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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-catalyzed phosphorylation of L13 at a mycobacterial specific site, T11, is required for mycobacterial biofilm growth.(A) Sequence alignment of the N-terminal 20 amino acids of L13 proteins from different bacteria. Residues marked with asterisks (T11, T12, and S14) are potential targets of phosphorylation by PknG. (B) Phosphorylation of L13 and its mutants by PknG. In L13(3A), all three residues (T11, T12, and S14) were mutated to alanine. Inhibition was achieved by pre-incubation of PknG in 1 mM AX20017 (AX). (C) Chromatograms confirming wild type and mutant alleles of rplM on M. smegmatis chromosomes. The chromosomal loci were amplified from genomic DNA of M. smegmatis strains by primers that anneal to DNA sequences outside the regions homologous to the allelic exchange substrates, followed by cloning and sequencing. (D) Biofilm of M. smegmatis strains. Similar to MsΔpknG and MsΔrenU, Ms.L13(T11A) exhibited defective biofilm growth, while biofilm of Ms.L13(T11E) was largely identical to wild type. In trans expression of an allele encoding L13(T11E) restored biofilm growth to Ms.L13(T11A) strain. (E) Quantitation of biofilm growth of M. smegmatis strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference. (F) Biofilm of Mtb strains. Similar to MtbΔpknG and MtbΔrenU, Mtb.L13(T11A) exhibited defective biofilm growth while in trans expression of an allele encoding L13(T11E) restored its biofilm growth. Addition of PknG inhibitor AX20017 (+AX) had no effect on the biofilm of the complemented strain. (G) Quantitation of biofilm growth of Mtb strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference.
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ppat.1004839.g005: PknG-catalyzed phosphorylation of L13 at a mycobacterial specific site, T11, is required for mycobacterial biofilm growth.(A) Sequence alignment of the N-terminal 20 amino acids of L13 proteins from different bacteria. Residues marked with asterisks (T11, T12, and S14) are potential targets of phosphorylation by PknG. (B) Phosphorylation of L13 and its mutants by PknG. In L13(3A), all three residues (T11, T12, and S14) were mutated to alanine. Inhibition was achieved by pre-incubation of PknG in 1 mM AX20017 (AX). (C) Chromatograms confirming wild type and mutant alleles of rplM on M. smegmatis chromosomes. The chromosomal loci were amplified from genomic DNA of M. smegmatis strains by primers that anneal to DNA sequences outside the regions homologous to the allelic exchange substrates, followed by cloning and sequencing. (D) Biofilm of M. smegmatis strains. Similar to MsΔpknG and MsΔrenU, Ms.L13(T11A) exhibited defective biofilm growth, while biofilm of Ms.L13(T11E) was largely identical to wild type. In trans expression of an allele encoding L13(T11E) restored biofilm growth to Ms.L13(T11A) strain. (E) Quantitation of biofilm growth of M. smegmatis strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference. (F) Biofilm of Mtb strains. Similar to MtbΔpknG and MtbΔrenU, Mtb.L13(T11A) exhibited defective biofilm growth while in trans expression of an allele encoding L13(T11E) restored its biofilm growth. Addition of PknG inhibitor AX20017 (+AX) had no effect on the biofilm of the complemented strain. (G) Quantitation of biofilm growth of Mtb strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference.

Mentions: L13 proteins, encoded by rplM genes from both M. smegmatis (Fig 4C and 4E) and Mtb (Fig 5), were equally phosphorylated by PknG, indicating that this phosphate transfer reaction serves an identical function in mycobacteria. To identify the specific amino acid residues that are phosphorylated by PknG, Mtb L13 protein purified from E. coli was subjected to a cold kinase assay catalyzed by PknG. L13 was then digested with trypsin and the derived peptides were analyzed by ISL-TOFF mass spectrometry (Taplin Biological Mass Spectrometry Facility, Harvard Medical School). This analysis suggested that one phosphorylated residue was present among the three amino acids closely situated at positions 11–14 of the N-terminus of L13. Among these, T12 is absolutely conserved in all available bacterial L13 protein sequences, whereas T11 and S14 are exclusively conserved across the Mycobacterium genus (Fig 5A, marked with asterisks). We first created a triple mutant of Mtb L13, termed L13(3A), in which all three residues T11, T12 and S14 were mutated to alanine. In vitro phosphorylation assays showed that this mutant protein was no longer phosphorylated by PknG (Fig 5B, lane 5), confirming that the phosphorylated amino acid is among these three residues. Next, mutant L13 proteins with single mutations were made and the purified proteins were individually re-tested in in vitro phosphorylation assays. Whereas L13(T12A) and L13(S14A) mutants were readily phosphorylated (Fig 5B, lanes 7 and 8), L13(T11A) completely failed to be phosphorylated by PknG (Fig 5B, lane 6), similar to the triple mutant L13(3A) (Fig 5B, lane 5). These results indicate that the mycobacterial conserved T11 of L13 is uniquely phosphorylated by PknG.


