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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

L13, a ribosomal protein associated with RenU, is phosphorylated by PknG.(A) Representative in vitro phosphorylation of RenU.6H preparations purified from M. smegmatis (left) or E. coli (right) by purified PknG. PI, phosphatase inhibitors. (B) In vitro phosphorylation of corresponding fractions eluted from ion exchange columns by PknG. Numbers indicate the NaCl concentrations used in elution buffer. Samples loaded to the ion exchange columns were obtained from an immobilized Cobalt affinity chromatography of M. smegmatis RenU.6H (+) cell lysates or control lysates (-). (C) In vitro phosphorylation of purified 6H.L13 or 6H.SmpB by PknG. (D) Co-purification of L13 from M. smegmatis lysates by exogenous RenU.6H. Another recombinant 6H-tagged protein (6H.SHMT) was used as a control. Blots were detected by Anti-L13 or Anti-6H antibodies. (E) In vitro phosphorylation of recombinant or native L13 protein associated with RenU by PknG kinase activity. (F) In vitro phosphorylation of purified 6H.L13 by M. smegmatis cell lysates, followed by pull-down using Nickel-agarose beads.
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ppat.1004839.g004: L13, a ribosomal protein associated with RenU, is phosphorylated by PknG.(A) Representative in vitro phosphorylation of RenU.6H preparations purified from M. smegmatis (left) or E. coli (right) by purified PknG. PI, phosphatase inhibitors. (B) In vitro phosphorylation of corresponding fractions eluted from ion exchange columns by PknG. Numbers indicate the NaCl concentrations used in elution buffer. Samples loaded to the ion exchange columns were obtained from an immobilized Cobalt affinity chromatography of M. smegmatis RenU.6H (+) cell lysates or control lysates (-). (C) In vitro phosphorylation of purified 6H.L13 or 6H.SmpB by PknG. (D) Co-purification of L13 from M. smegmatis lysates by exogenous RenU.6H. Another recombinant 6H-tagged protein (6H.SHMT) was used as a control. Blots were detected by Anti-L13 or Anti-6H antibodies. (E) In vitro phosphorylation of recombinant or native L13 protein associated with RenU by PknG kinase activity. (F) In vitro phosphorylation of purified 6H.L13 by M. smegmatis cell lysates, followed by pull-down using Nickel-agarose beads.

Mentions: To investigate how PknG and RenU interact, we first tested if PknG phosphorylates RenU. The encoding genes were cloned for expression in M. smegmatis or E. coli as 6xHistidine-(6H) tagged proteins. Purified RenU.6H preparations were subjected to in vitro kinase assays using radioactive [γ-P32]-ATP as the phosphate donor. Whereas the M. smegmatis-derived RenU.6H displayed a protein species phosphorylated by PknG, the E. coli-derived equivalent did not show phosphorylation (Fig 4A). This was unlikely due to contaminated phosphatases because addition of phosphatase inhibitors (PI) did not reverse the phosphorylation pattern (Fig 4A).


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)

L13, a ribosomal protein associated with RenU, is phosphorylated by PknG.(A) Representative in vitro phosphorylation of RenU.6H preparations purified from M. smegmatis (left) or E. coli (right) by purified PknG. PI, phosphatase inhibitors. (B) In vitro phosphorylation of corresponding fractions eluted from ion exchange columns by PknG. Numbers indicate the NaCl concentrations used in elution buffer. Samples loaded to the ion exchange columns were obtained from an immobilized Cobalt affinity chromatography of M. smegmatis RenU.6H (+) cell lysates or control lysates (-). (C) In vitro phosphorylation of purified 6H.L13 or 6H.SmpB by PknG. (D) Co-purification of L13 from M. smegmatis lysates by exogenous RenU.6H. Another recombinant 6H-tagged protein (6H.SHMT) was used as a control. Blots were detected by Anti-L13 or Anti-6H antibodies. (E) In vitro phosphorylation of recombinant or native L13 protein associated with RenU by PknG kinase activity. (F) In vitro phosphorylation of purified 6H.L13 by M. smegmatis cell lysates, followed by pull-down using Nickel-agarose beads.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004839.g004: L13, a ribosomal protein associated with RenU, is phosphorylated by PknG.(A) Representative in vitro phosphorylation of RenU.6H preparations purified from M. smegmatis (left) or E. coli (right) by purified PknG. PI, phosphatase inhibitors. (B) In vitro phosphorylation of corresponding fractions eluted from ion exchange columns by PknG. Numbers indicate the NaCl concentrations used in elution buffer. Samples loaded to the ion exchange columns were obtained from an immobilized Cobalt affinity chromatography of M. smegmatis RenU.6H (+) cell lysates or control lysates (-). (C) In vitro phosphorylation of purified 6H.L13 or 6H.SmpB by PknG. (D) Co-purification of L13 from M. smegmatis lysates by exogenous RenU.6H. Another recombinant 6H-tagged protein (6H.SHMT) was used as a control. Blots were detected by Anti-L13 or Anti-6H antibodies. (E) In vitro phosphorylation of recombinant or native L13 protein associated with RenU by PknG kinase activity. (F) In vitro phosphorylation of purified 6H.L13 by M. smegmatis cell lysates, followed by pull-down using Nickel-agarose beads.
Mentions: To investigate how PknG and RenU interact, we first tested if PknG phosphorylates RenU. The encoding genes were cloned for expression in M. smegmatis or E. coli as 6xHistidine-(6H) tagged proteins. Purified RenU.6H preparations were subjected to in vitro kinase assays using radioactive [γ-P32]-ATP as the phosphate donor. Whereas the M. smegmatis-derived RenU.6H displayed a protein species phosphorylated by PknG, the E. coli-derived equivalent did not show phosphorylation (Fig 4A). This was unlikely due to contaminated phosphatases because addition of phosphatase inhibitors (PI) did not reverse the phosphorylation pattern (Fig 4A).

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