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. Author manuscript; available in PMC: 2016 Mar 19.
Published in final edited form as: Mol Cell. 2015 Feb 26;57(6):984–994. doi: 10.1016/j.molcel.2015.01.024

Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide

Marie I Samanovic 1, Shengjiang Tu 2, Ondřej Novák 3, Lakshminarayan M Iyer 4, Fiona E McAllister 5, L Aravind 4, Steven P Gygi 5, Stevan R Hubbard 6, Miroslav Strnad 3, K Heran Darwin 1,*
PMCID: PMC4369403  NIHMSID: NIHMS657975  PMID: 25728768

Summary

One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO-resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homologue of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report for the first time that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO.

Introduction

Mycobacterium tuberculosis (M. tuberculosis) is one of the world’s most devastating pathogens, killing nearly 1.5 million people yearly (WHO, 2014). Although many people are infected with this bacterium most are able to suppress its growth unless immunity wanes for reasons including old age and immunosuppression due to human immunodeficiency virus infection. M. tuberculosis resides in macrophages where the release of nitric oxide (NO) by the inducible NO synthase acts as a powerful anti-mycobacterial defense system (Chan et al., 1995; Chan et al., 1992; MacMicking et al., 1997). NO has the potential to damage nucleic acids, lipids and proteins as well as displace metal co-factors from essential metabolic enzymes [reviewed in (Bowman et al., 2011; Shiloh and Nathan, 2000)]. Despite the production of this important innate immune defense, M. tuberculosis remains an incredibly successful pathogen.

In order to understand how M. tuberculosis persists despite the macrophage production of NO, Carl Nathan’s laboratory performed a screen for pathways that protect against NO-mediated toxicity, and identified genes required for proteasome-dependent protein degradation in M. tuberculosis (Darwin et al., 2003). Proteasomes are barrel-shaped compartmentalized proteases that degrade proteins in a highly regulated manner [reviewed in (Schmidt and Finley, 2014; Tomko and Hochstrasser, 2013)]. In eukaryotes, the best-characterized substrates of the proteasome are post-translationally modified with the small protein ubiquitin [reviewed in (Komander and Rape, 2012)]. In M. tuberculosis, most known substrates are modified with the small protein Pup (prokaryotic ubiquitin-like protein) (Pearce et al., 2008). Although functionally analogous to ubiquitylation, the biochemistry of pupylation is strikingly different. In M. tuberculosis, Pup is translated with a carboxyl-terminal glutamine that must be converted to glutamate by the enzyme Dop (deamidase of Pup) prior to substrate attachment by the only known Pup ligase PafA (proteasome accessory factor A) (Striebel et al., 2009). Pup then interacts with the hexameric ATPase Mpa (mycobacterial proteasome ATPase), which translocates doomed proteins into proteasome core proteases for degradation (Pearce et al., 2008; Striebel et al., 2009; Wang et al., 2010). In addition to the deamidation of Pup, Dop can also remove Pup from substrates prior to degradation (Burns et al., 2010; Cerda-Maira et al., 2010; Delley et al., 2012; Imkamp et al., 2010). Mutations in dop, pafA, mpa or the genes encoding the M. tuberculosis 20S proteasome core protease (prcBA) sensitize bacteria to NO and attenuate bacterial growth in vivo, making the Pup-proteasome system (PPS) an attractive target for developing new tuberculosis therapeutics (Cerda-Maira et al., 2010; Darwin et al., 2003; Gandotra et al., 2010; Gandotra et al., 2007; Lamichhane et al., 2006; Lin et al., 2009). However, despite recent progress in characterizing the biochemistry of the mycobacterial PPS, the identification of the “pupylome” (Festa et al., 2010), and the transcriptional analysis of PPS mutants (Festa et al., 2011), the mechanism whereby proteolysis protects M. tuberculosis against NO stress had not been elucidated.

In this study, we performed an unbiased, genetic screen using an mpa null mutant to identify mutations that could suppress the NO-hypersensitivity phenotype of this strain. We found that disruption of Rv1205 suppressed the NO-sensitive phenotype of an mpa mutant. In addition, the Rv1205 mutation partially rescued the defective growth of the mpa mutant in mice. We found that Rv1205 is a new Pup-proteasome substrate, the accumulation of which leads to NO sensitivity. We determined that Rv1205 is homologous to the plant enzyme LONELY GUY (LOG), which is a phosphoribohydrolase that catalyzes the final step in the production of cytokinins. Cytokinins are plant hormones that are crucial to the development of plants. Not only did Rv1205 have LOG activity, we found that M. tuberculosis secreted several cytokinins. Using an unbiased metabolomics analysis, we determined that the accumulation of cytokinin breakdown products was likely responsible for the sensitization of M. tuberculosis PPS mutants to NO. Thus our data finally propose a mechanistic model for how proteasome activity can minimize the damaging effects of NO in M. tuberculosis.

