1. Microbiology and Infectious Disease
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Integrated sensing of host stresses by inhibition of a cytoplasmic two-component system controls M. tuberculosis acute lung infection

  1. John A Buglino
  2. Gaurav D Sankhe
  3. Nathaniel Lazar
  4. James M Bean
  5. Michael S Glickman  Is a corresponding author
  1. Immunology Program Sloan Kettering Institute, United States
  2. Immunology and Microbial Pathogenesis Graduate Program, Weill Cornell Graduate School, United States
  3. Division of Infectious Diseases, Memorial Sloan Kettering Cancer Center, United States
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Cite this article as: eLife 2021;10:e65351 doi: 10.7554/eLife.65351

Abstract

Bacterial pathogens that infect phagocytic cells must deploy mechanisms that sense and neutralize host microbicidal effectors. For Mycobacterium tuberculosis, the causative agent of tuberculosis, these mechanisms allow the bacterium to rapidly adapt from aerosol transmission to initial growth in the lung alveolar macrophage. Here, we identify a branched signaling circuit in M. tuberculosis that controls growth in the lung through integrated direct sensing of copper ions and nitric oxide by coupled activity of the Rip1 intramembrane protease and the PdtaS/R two-component system. This circuit uses a two-signal mechanism to inactivate the PdtaS/PdtaR two-component system, which constitutively represses virulence gene expression. Cu and NO inhibit the PdtaS sensor kinase through a dicysteine motif in the N-terminal GAF domain. The NO arm of the pathway is further controlled by sequestration of the PdtaR RNA binding response regulator by an NO-induced small RNA, controlled by the Rip1 intramembrane protease. This coupled Rip1/PdtaS/PdtaR circuit controls NO resistance and acute lung infection in mice by relieving PdtaS/R-mediated repression of isonitrile chalkophore biosynthesis. These studies identify an integrated mechanism by which M. tuberculosis senses and resists macrophage chemical effectors to achieve pathogenesis

Introduction

Intracellular bacterial pathogens such as Mycobacterium tuberculosis and Salmonella that infect macrophages have evolved elaborate mechanisms to respond to the toxic environment of the macrophage phagosome and associated endocytic compartments (Olive and Sassetti, 2016; Stallings and Glickman, 2019). Phagosomal pathogens must contend with such host-inflicted stresses as oxidative stress (Bustamante et al., 2011), nitrosative stress (Darwin et al., 2003; Fang and Vázquez-Torres, 2019), iron deprivation, copper and zinc toxicity (Botella et al., 2011; Sheldon and Skaar, 2019; Shi and Darwin, 2015), and low pH (Vandal et al., 2009), among others. The pathogen response to these molecules is multifaceted and must be dynamic and graded to respond both to rapidly changing environments and the prospect of combinations of stresses that vary in intensity and composition over time. When expelled by coughing and inhaled by the naïve host, M. tuberculosis must transition from the nutrient-rich environment of the pulmonary cavity to deposition in the alveolus and engulfment by an alveolar macrophage. Such rapid transitions in environment require rapid changes in gene expression for successful adaptation.

Bacterial two-component systems (TCS) are widespread sensing systems that respond to a wide variety of ligands, including ions, gases, and metabolites. For pathogenic bacteria, TCS promote pathogenesis by modifying bacterial gene expression in response to host-inflicted toxic stresses or metabolic environments (Bretl et al., 2011; Groisman, 2016). The classic TCS sensing system consists of a membrane-bound sensor kinase that senses an extracellular ligand and activates through autophosphorylation on a cytoplasmic histidine. Transfer of this phosphate to an aspartate in the receiver domain of the cognate response regulator (RR) activates the RR to bind DNA of its target genes, thereby controlling gene expression (Sankhe et al., 2018; Zschiedrich et al., 2016).

