) Effect of L13(T11E), a phosphorylation-mimic form of L13, on in vitro NADH hydrolytic activity of RenU. Initial rates from a continuous fluorescence excitation assay were fit by nonlinear least squares to the Michaelis-Menten equation to determine Km and Vmax values for RenU. Reaction was performed at 37°C. Error bars represent standard deviations of triplicates. The extent of the uncatalyzed reaction was ~10% of the RenU catalyzed reaction. (G) Effect of L13(T11E) on the catalytic activity of RenU. In the presence of L13(T11E), a 20.6% increase in Vmax was observed (p < 0.05x10-3), whereas Km, reflecting the binding affinity of RenU to NADH, was not affected by L13(T11E).
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ppat.1004839.g006: Correlation of NADH and RHOCS, and role of L13 phosphorylation by PknG.(A) Induction of PknG expression in M. smegmatis. Western analysis was used to detect PknG expression following the exposure of wild type M. smegmatis cultures (OD600 of 2) to various oxidative stimuli including NADH (upper) and FAD (lower) for 30 minutes. All chemicals were used at 10 mM except for bleomycin, which was used at 10 μg/ml. Samples were separated on SDS-PAGE, followed by immunodetection using an anti-PknG antibody or an anti-DivIVA antibody, as a control. Non-induced lysates from wild type M. smegmatis and MsΔpknG were used as controls. NADH uniquely induced expression of PknG. (B) Titration of the induced PknG expression by increasing NADH concentrations (0–30 mM) for 30 minutes, followed by Western analysis using anti-PknG antibody. (C) Time course of PknG expression (0–60 minutes) following cell exposure to 10 mM NADH. Detection of PknG was similar to (A) and (B). (D) Quantitation of cellular NADH (top), NAD+ (middle), and FAD (bottom) levels following oxidative stress induced by H2O2. M. smegmatis cells were exposed to 1 mM H2O2 for 1 hour. Bars show means with standard deviations from 3–6 biological repeats. *, p < 0.0001; ns, not significant relative to wild type M. smegmatis). (E) Effect of PknG-catalyzed phosphorylation of L13 on its association with RenU in the cytoplasm. Expression of PknG in M. smegmatis strains was induced by NADH. Cells were disintegrated by French Press, followed by ultracentrifugation to remove ribosomes. RenU.6H was added to the non-ribosomal fraction, followed by pull-down using Cobalt-agarose beads. The presence of L13 in the pulled down materials was detected by Western analysis using anti-L13 antibody. (F) Effect of L13(T11E), a phosphorylation-mimic form of L13, on in vitro NADH hydrolytic activity of RenU. Initial rates from a continuous fluorescence excitation assay were fit by nonlinear least squares to the Michaelis-Menten equation to determine Km and Vmax values for RenU. Reaction was performed at 37°C. Error bars represent standard deviations of triplicates. The extent of the uncatalyzed reaction was ~10% of the RenU catalyzed reaction. (G) Effect of L13(T11E) on the catalytic activity of RenU. In the presence of L13(T11E), a 20.6% increase in Vmax was observed (p < 0.05x10-3), whereas Km, reflecting the binding affinity of RenU to NADH, was not affected by L13(T11E).

Mentions: A recent study showed that expression of PknG is tightly regulated by unknown mechanisms related to the pathogenicity of Mtb [20]. Whereas PknG is highly expressed in slow growing mycobacteria such as Mtb and M. bovis BCG, the expression in M. smegmatis is extremely low [4,20]. To investigate the conditions that trigger PknG expression in M. smegmatis, the bacterium was treated with various redox stimuli, followed by analysis of PknG levels by Western analysis using a specific polyclonal antibody [3,4,20]. Interestingly, we found that PknG expression is uniquely induced when M. smegmatis cells are exposed to high levels of NADH (Fig 6A). None of the other tested chemicals, including FAD (lower panel), induced PknG expression. The induction of PknG expression by NADH, in both concentration- (Fig 6B) and time-dependent (Fig 6C) manners, may suggest a specific regulatory mechanism, similar to the Rex system originally described in Streptomyces [21–24], or an indirect effect due to changes in cellular metabolism or physiology caused by NADH exposure.


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)