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-catalyzed phosphorylation of L13 at a mycobacterial specific site, T11, is required for mycobacterial biofilm growth.(A) Sequence alignment of the N-terminal 20 amino acids of L13 proteins from different bacteria. Residues marked with asterisks (T11, T12, and S14) are potential targets of phosphorylation by PknG. (B) Phosphorylation of L13 and its mutants by PknG. In L13(3A), all three residues (T11, T12, and S14) were mutated to alanine. Inhibition was achieved by pre-incubation of PknG in 1 mM AX20017 (AX). (C) Chromatograms confirming wild type and mutant alleles of rplM on M. smegmatis chromosomes. The chromosomal loci were amplified from genomic DNA of M. smegmatis strains by primers that anneal to DNA sequences outside the regions homologous to the allelic exchange substrates, followed by cloning and sequencing. (D) Biofilm of M. smegmatis strains. Similar to MsΔpknG and MsΔrenU, Ms.L13(T11A) exhibited defective biofilm growth, while biofilm of Ms.L13(T11E) was largely identical to wild type. In trans expression of an allele encoding L13(T11E) restored biofilm growth to Ms.L13(T11A) strain. (E) Quantitation of biofilm growth of M. smegmatis strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference. (F) Biofilm of Mtb strains. Similar to MtbΔpknG and MtbΔrenU, Mtb.L13(T11A) exhibited defective biofilm growth while in trans expression of an allele encoding L13(T11E) restored its biofilm growth. Addition of PknG inhibitor AX20017 (+AX) had no effect on the biofilm of the complemented strain. (G) Quantitation of biofilm growth of Mtb strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference.
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ppat.1004839.g005: PknG-catalyzed phosphorylation of L13 at a mycobacterial specific site, T11, is required for mycobacterial biofilm growth.(A) Sequence alignment of the N-terminal 20 amino acids of L13 proteins from different bacteria. Residues marked with asterisks (T11, T12, and S14) are potential targets of phosphorylation by PknG. (B) Phosphorylation of L13 and its mutants by PknG. In L13(3A), all three residues (T11, T12, and S14) were mutated to alanine. Inhibition was achieved by pre-incubation of PknG in 1 mM AX20017 (AX). (C) Chromatograms confirming wild type and mutant alleles of rplM on M. smegmatis chromosomes. The chromosomal loci were amplified from genomic DNA of M. smegmatis strains by primers that anneal to DNA sequences outside the regions homologous to the allelic exchange substrates, followed by cloning and sequencing. (D) Biofilm of M. smegmatis strains. Similar to MsΔpknG and MsΔrenU, Ms.L13(T11A) exhibited defective biofilm growth, while biofilm of Ms.L13(T11E) was largely identical to wild type. In trans expression of an allele encoding L13(T11E) restored biofilm growth to Ms.L13(T11A) strain. (E) Quantitation of biofilm growth of M. smegmatis strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference. (F) Biofilm of Mtb strains. Similar to MtbΔpknG and MtbΔrenU, Mtb.L13(T11A) exhibited defective biofilm growth while in trans expression of an allele encoding L13(T11E) restored its biofilm growth. Addition of PknG inhibitor AX20017 (+AX) had no effect on the biofilm of the complemented strain. (G) Quantitation of biofilm growth of Mtb strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students t-test; ns, not significant difference.
Mentions: L13 proteins, encoded by rplM genes from both M. smegmatis (Fig 4C and 4E) and Mtb (Fig 5), were equally phosphorylated by PknG, indicating that this phosphate transfer reaction serves an identical function in mycobacteria. To identify the specific amino acid residues that are phosphorylated by PknG, Mtb L13 protein purified from E. coli was subjected to a cold kinase assay catalyzed by PknG. L13 was then digested with trypsin and the derived peptides were analyzed by ISL-TOFF mass spectrometry (Taplin Biological Mass Spectrometry Facility, Harvard Medical School). This analysis suggested that one phosphorylated residue was present among the three amino acids closely situated at positions 11–14 of the N-terminus of L13. Among these, T12 is absolutely conserved in all available bacterial L13 protein sequences, whereas T11 and S14 are exclusively conserved across the Mycobacterium genus (Fig 5A, marked with asterisks). We first created a triple mutant of Mtb L13, termed L13(3A), in which all three residues T11, T12 and S14 were mutated to alanine. In vitro phosphorylation assays showed that this mutant protein was no longer phosphorylated by PknG (Fig 5B, lane 5), confirming that the phosphorylated amino acid is among these three residues. Next, mutant L13 proteins with single mutations were made and the purified proteins were individually re-tested in in vitro phosphorylation assays. Whereas L13(T12A) and L13(S14A) mutants were readily phosphorylated (Fig 5B, lanes 7 and 8), L13(T11A) completely failed to be phosphorylated by PknG (Fig 5B, lane 6), similar to the triple mutant L13(3A) (Fig 5B, lane 5). These results indicate that the mycobacterial conserved T11 of L13 is uniquely phosphorylated by PknG.

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