In addition to TCS signaling, proteolysis is another widespread mechanism of bacterial signal transduction in which membrane-embedded proteases process membrane-embedded proteins, often anti-sigma factors (Schneider and Glickman, 2013; Urban, 2009). The S2P class of intramembrane proteases is widely distributed in bacteria, and several have been implicated in controlling virulence functions of bacterial and fungal pathogens, including Vibrio cholerae (Almagro-Moreno et al., 2015; Matson and DiRita, 2005), Cryptococcus (Bien et al., 2009), and M. tuberculosis. M. tuberculosis Rip1 is an important virulence determinant required for both acute growth in the lung and long-term persistence during chronic infection (Makinoshima and Glickman, 2005). Rip1 controls four independent sigma factor pathways through four anti-sigma factors substrates (Schneider et al., 2014; Sklar et al., 2010), but the virulence function of Rip1 appears to be independent of these pathways (Sklar et al., 2010). Given the importance of this pathway for M. tuberculosis pathogenesis, we sought to determine the virulence pathway(s) controlled by Rip1. These investigations, described below, uncover a new signaling system that integrates the bacterial response to Cu and NO and thereby controls growth in the host lung. The hub of this signaling system is the PdtaS/R cytoplasmic TCS, which constitutively represses virulence gene expression until inactivation by Cu and NO. The NO arm of the pathway is further controlled by titration of PdtaR from its RNA targets by an NO-induced, Rip1-controlled, small RNA, which binds directly to PdtaR and controls expression of isonitrile chalkophores. The ultimate Cu resistance mechanism controlled by Rip1/PdtaS/R is independent of chalkophores and remains to be identified.

Results

The Rip1 pathway defends against metal and nitrosative stress

To understand the basis for the severe virulence defect of M. tuberculosis lacking the Rip1 protease (Makinoshima and Glickman, 2005), a phenotype that is independent of the four identified Rip1-controlled sigma factor pathways (Schneider et al., 2014; Sklar et al., 2010), we hypothesized that Rip1 defends against specific host-imposed bactericidal effector molecules. We performed bacterial killing assays of wild-type (WT) M. tuberculosis and Δrip1 with nitric oxide, hydrogen peroxide, copper, zinc, low pH, detergent, lysozyme, and nutrient starvation. We observed no Rip1-dependent sensitization to starvation, lysozyme, oxidative stress, detergent, or low pH (Figure 1—figure supplement 1A–C). However, we observed that Rip1 is required for resistance to copper ions, nitric oxide, and zinc. M. tuberculosis Δrip1 is 10,000-fold more sensitive to copper on agar media than wild-type Mtb, and this phenotype is restored by genetic complementation by a plasmid encoding a wild-type copy of Rip1, but not a proteolytically inactive Rip1 (Figure 1A). Sensitivity was also observed for Δrip1 cells grown in liquid media with Cu supplementation (Figure 1—figure supplement 1D). Similarly, Δrip1 growth is inhibited by 100 μM Zn (Figure 1B), but not iron (Figure 1C). Rip1 is also required for resistance to nitric oxide. Treatment of wild-type Mtb for 3 days with 200 μM of the NO donor diethylenetriamine nitric oxide (DETA-NO) minimally reduced bacterial viability, whereas Δrip1 titers were reduced 100-fold, a phenotype that was also complemented with wild-type Rip1 but not proteolytically inactive Rip1 (Figure 1D). Importantly, similar metal nor NO sensitivity was observed for M. tuberculosis strains lacking individual or any tested combination of Rip1-controlled sigma factor pathways (Figure 1—figure supplement 2A,B), indicating that the Rip1-controlled metal/NO resistance pathway is a previously unrecognized, sigma factor-independent arm of the Rip1 pathway.

Figure 1 with 3 supplements see all
The Rip1 protease controls copper and nitric oxide resistance.