Results

A suppressor screen identifies mutations that restore NO resistance to a proteasomal-degradation deficient M. tuberculosis strain

To elucidate the link between the PPS and NO resistance, we screened for genetic suppressors of the NO-hypersensitivity phenotype of a Δmpa::hyg mutant. We made 18 ΦMycoMarT7 transposon (Sassetti et al., 2001) mutant pools resulting in ~72,000 double mutants. Mutant pools were exposed to acidified nitrite, a source of NO, to enrich for NO-resistant clones. Three of the 18 pools yielded bacteria that survived two rounds of acidified nitrite exposure and 360 clones were tested individually for NO resistance. After Southern blot analysis of 62 representative clones with increased NO resistance, we identified three allelic groups based on their BamHI restriction patterns (not shown). We identified the transposon insertion sites (Figure 1A) and quantified NO resistance of four representative mutants (Figure 1B). We complemented all of the suppressor mutations in single copy, resulting in the (re)sensitization of the mutants to NO (Figure 1B). An integrated plasmid encoding Rv1205 under the control of its native promoter complemented the sup1 and sup2 strains. Strain sup3 had a mutation between the divergently expressed Rv2699c and Rv2700 genes and was complemented by Rv2700 but not Rv2699c. Rv2700 encodes a putative secreted protein with a predicted lipid-binding domain related to TraT, LytR and CpsA (Pfam: PF03816) and is predicted to be essential for optimal growth in vitro (Sassetti et al., 2003); however, the sup3 mutant grew normally under standard conditions (Figure 1C). Mutant sup4 was complemented by glpK, which encodes a probable glycerol kinase, and was the only mutant with a growth defect under normal culture conditions (Figure 1C). We also tested a strain with a transposon mutation in Rv1205 in an mpa+ background; this strain exhibited wild type (wt) NO resistance (Figure 1D), suggesting that disruption of Rv1205 alone does not cause NO hyper-resistance under the conditions tested.

Figure 1. Identification of NO-resistant suppressor mutants.

Figure 1

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(A) Genomic regions surrounding transposon insertions that suppressed the NO-sensitive phenotype of a Δmpa::hyg mutant. Black arrows on triangles represent the direction of neo expression in the transposon. Gene data are from http://genolist.pasteur.fr/TubercuList/. (B) NO susceptibility and complementation of suppressor mutants. All strains harbor an integrated pMV306.strep vector with or without the indicated gene. (−) indicates empty vector. Colony forming units (CFU) of surviving bacteria after exposure to acidified media without (grey bars) or with (black bars) NaNO2. Open bars show input CFU. Data show means ± standard deviation (SD), n = 3, from one of three independent experiments with similar results. All samples exposed to NaNO2 were compared to wt with two-tailed Student’s t-test; * p < 0.05; ** p < 0.01; ns: not statistically significantly different. (C) Growth curves comparing wt, mpa and sup1–4 strains in 7H9 broth. (D) NO susceptibility assay as described in (B) of the Rv1205 mutant and the complemented strain. (E) and (F) Disruption of Rv1205 partially restored virulence to an mpa mutant. Bacterial CFU after infection of wt C57BL6/J mice with wt, mpa, sup1 and Rv1205 strains. CFU from the lungs (E) and spleens (F) from two independent experiments at days 1 (n = 6), 21 (n = 8) and 49 (n = 8). Data were analyzed for statistical significance by a two-way ANOVA followed by a Bonferroni post-test. For spleens: day 21 CFU were not significantly different; day 49, p <0.05 between mpa and sup1. For lungs: day 21 and day 49 p <0.001 and p <0.01, respectively, between mpa and sup1.

PPS defective M. tuberculosis strains are highly attenuated in mice, partly due to their increased sensitivity to NO (Darwin et al., 2003). In addition to protecting against NO toxicity, the M. tuberculosis proteasome is required for the regulation of several metabolic pathways (Festa et al., 2010), including a copper homeostasis system that is essential for the full virulence of M. tuberculosis (Festa et al., 2011; Shi et al., 2014). Notwithstanding the pleiotropic role of Mpa and the proteasome in M. tuberculosis physiology, we tested if disruption of Rv1205 could reverse the attenuated phenotype of an mpa mutant in mice. We selected Rv1205 because it was identified twice in our screen and had a potentially interesting enzymatic activity that had not yet been described in M. tuberculosis (to be discussed below). We infected wt C57BL6/J mice by the aerosol inhalation route with wt, Δmpa::hyg, Rv1205::ΦMycoMarT7 or sup1 strains. The disruption mutation in Rv1205 partially restored the growth defect in the lungs and spleens of mice caused by the Δmpa::hyg mutation (Figures 1E and 1F). The Rv1205 single mutant was as virulent as the wt strain at the observed time points, suggesting that Rv1205 activity is not required for virulence under the conditions tested. In one experiment, the Rv1205 mutant grew to higher numbers in the spleens compared to wt M. tuberculosis; however, this effect was not observed in both experiments.

Rv1205 is a newly identified pupylated proteasome substrate

At this point, we hypothesized that Rv1205 protein accumulated in the mpa mutant, resulting in NO sensitivity. We tested if Rv1205, or the other genes identified in the suppressor screen, encoded proteasome substrates. Rv1205, Rv2700 and glpK had not previously been identified to encode PPS substrates (Festa et al., 2010; Poulsen et al., 2010; Watrous et al., 2010) and none of these genes was differentially expressed in PPS mutants under routine culture conditions (Festa et al., 2011). We raised antibodies against hexahistidine-tagged Rv1205 and GlpK to determine if either accumulated in PPS-defective strains; attempts to produce recombinant Rv2700 have so far been unsuccessful. GlpK did not accumulate in mutants lacking the proteasome ATPase (mpa), the Pup ligase (pafA) or the proteasome core protease (prcBA) genes (Figure 2A). In contrast, Rv1205 accumulated in all three strains (Figure 2B). Rv1205 transcript levels were unchanged between the wt and mpa strains (Figure 2C), demonstrating that the accumulation of wt Rv1205 protein in the mpa, pafA and prcBA mutants was likely due to failed degradation, and not due to increased transcription of the Rv1205 gene. We pupylated Rv1205-His6 in vitro and found that Pup modified lysine 74 (Figure 2D and Figure S1). Mutagenesis of lysine 74 to alanine (Rv1205K74A) abrogated Rv1205-His6 pupylation in vitro (Figure 2D). Importantly, Rv1205K74A accumulated in M. tuberculosis with a functional proteasome system (Figure 2E, far right). Thus, Rv1205 appears to be a newly discovered substrate of the M. tuberculosis PPS.

