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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

PknG kinase activity is required for biofilm growth in mycobacteria.(A) Role of pknG in M. smegmatis planktonic growth. Wild type M. smegmatis mc2155 (red filled circles), its derived MsΔpknG mutant (blue filled triangles), and the complemented strain MsΔpknG/pknG (green filled squares) were grown in 7H9 medium supplemented with 0.2% glucose with shaking at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and MsΔpknG in stationary phase are not significant. (B) Role of pknG in Mtb planktonic growth. Wild type Mtb H37Rv (open circles) and its derived MtbΔpknG mutant (open triangles) were grown in 7H9-OADC medium with 0.2% glucose (blue) or 1% glucose (red). Cultures were shaken at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and MtbΔpknG in stationary phase (5–11 hours) are statistically significant (two-tailed t-test, p<0.05). (C) pknG is required for M. smegmatis biofilm growth. Wild type M. smegmatis, MsΔpknG, and the mutant strains complemented with the M. smegmatis (Ms-pknG) or M. tuberculosis (Mtb-pknG) gene. Pictures were taken after 7 days of static growth at 30°C. Shown images are representatives of biological triplicates. (D) pknG is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔpknG, and the complemented strain were assayed as previously described [10]. Pictures were taken after 6 weeks of growth at a static humidified condition of 37°C and 5% CO2. Shown images are representatives of biological triplicates. (E) Quantitation of biofilm growth of Mtb strains. Biofilms were harvested and quantified as described in Experimental Procedures. Error bars represent standard deviation of biological triplicates (*, p<0.0001; ns, not significant difference between wild type H37Rv and the complemented strain). (F) PknG kinase activity is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔpknG, and the complemented strain were assayed in the absence (-) or presence (+) of 1 mM AX20017, a specific inhibitor of PknG. Pictures were taken after 6 weeks of growth at static humidified condition of 37°C and 5% CO2. Shown images are representatives of biological triplicates.
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ppat.1004839.g001: PknG kinase activity is required for biofilm growth in mycobacteria.(A) Role of pknG in M. smegmatis planktonic growth. Wild type M. smegmatis mc2155 (red filled circles), its derived MsΔpknG mutant (blue filled triangles), and the complemented strain MsΔpknG/pknG (green filled squares) were grown in 7H9 medium supplemented with 0.2% glucose with shaking at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and MsΔpknG in stationary phase are not significant. (B) Role of pknG in Mtb planktonic growth. Wild type Mtb H37Rv (open circles) and its derived MtbΔpknG mutant (open triangles) were grown in 7H9-OADC medium with 0.2% glucose (blue) or 1% glucose (red). Cultures were shaken at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and MtbΔpknG in stationary phase (5–11 hours) are statistically significant (two-tailed t-test, p<0.05). (C) pknG is required for M. smegmatis biofilm growth. Wild type M. smegmatis, MsΔpknG, and the mutant strains complemented with the M. smegmatis (Ms-pknG) or M. tuberculosis (Mtb-pknG) gene. Pictures were taken after 7 days of static growth at 30°C. Shown images are representatives of biological triplicates. (D) pknG is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔpknG, and the complemented strain were assayed as previously described [10]. Pictures were taken after 6 weeks of growth at a static humidified condition of 37°C and 5% CO2. Shown images are representatives of biological triplicates. (E) Quantitation of biofilm growth of Mtb strains. Biofilms were harvested and quantified as described in Experimental Procedures. Error bars represent standard deviation of biological triplicates (*, p<0.0001; ns, not significant difference between wild type H37Rv and the complemented strain). (F) PknG kinase activity is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔpknG, and the complemented strain were assayed in the absence (-) or presence (+) of 1 mM AX20017, a specific inhibitor of PknG. Pictures were taken after 6 weeks of growth at static humidified condition of 37°C and 5% CO2. Shown images are representatives of biological triplicates.

Mentions: Studies in other bacteria have established the phenotypic relationship between host persistence, antibiotic resistance and biofilm growth [6,9]. PknG was previously shown to be required for two of these three phenotypes, namely survival in macrophages and resistance to multiple antibiotics [3,4]. In addition, phenotypic characterization of a M. smegmatis ΔpknG mutant revealed profound alterations in surface charge and hydrophobicity of the cell wall (S1 Table) [4], suggesting that PknG might be involved in mycobacterial biofilm growth. Wild type strains of M. smegmatis, M. bovis BCG, and Mtb, together with their derived ΔpknG mutants and complemented strains were assayed for growth in both planktonic cultures and static biofilms (Figs 1 and S1). Similar to M. bovis BCG [3,13], absence of PknG did not affect planktonic growth of M. smegmatis (Fig 1A). However, the MtbΔpknG displayed a slow growth defect in stationary phase (Fig 1B), as previously reported [14]. These phenotypic variations suggest a complexity of PknG function among mycobacterial species that warrants further investigation.