Correlation of NADH and RHOCS, and role of L13 phosphorylation by PknG.(A) Induction of PknG expression in M. smegmatis. Western analysis was used to detect PknG expression following the exposure of wild type M. smegmatis cultures (OD600 of 2) to various oxidative stimuli including NADH (upper) and FAD (lower) for 30 minutes. All chemicals were used at 10 mM except for bleomycin, which was used at 10 μg/ml. Samples were separated on SDS-PAGE, followed by immunodetection using an anti-PknG antibody or an anti-DivIVA antibody, as a control. Non-induced lysates from wild type M. smegmatis and MsΔpknG were used as controls. NADH uniquely induced expression of PknG. (B) Titration of the induced PknG expression by increasing NADH concentrations (0–30 mM) for 30 minutes, followed by Western analysis using anti-PknG antibody. (C) Time course of PknG expression (0–60 minutes) following cell exposure to 10 mM NADH. Detection of PknG was similar to (A) and (B). (D) Quantitation of cellular NADH (top), NAD+ (middle), and FAD (bottom) levels following oxidative stress induced by H2O2. M. smegmatis cells were exposed to 1 mM H2O2 for 1 hour. Bars show means with standard deviations from 3–6 biological repeats. *, p < 0.0001; ns, not significant relative to wild type M. smegmatis). (E) Effect of PknG-catalyzed phosphorylation of L13 on its association with RenU in the cytoplasm. Expression of PknG in M. smegmatis strains was induced by NADH. Cells were disintegrated by French Press, followed by ultracentrifugation to remove ribosomes. RenU.6H was added to the non-ribosomal fraction, followed by pull-down using Cobalt-agarose beads. The presence of L13 in the pulled down materials was detected by Western analysis using anti-L13 antibody. (F) Effect of L13(T11E), a phosphorylation-mimic form of L13, on in vitro NADH hydrolytic activity of RenU. Initial rates from a continuous fluorescence excitation assay were fit by nonlinear least squares to the Michaelis-Menten equation to determine Km and Vmax values for RenU. Reaction was performed at 37°C. Error bars represent standard deviations of triplicates. The extent of the uncatalyzed reaction was ~10% of the RenU catalyzed reaction. (G) Effect of L13(T11E) on the catalytic activity of RenU. In the presence of L13(T11E), a 20.6% increase in Vmax was observed (p < 0.05x10-3), whereas Km, reflecting the binding affinity of RenU to NADH, was not affected by L13(T11E).
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ppat.1004839.g006: Correlation of NADH and RHOCS, and role of L13 phosphorylation by PknG.(A) Induction of PknG expression in M. smegmatis. Western analysis was used to detect PknG expression following the exposure of wild type M. smegmatis cultures (OD600 of 2) to various oxidative stimuli including NADH (upper) and FAD (lower) for 30 minutes. All chemicals were used at 10 mM except for bleomycin, which was used at 10 μg/ml. Samples were separated on SDS-PAGE, followed by immunodetection using an anti-PknG antibody or an anti-DivIVA antibody, as a control. Non-induced lysates from wild type M. smegmatis and MsΔpknG were used as controls. NADH uniquely induced expression of PknG. (B) Titration of the induced PknG expression by increasing NADH concentrations (0–30 mM) for 30 minutes, followed by Western analysis using anti-PknG antibody. (C) Time course of PknG expression (0–60 minutes) following cell exposure to 10 mM NADH. Detection of PknG was similar to (A) and (B). (D) Quantitation of cellular NADH (top), NAD+ (middle), and FAD (bottom) levels following oxidative stress induced by H2O2. M. smegmatis cells were exposed to 1 mM H2O2 for 1 hour. Bars show means with standard deviations from 3–6 biological repeats. *, p < 0.0001; ns, not significant relative to wild type M. smegmatis). (E) Effect of PknG-catalyzed phosphorylation of L13 on its association with RenU in the cytoplasm. Expression of PknG in M. smegmatis strains was induced by NADH. Cells were disintegrated by French Press, followed by ultracentrifugation to remove ribosomes. RenU.6H was added to the non-ribosomal fraction, followed by pull-down using Cobalt-agarose beads. The presence of L13 in the pulled down materials was detected by Western analysis using anti-L13 antibody. (F) Effect of L13(T11E), a phosphorylation-mimic form of L13, on in vitro NADH hydrolytic activity of RenU. Initial rates from a continuous fluorescence excitation assay were fit by nonlinear least squares to the Michaelis-Menten equation to determine Km and Vmax values for RenU. Reaction was performed at 37°C. Error bars represent standard deviations of triplicates. The extent of the uncatalyzed reaction was ~10% of the RenU catalyzed reaction. (G) Effect of L13(T11E) on the catalytic activity of RenU. In the presence of L13(T11E), a 20.6% increase in Vmax was observed (p < 0.05x10-3), whereas Km, reflecting the binding affinity of RenU to NADH, was not affected by L13(T11E).
Mentions: A recent study showed that expression of PknG is tightly regulated by unknown mechanisms related to the pathogenicity of Mtb [20]. Whereas PknG is highly expressed in slow growing mycobacteria such as Mtb and M. bovis BCG, the expression in M. smegmatis is extremely low [4,20]. To investigate the conditions that trigger PknG expression in M. smegmatis, the bacterium was treated with various redox stimuli, followed by analysis of PknG levels by Western analysis using a specific polyclonal antibody [3,4,20]. Interestingly, we found that PknG expression is uniquely induced when M. smegmatis cells are exposed to high levels of NADH (Fig 6A). None of the other tested chemicals, including FAD (lower panel), induced PknG expression. The induction of PknG expression by NADH, in both concentration- (Fig 6B) and time-dependent (Fig 6C) manners, may suggest a specific regulatory mechanism, similar to the Rex system originally described in Streptomyces [21–24], or an indirect effect due to changes in cellular metabolism or physiology caused by NADH exposure.

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