Figure 2. Rv1205 is a newly identified proteasome substrate.

Figure 2

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(A) Immunoblot for GlpK in total cell lysates of wt, prcBA, pafA, mpa, and sup4 strains. (B) Immunoblot for Rv1205 (20 kD) in total cell lysates of wt, mpa, pafA, and prcBA strains. (C) Quantitative-real time-PCR (qRTPCR) shows Rv1205 transcripts were unchanged in the mpa mutant when compared with wt M. tuberculosis. Fold changes relative to wt M. tuberculosis are plotted on the y-axis. This experiment represents one biological replicate performed in triplicate. Data show means ± SD. dlaT (dihydrolipoamide acyltransferase) was used as a control for a gene whose expression does not change in PPS mutants. (D) In vitro pupylation assay of recombinant histidine-tagged Rv1205 and Rv1205K74A. See also Figure S1. Proteins were detected with polyclonal antibodies to Rv1205-His6. (E) Immunoblot for Rv1205 in total cell lysates of wt, mpa, and Rv1205 strains harboring integrated vector with or without the Rv1205 gene. (−) indicates empty vector. The loading control for all immunoblots is DlaT. For all panels the molecular weight (MW) markers are indicated on the left. The asterisk represents a cross-reactive protein in panels (B) and (E).

Rv1205 is a homologue of the plant cytokinin-activating enzyme LONELY GUY

Rv1205 function had not been previously characterized, but is annotated as a possible lysine decarboxylase (LDC) (Pfam: PF03641). However, we detected no LDC activity (L-lysine conversion to cadaverine) with Rv1205-His6 under conditions in which Escherichia coli (E. coli) LDC was strongly active (not shown). However, using sequence profile searches with the PSI-BLAST and JACKHMMER programs we established that Rv1205 is related to the rice Oryza sativa protein LONELY GUY (LOG) (e-value = 10−24 in iteration 2), an enzyme that catalyzes the hydrolytic removal of ribose 5′-monophosphate from nitrogen (N)6-modified adenosines, the final step of bioactive cytokinin synthesis (Figure 3A) (Kurakawa et al., 2007). Furthermore, these searches, as well as profile-profile comparisons with HHpred (Soding et al., 2005), revealed no significant relationship between LOG family members and the LDC superfamily. The crystal structure of the Rv1205 orthologue in the close M. tuberculosis relative Mycobacterium marinum (M. marinum) was previously crystallized (PDB ID: 3SBX) in an effort to characterize mycobacterial proteins of unknown function by the Seattle Structural Genomics Center for Infectious Disease. HHPred analysis revealed that this protein, MMAR_4233, looks virtually identical to Arabidopsis thaliana (A. thaliana) LOG (Figure 3B).

Figure 3. Rv1205 is a homologue of LOG.

Figure 3

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(A) LOG activity and structures of select cytokinins. R = isoprene-derived or aromatic side chain. iP: isopentenyladenine; tZ: trans-zeatin. p-topolin: para-topolin. (B) Comparison of the crystal structures of proteins from M. marinum (left, PDB code 3SBX) and A. thaliana (right, PDB code 1YDH). The two subunits in the homodimers are colored green and yellow. AMP molecules in the M. marinum structure are shown in stick representation with carbon atoms colored orange, oxygen atoms red, nitrogen atoms blue, and phosphorus atoms black. (C) Phylogenetic analysis of LOG-like genes. See also Figure S2. Clades with bootstrap values greater than 90% have a red circle. Well-supported monophyletic clades are shown as filled triangles. Predicted operons and domain architectures are illustrated to the right. Names of the LOG-like genes are indicated below.

Cytokinins are hormones that are critical for plant development [reviewed in (Argueso et al., 2009)] and are also produced by bacterial phytopathogens and phytosymbionts to promote their growth in plants [reviewed in (Frebort et al., 2011)]. Cytokinins are adenine derivatives that usually have either an isoprene-derived or aromatic side chain at the N6 position. In plants, isoprenoid cytokinins [N6-(Δ2-isopentenyl) adenine or isopentenyladenine (iP); trans-zeatin (tZ); cis-zeatin (cZ) and dihydrozeatin (DHZ)] are more typically observed and found in greater abundance than aromatic cytokinins (meta-, ortho- and para-topolins) [reviewed in (Sakakibara, 2006)]. For cytokinin synthesis, two pathways have been described: the direct de novo biosynthesis of free cytokinins (prenylated adenylic nucleotides) and the salvage of cytokinins from transfer RNAs (tRNAs), which typically have prenyl modifications [reviewed in (Kamada-Nobusada and Sakakibara, 2009)].

