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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

Both pknG and its adjacent gene renU are each required for oxidative stress resistance.(A) Alignment of the pknG loci from Mtb and M. smegmatis. renU (previously annotated as mutT3) shares the same intergenic region with the operon encoding pknG. Bar, 1kb. (B) Both pknG and renU are each required for M. smegmatis resistance to H2O2 (left) and diamide (right). Wild type M. smegmatis (circles), MsΔpknG (triangles) and MsΔrenU (squares) were grown in 7H9 medium. At the indicated times (arrows), 10mM H2O2 or 15mM diamide was added. Growth was estimated through optical absorbance at 600 nm (OD600nm). Error bars represent standard deviation of biological triplicates. (C) pknG and renU are each required for Mtb resistance to H2O2 (left) and diamide (right). Wild type Mtb (circles or striped bars), MtbΔpknG (triangles or black filled bars) and MtbΔrenU (squares or grey filled bars) of were grown in 7H9-OADC medium. At the indicated times (arrows), 20 mM H2O2 or 10 mM diamide was added. Growth was estimated through measuring optical absorbance at 600 nm (OD600nm, top) or determining colony forming units (CFU, bottom) by serial dilution plating. Error bars represent standard deviation of biological triplicates.
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ppat.1004839.g002: Both pknG and its adjacent gene renU are each required for oxidative stress resistance.(A) Alignment of the pknG loci from Mtb and M. smegmatis. renU (previously annotated as mutT3) shares the same intergenic region with the operon encoding pknG. Bar, 1kb. (B) Both pknG and renU are each required for M. smegmatis resistance to H2O2 (left) and diamide (right). Wild type M. smegmatis (circles), MsΔpknG (triangles) and MsΔrenU (squares) were grown in 7H9 medium. At the indicated times (arrows), 10mM H2O2 or 15mM diamide was added. Growth was estimated through optical absorbance at 600 nm (OD600nm). Error bars represent standard deviation of biological triplicates. (C) pknG and renU are each required for Mtb resistance to H2O2 (left) and diamide (right). Wild type Mtb (circles or striped bars), MtbΔpknG (triangles or black filled bars) and MtbΔrenU (squares or grey filled bars) of were grown in 7H9-OADC medium. At the indicated times (arrows), 20 mM H2O2 or 10 mM diamide was added. Growth was estimated through measuring optical absorbance at 600 nm (OD600nm, top) or determining colony forming units (CFU, bottom) by serial dilution plating. Error bars represent standard deviation of biological triplicates.

Mentions: Structural studies revealed a rubredoxin-like domain at the N-terminus of PknG, suggesting a possible involvement of this kinase in redox homeostasis [15]. In fact, our in vitro phosphorylation assays supported the hypothesis that PknG kinase activity may be regulated by the redox state of the mycobacterial cytoplasm (S3 Fig). We studied the role of the pknG locus in mycobacterial redox homeostasis. Interestingly, these studies revealed that pknG and a neighboring gene (msmeg_0790/rv0413), previously annotated as mutT3 (Fig 2A), were each required for oxidative stress resistance in M. smegmatis and Mtb (Fig 2B–2C). The deduced amino acid sequences encoded by msmeg_0790 and rv0413 show the motif GX5EX7REUXEEXGU (where U = L,V,I), typical of Nudix (Nucleoside diphosphate linked moiety X) hydrolases [16]. Nudix hydrolases are low molecular weight (MW) “housecleaning” phosphohydrolases that provide control over cellular levels of deleterious metabolic intermediates [16]. The proteins encoded by msmeg_0790 and rv0413 had been wrongly annotated as MutT3 because they were thought to be anti-mutators, prototypical Nudix hydrolases that degrade and prevent misincorporation of 8-oxo-guanosine triphosphate into nucleic acids. However, recent studies showed that msmeg_0790 and rv0413 are not involved in anti-mutation activity [17].


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)

Both pknG and its adjacent gene renU are each required for oxidative stress resistance.(A) Alignment of the pknG loci from Mtb and M. smegmatis. renU (previously annotated as mutT3) shares the same intergenic region with the operon encoding pknG. Bar, 1kb. (B) Both pknG and renU are each required for M. smegmatis resistance to H2O2 (left) and diamide (right). Wild type M. smegmatis (circles), MsΔpknG (triangles) and MsΔrenU (squares) were grown in 7H9 medium. At the indicated times (arrows), 10mM H2O2 or 15mM diamide was added. Growth was estimated through optical absorbance at 600 nm (OD600nm). Error bars represent standard deviation of biological triplicates. (C) pknG and renU are each required for Mtb resistance to H2O2 (left) and diamide (right). Wild type Mtb (circles or striped bars), MtbΔpknG (triangles or black filled bars) and MtbΔrenU (squares or grey filled bars) of were grown in 7H9-OADC medium. At the indicated times (arrows), 20 mM H2O2 or 10 mM diamide was added. Growth was estimated through measuring optical absorbance at 600 nm (OD600nm, top) or determining colony forming units (CFU, bottom) by serial dilution plating. Error bars represent standard deviation of biological triplicates.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004839.g002: Both pknG and its adjacent gene renU are each required for oxidative stress resistance.(A) Alignment of the pknG loci from Mtb and M. smegmatis. renU (previously annotated as mutT3) shares the same intergenic region with the operon encoding pknG. Bar, 1kb. (B) Both pknG and renU are each required for M. smegmatis resistance to H2O2 (left) and diamide (right). Wild type M. smegmatis (circles), MsΔpknG (triangles) and MsΔrenU (squares) were grown in 7H9 medium. At the indicated times (arrows), 10mM H2O2 or 15mM diamide was added. Growth was estimated through optical absorbance at 600 nm (OD600nm). Error bars represent standard deviation of biological triplicates. (C) pknG and renU are each required for Mtb resistance to H2O2 (left) and diamide (right). Wild type Mtb (circles or striped bars), MtbΔpknG (triangles or black filled bars) and MtbΔrenU (squares or grey filled bars) of were grown in 7H9-OADC medium. At the indicated times (arrows), 20 mM H2O2 or 10 mM diamide was added. Growth was estimated through measuring optical absorbance at 600 nm (OD600nm, top) or determining colony forming units (CFU, bottom) by serial dilution plating. Error bars represent standard deviation of biological triplicates.
Mentions: Structural studies revealed a rubredoxin-like domain at the N-terminus of PknG, suggesting a possible involvement of this kinase in redox homeostasis [15]. In fact, our in vitro phosphorylation assays supported the hypothesis that PknG kinase activity may be regulated by the redox state of the mycobacterial cytoplasm (S3 Fig). We studied the role of the pknG locus in mycobacterial redox homeostasis. Interestingly, these studies revealed that pknG and a neighboring gene (msmeg_0790/rv0413), previously annotated as mutT3 (Fig 2A), were each required for oxidative stress resistance in M. smegmatis and Mtb (Fig 2B–2C). The deduced amino acid sequences encoded by msmeg_0790 and rv0413 show the motif GX5EX7REUXEEXGU (where U = L,V,I), typical of Nudix (Nucleoside diphosphate linked moiety X) hydrolases [16]. Nudix hydrolases are low molecular weight (MW) “housecleaning” phosphohydrolases that provide control over cellular levels of deleterious metabolic intermediates [16]. The proteins encoded by msmeg_0790 and rv0413 had been wrongly annotated as MutT3 because they were thought to be anti-mutators, prototypical Nudix hydrolases that degrade and prevent misincorporation of 8-oxo-guanosine triphosphate into nucleic acids. However, recent studies showed that msmeg_0790 and rv0413 are not involved in anti-mutation activity [17].

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