(A) The Rip1 pathway confers resistance to copper. Bacterial colony-forming units (CFUs) of the indicated strains grown on agar plates supplemented with 200 or 500 µM copper sulfate. Relative survival is normalized to untreated controls, which are set at 100 = 1. Each value is the average of technical duplicate measurements for n = 3 biological replicates. Statistical analysis by two-way ANOVA with Tukey’s multi-comparison correction *p<0.01 WT + vector 200 µM vs. Δrip1 +H21A rip1; ***p<0.001 WT + vector 500 µM vs. Δrip1 +vector; WT + vector 500 µM vs. Δrip1 +H21A rip1. (B) The Rip1 pathway confers resistance to zinc. Growth (OD600) of the indicated strains in liquid culture supplemented with 100 µM zinc sulfate. Data plotted is SEM of n = 3 biological replicates. Statistical analysis by ordinary one-way ANOVA with Tukey’s multi-comparison correction. *p<0.05, **p<0.01; day 14 WT + vector vs. Δrip1 + vector p=0.027; day 14 Δrip1 +vector vs. Δrip1 + H21A rip1 p=0.032; day 14 WT + vector vs. Δrip1 + H21A rip1 p=0.002. (C) The Rip1 pathway does not control iron sensitivity. Growth on agar plates supplemented with 300 or 600 µM iron chloride normalized to untreated controls as in (A). Each value is the average of technical duplicate measurements for n = 2 biological replicates. (D) The Rip1 pathway confers resistance to nitric oxide. CFU counts of the indicated strains post treatment with vehicle or 200 µM diethylenetriamine nitric oxide adduct (DETA-NO) for 3 days. Relative survival = CFU day 3 post treatment/CFU at day 0 for each condition n = 8 biological replicates for WT + vector, Δrip1 + vector, and Δrip1 + WT rip1, and n = 2 for Δrip1 + H21A rip1. Statistical analysis by unpaired t-test. *p<0.05; DETA-NO WT + vector vs. Δrip1 + vector p=0.041; DETA-NO Δrip1 + vector vs. Δrip1 + WT rip1 p=0.031. (E) Loss of Rip1 does not affect Cu-induced transcription. RT-qPCR quantitation of known copper-regulated transcripts ctpV, lpqS, and mymT between WT (blue) and Δrip1 (red) cells in resting (filled symbols) or 200 µM copper sulfate (open symbols) conditions for 2 hr. Values are normalized to sigA transcript levels. Values represent average of three technical replicate measurements of three biological replicates. (F) Loss of Rip1 does not affect nitric oxide-regulated gene expression through DosR. RT-qPCR comparison of nitric oxide-induced, DosR-regulated transcripts hspX, rv2625c, and dosR between WT and Δrip1 cells following 3 hr of treatment with vehicle (filled symbols) or 200 μM DETA-NO (empty symbols). Values are normalized to sigA transcript levels. Mean of biological triplicates reported. (G) NO mediates attenuation of M. tuberculosis Δrip1 in mouse lung infection. Bacterial burden (CFU) in lungs of C57bl/6 (broken lines), and age-matched Nos2-/- (solid lines) mice infected with WT M. tuberculosis (blue) or M. tuberculosis Δrip1 (red) at the indicated times (days) post infection. Data plotted is mean and SD of n = 4 biological replicates. ***p=0.0003 by unpaired t-test at 21-day time point for Δrip1 in WT vs. Nos2 -/-. (H) Repeat experiment as in (G), but with lower initial inoculum of Δrip1 M. tuberculosis in WT (open triangles) and Nos2-/- (closed squares) mice. For all strains/host day 1 n = 3 biological replicates; n = 4 biological replicates for all other time points. Statistical significance by unpaired t-test analysis is represented as *p<0.05, **p<0.01, ***p<0.001.

Multiple prior studies have investigated copper and NO response and resistance mechanisms in M. tuberculosis. Copper is sensed by the metal binding repressors RicR and CsoR (Festa et al., 2011; Liu et al., 2007; Marcus et al., 2016; Shi and Darwin, 2015), which control regulons involved in copper binding and export (Rowland and Niederweis, 2012; Shi et al., 2014; Ward et al., 2010). We tested whether relief of RicR or CsoR repression was impaired in Δrip1 by measuring copper-induced transcription of their target genes. We found no defect in the copper-induced transcription of mymT or lpqS (RicR targets; Festa et al., 2011) or CtpV (CsoR target) in Δrip1 cells, indicating functionality of these systems (Figure 1E, and Figure 1—figure supplement 3A,B). To determine whether a Rip1-dependent post-translational modification of copper export or chelation by CtpV (Ward et al., 2010) or MymT (Gold et al., 2008) might explain the copper sensitivity of Rip1, we performed genetic epistasis tests in Δrip1ctpV and Δrip1mymT. We found minimal copper sensitivity of ΔctpV or ΔmymT, and no enhanced copper sensitivity of ΔripctpV or Δrip1ΔmymT compared to Δrip1 (Figure 1—figure supplement 3C). Additionally, de-repression of the CsoR regulon in the Δrip1 background (through Δrip1ΔcsoR) had no effect on Δrip1 copper sensitivity (Figure 1—figure supplement 3C). These results indicate that the Rip1 copper sensitivity is not due to known pathways of copper chelation or efflux. We similarly examined known NO response regulons, but found that NO-induced transcription of three DosS/T/R target genes, which controls the dominant transcriptional response to NO (Voskuil et al., 2003), was intact in Δrip1, indicating intact NO sensing at the cell surface through this TCS (Figure 1F). Our results indicate that Rip1 controls a pathway of resistance to Cu and NO, which is likely distinct from previously defined pathways.