Some phytobacteria deploy cytokinins synthesized by dedicated operons, which include a LOG-like gene, against their plant hosts. We observed LOG homologues in all major lineages of prokaryotes (Figure 3C and Figure S2), but not in species such as E. coli and most obligate intracellular pathogens with reduced genomes. Several Actinobacteria, but not Mycobacteria, contain two adjacent LOG-like genes. The presence of LOG-like genes often correlates with the presence of miaA, a gene involved in the prenylation of a highly conserved adenine at the 3′ end of tRNA anticodons that facilitates translational fidelity (Neidhardt, 1996; Urbonavicius et al., 2001). LOG-like genes can also be found in operons with mazG in several bacterial species (e.g. M. marinum). MazG proteins, which have pyrophosphatase activity, could potentially hydrolyze N6-prenylated ATP to generate N6-prenylated adenosine monophosphate (AMP), a LOG-like enzyme substrate. In some species, LOG domains are fused to a MutT/Nudix-type hydrolase, which could also possibly catalyze pyrophosphate hydrolysis of nucleotide triphosphates to create LOG substrates (McLennan, 2006).

Rv1205 is a phosphoribohydrolase that produces cytokinins

We next determined if M. tuberculosis produced cytokinins, an activity that had never been reported before in a mammalian pathogen or symbiont. Using a mass-spectrophotometric protocol developed specifically for the analysis of plant cytokinins, we quantified several cytokinins and their derivatives in the supernatants and cellular extracts of M. tuberculosis cultures (Table 1 and Table S2, Figure S3). iP and 2-methylthio-iP (2MeS-iP) were the most abundant cytokinins detected in M. tuberculosis lysates and supernatants (Table 1, cytokinins highlighted in yellow; Figure S3). Importantly, we observed a significant accumulation of 2MeS-iP in the mpa mutant supernatants with a concomitant reduction of the dephosphorylated form (2MeS-iPR) of its presumed precursor (2MeS-iPRMP) in the cell pellet (Table 1). These results were consistent with the notion that the mpa mutant had more Rv1205 protein and thus more LOG-like activity. It is notable that the cytokinin ribosides (iPR and 2MeS-iPR) were more apparent than the nucleotides (iPRMP or 2MeS-iPRMP, which was undetectable). Although it is the nucleotide forms (Table 1, highlighted in blue) that are substrates of LOG-like enzymes, we believe that these are rapidly dephosphorylated into the riboside forms and thus both nucleosides and nucleotides could be considered cytokinin precursors. Thus, when one considers both the phosphorylated and unphosphorylated ribosides in total, it is apparent that these are reduced in the mpa mutant. We did not observe accumulation of the cytokinin iP in the mpa strain samples as we would have expected; however, iP may possibly be turned over upon accumulation, or rapidly modified to make 2MeS-iP, which accumulates in the mpa strain as one might expect.

Table 1. Quantification of cytokinins in M. tuberculosis.

Cytokinin and cytokinin precursor levels in the supernatants of M. tuberculosis culture (“MEDIA”) and in 1 g (wet weight) of cells (“LYSATE”) grown to an OD580 = 1. Cytokinins are highlighted in yellow and LOG-like enzyme substrates are highlighted in blue. Ribosides are likely dephosphorylated LOG-like substrates. See also Table S2 and Figure S3.

LYSATE wt mpa sup1 Rv1205 sup1 + Rv1205D120A,E121A
iP 121.09 ± 21.88 89.66 ± 6.75 30.17 ± 4.01 ** 27.11 ± 3.33 ** 21.65 ± 1.23 **
iPR 125.81 ± 7.55 39.12 ± 8.23 *** 822.85 ± 177.87 ** 983.59 ± 169.25 ** 352.49 ± 76.72 *
iPRMP 298.74 ± 49.81 185.72 ± 18.68 * 3595.85 ± 640.44 ** 2021.84 ± 319.05 ** 1750.04 ± 379.28 **
2MeS-iP 11916.7 ± 1620.4 12130.6 ± 1091.8 183.7 ± 59.1 *** 141.5 ± 38.6 *** 222.8 ± 75.0 ***
2MeS-iPR 19895.9 ± 2441.2 8164.8 ± 195.8 ** 29614.2 ± 5138.7 40724.0 ± 6622.2 * 29187.0 ± 2727.8 *
tZ 1.33 ± 0.24 1.23 ± 0.19 0.65 ± 0.17 * 3.60 ± 0.91 * 2.48 ± 0.24 **
tZR 0.71 ± 0.16 0.95 ± 0.19 0.92 ± 0.10 1.56 ± 0.31 * 0.79 ± 0.05
2MeS-tZR 49.28 ± 10.46 89.20 ± 30.49 58.69 ± 15.89 33.72 ± 11.48 55.01 ± 4.27
MEDIA wt mpa sup1 sup1 + Rv1205D120A,E121A
iP 234.39 ± 18.03 258.44 ± 36.17 7.8 ± 1.7 *** 10.57 ± 1.49 ***
iPR 47.52 ± 5.65 10.92 ± 1.00 *** 121.10 ± 20.06 ** 260.18 ± 24.66 ***
iPRMP 11.15 ± 1.17 9.29 ± 0.27 10.05 ± 0.31 6.59 ± 0.46 **
2MeS-iP 6184.89 ± 191.77 10293.56 ± 1441.70 * 61.4 ± 15.8 *** 17.51 ± 0.55 ***
2MeS-iPR 5830.93 ± 141.05 3013.1 ± 523.0 ** 10243.2 ± 1671.2 * 12354.6 ± 1616.2 **
tZ 0.16 ± 0.04 0.145 ± 0.038 0.015 ± 0.003 ** 0.029 ± 0.004 **
tZR 1.36 ± 0.30 1.677 ± 0.356 1.758 ± 0.450 <LOD
2MeS-tZR 86.19 ± 6.08 40.175 ± 4.706 ** 132.855 ± 17.364 * 54.042 ± 15.142 *
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Values are in pmol/l; mean ± SD, n=3. Statistically significant values are indicated in bold. Asterisks indicate statistically significant difference in mutant lines versus wt with an ANOVA analysis (*, **, and *** correspond to p-values of 0.05 > p > 0.01, 0.01 > p > 0.001, and p < 0.001, respectively). LOD: limit of detection, defined as a signal to noise ratio of 3:1. iP: isopentenyladenine; iPR: isopentenyladenosine; iPRMP: isopentenyladenosine monophosphate; 2MeS-iP: 2-methylthio-isopentenyladenine; 2MeS-iPR: 2-methylthio-isopentenyladenosine; tZ: trans-zeatin; tZR: trans-zeatin-riboside; 2MeS-tZR: 2-methylthio-trans-zeatin-riboside.