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)

PknG kinase activity is required for biofilm growth in mycobacteria.(A) Role of pknG in M. smegmatis planktonic growth. Wild type M. smegmatis mc2155 (red filled circles), its derived MsΔpknG mutant (blue filled triangles), and the complemented strain MsΔpknG/pknG (green filled squares) were grown in 7H9 medium supplemented with 0.2% glucose with shaking at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and MsΔpknG in stationary phase are not significant. (B) Role of pknG in Mtb planktonic growth. Wild type Mtb H37Rv (open circles) and its derived MtbΔpknG mutant (open triangles) were grown in 7H9-OADC medium with 0.2% glucose (blue) or 1% glucose (red). Cultures were shaken at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and MtbΔpknG in stationary phase (5–11 hours) are statistically significant (two-tailed t-test, p<0.05). (C) pknG is required for M. smegmatis biofilm growth. Wild type M. smegmatis, MsΔpknG, and the mutant strains complemented with the M. smegmatis (Ms-pknG) or M. tuberculosis (Mtb-pknG) gene. Pictures were taken after 7 days of static growth at 30°C. Shown images are representatives of biological triplicates. (D) pknG is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔpknG, and the complemented strain were assayed as previously described [10]. Pictures were taken after 6 weeks of growth at a static humidified condition of 37°C and 5% CO2. Shown images are representatives of biological triplicates. (E) Quantitation of biofilm growth of Mtb strains. Biofilms were harvested and quantified as described in Experimental Procedures. Error bars represent standard deviation of biological triplicates (*, p<0.0001; ns, not significant difference between wild type H37Rv and the complemented strain). (F) PknG kinase activity is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔpknG, and the complemented strain were assayed in the absence (-) or presence (+) of 1 mM AX20017, a specific inhibitor of PknG. Pictures were taken after 6 weeks of growth at static humidified condition of 37°C and 5% CO2. Shown images are representatives of biological triplicates.
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Related In: Results  -  Collection

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ppat.1004839.g001: PknG kinase activity is required for biofilm growth in mycobacteria.(A) Role of pknG in M. smegmatis planktonic growth. Wild type M. smegmatis mc2155 (red filled circles), its derived MsΔpknG mutant (blue filled triangles), and the complemented strain MsΔpknG/pknG (green filled squares) were grown in 7H9 medium supplemented with 0.2% glucose with shaking at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and MsΔpknG in stationary phase are not significant. (B) Role of pknG in Mtb planktonic growth. Wild type Mtb H37Rv (open circles) and its derived MtbΔpknG mutant (open triangles) were grown in 7H9-OADC medium with 0.2% glucose (blue) or 1% glucose (red). Cultures were shaken at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and MtbΔpknG in stationary phase (5–11 hours) are statistically significant (two-tailed t-test, p<0.05). (C) pknG is required for M. smegmatis biofilm growth. Wild type M. smegmatis, MsΔpknG, and the mutant strains complemented with the M. smegmatis (Ms-pknG) or M. tuberculosis (Mtb-pknG) gene. Pictures were taken after 7 days of static growth at 30°C. Shown images are representatives of biological triplicates. (D) pknG is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔpknG, and the complemented strain were assayed as previously described [10]. Pictures were taken after 6 weeks of growth at a static humidified condition of 37°C and 5% CO2. Shown images are representatives of biological triplicates. (E) Quantitation of biofilm growth of Mtb strains. Biofilms were harvested and quantified as described in Experimental Procedures. Error bars represent standard deviation of biological triplicates (*, p<0.0001; ns, not significant difference between wild type H37Rv and the complemented strain). (F) PknG kinase activity is required for Mtb biofilm growth. Wild type Mtb H37Rv, MtbΔpknG, and the complemented strain were assayed in the absence (-) or presence (+) of 1 mM AX20017, a specific inhibitor of PknG. Pictures were taken after 6 weeks of growth at static humidified condition of 37°C and 5% CO2. Shown images are representatives of biological triplicates.
Mentions: Studies in other bacteria have established the phenotypic relationship between host persistence, antibiotic resistance and biofilm growth [6,9]. PknG was previously shown to be required for two of these three phenotypes, namely survival in macrophages and resistance to multiple antibiotics [3,4]. In addition, phenotypic characterization of a M. smegmatis ΔpknG mutant revealed profound alterations in surface charge and hydrophobicity of the cell wall (S1 Table) [4], suggesting that PknG might be involved in mycobacterial biofilm growth. Wild type strains of M. smegmatis, M. bovis BCG, and Mtb, together with their derived ΔpknG mutants and complemented strains were assayed for growth in both planktonic cultures and static biofilms (Figs 1 and S1). Similar to M. bovis BCG [3,13], absence of PknG did not affect planktonic growth of M. smegmatis (Fig 1A). However, the MtbΔpknG displayed a slow growth defect in stationary phase (Fig 1B), as previously reported [14]. These phenotypic variations suggest a complexity of PknG function among mycobacterial species that warrants further investigation.

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