As expected with strains lacking Rv1205, we observed a significant reduction in the amount of several cytokinins. iP levels were almost 30 times lower in the sup1 and Rv1205 mutant supernatants, along with a corresponding increase in the concentration of the cytokinin precursors (e.g. iPRMP increased in the Rv1205 mutants almost 10-fold in cell lysates). Remarkably, the level of 2MeS-iP was reduced by almost two orders of magnitude in strains lacking Rv1205. It was notable that in contrast to iP and 2MeS-iP, tZ, cZ, DHZ and their derivatives, which are important cytokinins in the plants, did not follow Rv1205 protein levels, suggesting possible alternative pathways for the biosynthesis of these cytokinins in M. tuberculosis.

Using a high performance liquid chromatography (HPLC)-based assay, we tested Rv1205-His6 for LOG activity and found that it hydrolyzed two different commercially available cytokinin nucleoside monophosphates: iP and tz (Figure 4A). Rv1205 had a KM = 5.6 μM, kcat = 434.3 min−1 and kcat/KM = 7.8 × 107 min−1M−1 for iPRMP (Figure 4B). As controls, Rv1205 did not hydrolyze adenosine triphosphate (ATP) (Figure 4A) and hydrolyzed AMP slowly with a KM = 73.1 μM, kcat = 2.3 min−1 and kcat/KM = 3.1 × 104 min−1M−1, suggesting neither was a natural substrate of this enzyme (Figure 4C).

Figure 4. Rv1205 is a phosphoribohydrolase.

Figure 4

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(A) Rv1205-His6 phosphoribohydrolase activity towards the substrates iPRMP and tZRMP as measured by HPLC, (means ± SD, n = 3). ATP and AMP were used as controls. (B) and (C) Determination of Rv1205 kinetic parameters for iPRMP (B) and AMP (C) conversion to iP and adenine, respectively. (D) Three-dimensional model of Rv1205 constructed using SWISS-MODEL (Arnold et al., 2006) based on M. marinum PDB ID: 3SBX, which has a sequence identity of 84% to Rv1205. The viewing angle is the same as in Figure 3B. The ribbon diagram is centered on the presumed active site with iPRMP placed according to the position of AMP in the M. marinum structure. Rv1205 is predicted to be a homodimer (subunits are colored green and yellow). Selected side chains are shown in stick representation. Atom colors: carbon in Rv1205 (green) or iPRMP (orange), oxygen (red), nitrogen (blue), sulfur (yellow) and phosphorus in iPRMP (black). (---) indicates potential hydrogen bonding. (E) Separation by native gel electrophoresis of recombinant wt or mutant Rv1205-His6 proteins. (F) Activity of point mutant alleles of Rv1205-His6 with indicated substrates. See also Figure S4. Data show means ± SD, n = 3. iPRMP: isopentenyladenosine monophosphate; iP: isopentenyladenine; tZRMP: trans-zeatin-riboside monophosphate; tZ: trans-zeatin; ATP: adenosine triphosphate and AMP: adenosine monophosphate.

Based on the crystal structure of the M. marinum protein, we mutated several conserved residues in Rv1205 that were predicted to coordinate or hydrolyze cytokinin precursors and tested the activity of the mutant proteins (Figure 4D). Although we cannot definitively be certain that the proteins were properly folded, all five mutant proteins were as soluble as the wt protein dimer and each migrated similarly as wt Rv1205 in native gels (Figure 4E). Mutagenesis of the conserved residues significantly reduced phosphoribohydrolase activity (Figure 4F, Figure S4). Additionally, Rv1205 with mutations at aspartate 120 and glutamate 121 (D120A E121A) failed to restore cytokinin secretion in the mpa Rv1205 (sup1) mutant (Table 1). Based on the strong conservation among different species, we predict glutamate 121 is a key catalytic residue of Rv1205 (Figure 4D).

We also performed NO-susceptibility assays on the sup1 strain transformed with the mutant Rv1205 alleles and found that the mpa mutant sensitivity to NO was linked to functional Rv1205 activity (Figure 5).

Figure 5. Site-directed mutagenesis of Rv1205 abrogates NO sensitization in M. tuberculosis.

Figure 5

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Point mutant alleles of Rv1205 could not fully restore NO-sensitivity to the sup1 mutant. Data show mean ± SD, n = 9 (upper panel). All samples exposed to NaNO2 were compared to wt with a two-tailed Student’s t-test and statistical differences are indicated with * p < 0.05; *** p < 0.001. ns: not statistically significantly different. Samples showing statistical difference to wt were also compared to mpa and significant differences are similarly indicated; these data suggest partial complementation. See also Figure S5. Immunoblot for Rv1205 (20 kD) in total cell lysates of indicated strains. DlaT is the loading control. MW markers are indicated on the right. The asterisk represents a cross-reactive protein. A: alanine; M: methionine; R: arginine; D: aspartate; E: glutamate; T: threonine.

In Figure 2B we showed that Rv1205 accumulated in PPS mutants and mutagenesis of Rv1205 Lys74 to Ala resulted in stabilization of the protein (Figure 2E). We tested the NO-susceptibility of this strain and found that it was significantly more sensitive to NO than wt bacteria, but not as sensitive as an mpa strain (Figure S5A). We measured phosphoribohydrolase activity of Rv1205K74A and found this protein had drastically reduced activity (Figure S5B), possibly explaining the lack of a robust NO-sensitization phenotype. We also tried to over-produce Rv1205 in wt bacteria with the overexpression plasmid pOLYG (Garbe et al., 1994), however, this was toxic to the bacteria and we could not use this strain in an NO assay (data not shown).

Because Rv1205 is a cytokinin-producing phosphoribohydrolase that is highly similar to plant LOG, we hereafter refer to Rv1205 and its mycobacterial orthologues as “Log”.

Accumulation of aldehydes leads to NO sensitivity

To gain some understanding of the impact of increased cytokinin levels in the mpa mutant, we examined the metabolites present in cell extracts of the wt, mpa, sup1 (mpa log) and log strains. We used a global screening approach and detected a total of 220 metabolites (155 compounds of known identity and 65 compounds of unknown structural identity), and examined metabolites that differed significantly between experimental groups (See Table S3 for statistical summary). The strongest observed change in the mpa strain relative to the wt strain was the accumulation of para-hydroxybenzaldehyde (pHBA) (Figure 6A and Table S4). pHBA can form via several pathways, including the enzymatic degradation of the aromatic cytokinin p-topolin by cytokinin oxidases (Popelkova et al., 2006). Aromatic cytokinins (meta-, ortho- and para-topolins) and their precursors were also detected in our M. tuberculosis lysates and supernatants analysis, but with levels near the limit of detection of our assay (data not shown). Using a spectrophotometric assay for the detection of aldehyde formation, we detected conversion of exogenously added p-topolin into pHBA in M. tuberculosis cell extracts, suggesting cytokinin oxidase activity is present in M. tuberculosis (Figure 6B).

Figure 6. Aldehyde accumulation in M. tuberculosis causes NO sensitivity.

Figure 6

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(A) Metabolomics analysis revealed several molecules at elevated levels in an mpa mutant. Results of relevant compounds from cell lysates of wt, mpa, sup1 and log M. tuberculosis strains (n = 4). HODE: hydroxy octadecadienoic acid. See also Tables S3 and S4. (B) Detection of pHBA, a degradation product of p-topolin, in M. tuberculosis lysates. Absorption spectra of the 4-aminophenol assay, which detects aldehyde release, are shown. p -topolin without M. tuberculosis lysate ( Inline graphic), 500 μM p-topolin with M. tuberculosis lysate (●), and 300 μM pHBA with M. tuberculosis lysate was used as a positive pHBA control (▲). M. tuberculosis lysate was used as a blank for the latter two experiments. (C) pHBA sensitized wt M. tuberculosis to NO in a dose-responsive manner. Data show mean ± SD, n = 3. (D) 2-methyl-3-butanal sensitized wt M. tuberculosis to NO in a dose-responsive manner. Data show mean ± SD, n = 3. Also See Figure S6.

In addition to pHBA, we observed a significant accumulation of several fatty acids (linoleate, palmitoleate, margarate, and oleate) and lipid peroxidation products (9-hydroxy-10,12-octadecadienoic acid, 9-HODE; and 13-hydroxy-9,11-octadecadienoic acid, 13-HODE) in the mpa mutant (Figure 6A). Disruption of log in the mpa mutant (sup1) restored wt levels of all of these metabolites as well as pHBA.

We next tested if exogenously added pHBA could kill M. tuberculosis in the presence or absence of NO. We found that in a dose-dependent manner, micromolar concentrations of pHBA dramatically killed wt M. tuberculosis in acidified nitrite but not in acidic media alone or acidified nitrate (Figure 6C, Figure S6). Increased nitrite sensitivity due to pHBA was also observed for M smegmatis but not for E. coli (Figure S6).

The breakdown of all cytokinins results in the appearance of adenine and an aldehyde derived from the N6 position of the adenine base (Frebort et al., 2011). Metabolomics analysis did not reveal aldehydes other than pHBA; however, the other aldehydes of cytokinin degradation are highly unstable. iP and 2MeS-iP were among the most abundant cytokinins observed in M. tuberculosis culture supernatants therefore we tested if the aldehyde breakdown product of these two molecules, 2-methyl-3-butenal, could sensitize M. tuberculosis to NO. While 2-methyl-3-butenal was highly volatile, it could nonetheless exacerbate killing of M tuberculosis by NO (Figure 6D). Thus it appears that at least two different cytokinin breakdown products could synergize with NO to kill M. tuberculosis.

Discussion

In this work we determined how the PPS protects M. tuberculosis against NO by an unbiased, genetic approach. We identified four independent mutants, two with insertions in Rv1205 or log. We showed that disruption of log in a proteasome-defective strain not only restored NO resistance to wt levels but also restored a significant amount of bacterial growth in mice. We further established that Log is a pupylated proteasome substrate that accumulates in proteasome degradation-defective strains. Log has high structural similarity to the plant enzyme LOG and has the same phosphoribohydrolase activity as plant LOGs, which convert N6-modified adenosine monophosphate derivatives into cytokinin free bases. Using metabolomics we found the accumulation of at least one cytokinin breakdown product, pHBA, in an mpa mutant. We determined that pHBA and another aldehyde product of cytokinin metabolism synergize with NO to kill M. tuberculosis. Aldehydes are known to target membranes (Schauenstein et al., 1977), which might explain the lipid peroxidation observed in our metabolomics analysis. Furthermore, NO and other reactive nitrogen intermediates could possibly exacerbate this process. Although we cannot rule out that the accumulation of active Log has additional effects that result in NO-sensitivity, our data currently support a model where the accumulation of aldehydes likely contributes to the NO-hypersusceptibility phenotype of PPS mutants. Thus our work suggests that the accumulation of a single protein, rather than bulk protein damage, is sufficient to sensitize proteasome-defective M. tuberculosis to NO. Importantly, this work demonstrates for the first time that a mammalian bacterial pathogen produces cytokinins. This observation is remarkable when one considers M. tuberculosis is never found in an environmental reservoir where it could encounter plants.

The M. tuberculosis proteasome was discovered in an effort to understand how this human-exclusive pathogen persists in the face of a robust immune response, which includes the production of NO. Although several groups purified and identified pupylated proteins in M. tuberculosis and M. smegmatis (Festa et al., 2010; Poulsen et al., 2010; Watrous et al., 2010), none of the studies was able to link a particular proteasome substrate to NO resistance. Incredibly, none of the proteomics approaches identified Log, presumably because under normal conditions Log must be kept at very low levels, as evidenced in our immunoblots where we could barely detect endogenous Log in wt M. tuberculosis (Figure 2B and 2E).

A log homologue was previously detected in a screen for M. marinum genes specifically expressed in the granulomas of infected frogs, but not in cultured macrophages (Ramakrishnan et al., 2000). This suggests that Log has a role during specific phases of mycobacterial infections that have yet to be determined. Another small adenosine-derived molecule (1-tuberculosinyladenosine or 1-TbAd) was recently discovered in M. tuberculosis with a probable involvement in virulence (Layre et al., 2014). However, to date there are no data suggesting that 1-TbAd is linked to cytokinin biosynthesis, and 1-TbAd levels were similar among our strains (Table S4).

The function of cytokinins in M. tuberculosis is unknown. In plants, cytokinins regulate numerous developmental processes and responses to the environment (Sakakibara, 2006) as well as promote defense against pathogens (Choi et al., 2011). Conversely, cytokinin production allows several phytopathogens to redirect plant development and nutrient distribution to facilitate microbial growth. The Actinomycete Rhodococcus fascians, a distant relative of M. tuberculosis, induces plant pathology via the local and persistent secretion of cytokinins (Pertry et al., 2009). Why would M. tuberculosis, a human-adapted pathogen, produce cytokinins? We speculate that cytokinins may be used as signalling molecules to communicate amongst mycobacteria and/or have an impact on the host to help bacteria establish a successful infection.

Finally, it is notable that M. tuberculosis produces Log and tightly controls its levels post-translationally, supporting the notion that the untimely presence of Log is detrimental for the lifestyle of this pathogen. Other bacterial species, in particular important animal pathogens like Staphylococcus aureus and Bordetella species harbor LOG homologues (Figure 3C). Therefore, it will be interesting to determine the function of what were once believed to be plant-exclusive hormones in other bacterial systems and their animal hosts.

Experimental procedures

Details of experimental procedures are described in the Extended Experimental Procedures

Bacterial strains, plasmids, primers, chemicals and culture conditions

Bacterial strains, plasmids and primer sequences used in this study are listed in Table S1. See Extended Experimental Procedures for details.

iPRMP, tZRMP, iP, tZ and p-topolin were purchased from OlChemim (Olomouc, Czech Republic). pHBA and 2-methyl-3-butenal were purchased from Sigma.

Transposon mutagenesis, nitrite treatment and suppressor screen

We mutagenized Δmpa::hyg mutant cultures with the mariner-based ΦMycoMarT7 transposon (KanR) (Sassetti et al., 2001). We generated 18 independent pools with ~4,000 double mutants per pool (HygR, KanR). Each pool was then exposed for six days to 3 mM sodium nitrite (NaNO2) in 7H9 at pH 5.5. Bacteria were allowed to recover in 7H9 at pH 6.8 until bacterial growth was observed, about three to five weeks depending on the pool. Three pools recovered and were subjected to another round of acidified nitrite treatment for six days and directly plated on 7H11 agar.

120 individual colonies were picked from each of the three pools and inoculated into 200 μl 7H9 until growth reached stationary phase. 10 μl aliquots of each mini-culture were then sub-cultured into 190 μl of acidified 7H9 containing a final concentration of 3 mM NaNO2. After six days, 50 μl from each well was mixed with 150 μl 7H9, pH 6.8 and recovery was monitored by optical density at 580 nm (OD580) two to three weeks later. Double mutant recovery was compared to the recovery of wt (NO-resistant) and Δmpa::hyg strains (NO-sensitive) undergoing the same treatment. Candidate suppressor mutants were selected if the OD580 were comparable to the wt control.

62 clones were selected through this protocol (24, 24 and 14 from pools 4, 6, and 12, respectively). Chromosomal DNA from all 62 was analyzed by Southern blotting (data not shown). See Extended Experimental Procedures for details.

Each mutant had a single, unique transposon insertion. We detected three different restriction patterns from the mutants and surmised that three different loci were affected. One pattern was found in two pools (4 and 6). Genomic DNA encompassing the transposon insertion site was cloned and sequenced as previously described (Darwin et al., 2003). DNA sequencing using a primer that annealed to the neo gene was performed by GENEWIZ (South Plainfield, NJ).

Nitrite-resistance was quantified by nitric oxide assay for each of the suppressor mutants exactly as described previously (Darwin et al., 2003).

Mouse infections

Mouse infections were performed essentially as described previously (Darwin et al., 2003). See Extended Experimental Procedures for details.

Protein purification and immunoblotting

Recombinant protein production and immunoblotting analysis of M. tuberculosis cell lysates was performed essentially as previously described (Festa et al., 2007). See Extended Experimental Procedures for details.

In vitro pupylation assay

In vitro conjugation of PupGlu (10 μM) to Log (2 μM) was carried out at room temperature (25°C) in 50 mM Tris pH 8, 150 mM NaCl, 20 mM MgCl2, 10% glycerol, 1 mM DTT and 5 mM ATP. The reaction was started by the addition of 0.5 μM recombinant PafA-His6 (gift from Huilin Li) and stopped by addition of 4 × SDS sample buffer and boiled for 5 min.

Phosphoribohydrolase assay

The assay was adapted from a previously described method (Kurakawa et al., 2007). See Extended Experimental Procedures for details.

Sequence and phylogenetic analysis of mycobacterial Log homologues

Homologues of Log were obtained by performing iterative profiles searches using the PSI-BLAST (Altschul et al., 1997) and JACKHMMER (Finn et al., 2011) programs, run against the non-redundant (NR) protein database of the National Center for Biotechnology Information (NCBI), with various LOG domain-containing proteins as queries. See Extended Experimental Procedures for details.

M. tuberculosis supernatant and intracellular cytokinin measurements

For supernatant analysis, cultures were grown in triplicate in 7H9 media to an OD580 of 1. 36 ml of supernatant for each replicate was filter-sterilized through 0.45 μm syringe filters and lyophilized (Labconco freeze-dry system). For cell lysates, cultures were grown in triplicate in Sauton’s minimal media (Festa et al., 2010) up to an OD580 of 1. 35 ml of cultures were pelleted and washed once in PBS. Each pellet was resuspended in 2 ml 70% ethanol + 0.004% of sodium diethyldithiocarbamic acid (DCC). Cells were lysed by bead beating with zirconia beads three times for 30 s. Supernatants were gently mixed with 2.5 ml 70% ethanol + 0.004% DCC for 24 hours at 4°C, before lyophilization. Measurement of cytokinins was performed as described previously (Novak et al., 2003; Svacinova et al., 2012; Tarkowski et al., 2010). See Extended Experimental Procedures for details.

Metabolomic analysis of M. tuberculosis cell lysates

Cultures were grown in quadruplicate up to an OD580 of 1. Sixty-five OD equivalents per replicate were processed by chloroform:methanol extraction (Layre et al., 2011). Metabolomic profiling was carried out by Metabolon® (Durham, North Carolina) as previously described (Dehaven et al., 2010; Evans et al., 2009; Evans et al., 2012).

Cytokinin oxidase activity assay

The assay was adapted from a method described previously (Frebort et al., 2002). See Extended Experimental Procedures for details.

Supplementary Material

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2
3

Acknowledgments

We are grateful to A. Darwin and C. Nathan for review of draft versions of this manuscript, V. DiRita, I. Mohr, T. Richardson and V. Torres for helpful suggestions. We thank D. Reinberg for generous support, W. Houry for E. coli LDC and advice, H. Li for purified PafA, S. Ehrt for the ΔprcBA::hyg mutant, B. Bennett and S. Butler-Wu for cloning assistance, J. McKinney for pMV306.Strep, H. Martínková and B. Pařízková for technical assistance in cytokinin detection, K. Rhee for metabolomics advice, R. Michalek at Metabolon for outstanding data analysis, and the Rockefeller Proteomics Resource Center for use of their facilities. We are eternally grateful to Charlie Rice and The Rockefeller University for providing laboratory space and support for the 15 months after Superstorm Sandy. This work was supported by NIH grant R01HL092774 awarded to K.H.D and the Jan T. Vilcek Endowed Fellowship fund awarded to M.I.S. L.A. and L.M.I. are funded by the intramural research program of the National Library of Medicine (NIH, US Department of Health and Human Services). O.N. and M.S. work is supported by the Centre of the Region Haná for Biotechnological and Agricultural Research (ED0007/01/01) and the Internal Grant Agency of Palacký University (PrF_2013_023). K.H.D holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.

Footnotes

Author contributions

M.I.S. performed the suppressor screen; isolated, cloned and biochemically characterized Rv1205, and performed all in vitro and in vivo M. tuberculosis work. S.T. performed all HPLC analysis. L.A. and L.M.I. carried out the genomic and evolutionary analysis. F.E.M. and S.P.G did the LC/MS/MS analysis of Pup~Log. S.R.H. modeled the M. tuberculosis Log active site. O.N. and M.S. quantified the cytokinins. M.I.S. and K.H.D. wrote the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS657975-supplement-1.pdf (3MB, pdf)
2
NIHMS657975-supplement-2.xls (298.5KB, xls)
3
NIHMS657975-supplement-3.xls (39.5KB, xls)

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