Nitric oxide-dependent attenuation of M. tuberculosis Δrip1

To test the importance of the Rip1-dependent NO resistance pathway in vivo, we infected NOS2-deficient mice. We reasoned that if NO is a significant attenuating pressure for M. tuberculosis Δrip1 in vivo, we would observe some reversal of the Δrip1 virulence defect when NO is removed, as has been reported for other NO-sensitive mutants (Darwin et al., 2003; Darwin and Nathan, 2005). NOS2-deficient mice infected with wild-type Mtb were highly susceptible to Mtb infection, as previously reported (MacMicking et al., 1997; Figure 1G). The impaired growth of M. tuberculosis Δrip1 in the lung was dramatically reversed in the absence of NO, such that Δrip1 lung titers in NOS2-deficient animals were 100-fold higher than in wild-type mice at 3 weeks and 10,000-fold higher at 8–10 weeks post infection (Figure 1G). A repeat infection at a lower inoculum confirmed these findings (Figure 1H). These data establish that the Rip1 pathway defends against NO in vivo during acute lung infection, possibly through a direct antimicrobial effect of NO.

The PdtaS/PdtaR TCS controls NO and Cu resistance downstream of Rip1

The data presented above indicate that the Rip1 protease controls a pathway that defends against Cu and NO, but that this phenotype is neither attributable to previously identified Rip1-controlled sigma factor pathways nor due to known mechanisms of Cu or NO sensing. To examine the molecular basis for these phenotypes, we executed a genetic suppressor screen for spontaneous chromosomal mutations that revert the Cu sensitivity phenotype of M. tuberculosis Δrip1 (Figure 2A). M. tuberculosis Δrip1 was selected on agar media containing 500 μM CuSO4, and surviving bacteria were clonally purified, and after confirming the acquisition of Cu resistance, examined by whole genome sequencing (Figure 2A). We detected two suppressor strains with distinct mutations in the rv3220 gene, encoding the PdtaS sensor kinase: a frameshift mutation at amino acid 37 of the encoded protein (F37X) and a substitution mutation V54F. Both mutations were confirmed by sequencing of chromosomal segments amplified by PCR from the suppressor strains (Figure 2—figure supplement 1A,B). In contrast to most sensor kinases that are membrane bound, PdtaS is a soluble sensor kinase with a C-terminal histidine kinase domain and N-terminal sensing GAF (in which the suppressor mutations were detected) and PAS domains (Figure 2B). Its structure is known (Preu et al., 2012), but its function is not understood.

Figure 2 with 1 supplement see all
The PdtaS/PdtaR two-component system controls copper and NO resistance downstream of Rip1.

(A) Flow chart of a genetic suppressor screen to isolate reversion mutations of the Rip1 Cu sensitivity and testing their reversion of NO sensitivity. WGS: whole genome sequencing; SNP: single nucleotide polymorphism. (B) Domain structure of PdtaS with GAF, PAS, and kinase domains. Identified suppressor mutations of PdtaS are shown in the N-terminal GAF domain. The reported phosphorylation target of PdtaS is the PdtaR response regulator (RR), which contains a C-terminal ANTAR RNA binding domain. (C) Protein levels of the indicated alleles of pdtaS reintroduced into the Δrip1pdtaS F37X background as C-terminal 10X His fusion proteins with Rpoβ levels as a loading control. For WT, V54F, and H302Q/H303Q, two independent strains are shown, whereas for vector and G443A/G445A, one strain is shown. (D) Loss of pdtaS suppresses copper sensitivity of Δrip1. Relative survival of the indicated strains grown on agar plates supplemented with 200 or 500 µM copper sulfate normalized as in Figure 1. H302Q/H303Q and G443A/G445A are kinase dead alleles. Each value is the average of technical duplicate measurements for n = 3 biological replicates. Statistical analysis by two-way ANOVA with Tukey’s multi-comparison correction. (E) Loss of pdtaR suppresses copper sensitivity of Δrip1. Cu sensitivity assay as noted in (D). PdtaR D65A lacks the receiver aspartate for PdtaS phosphorylation. Each value is the average of technical duplicate measurements for n = 3 biological replicates. (F) Loss of pdtaS suppresses NO sensitivity of Δrip1. Colony-forming unit (CFU) counts of the indicated strains post treatment with vehicle or 200 µM diethylenetriamine nitric oxide (DETA-NO) for 3 days. Color coding of strain genotypes is the same as in (D). n = 8 biological replicates for WT, Δrip1, Δrip1pdtaS F37X; n = 6 biological replicates for Δrip1pdtaS V54F; n = 4 biological replicates for Δrip1pdtaS F37X + pdtaS, Δrip1pdtaS F37X + pdtaS H302Q/H303Q, Δrip1ΔpdtaS, ΔpdtaS; n = 3 biological replicates for Δrip1pdtaS F37X + pdtaS G443A/G445A; n = 2 for Δrip1pdtaS F37X + pdtaS V54F. Statistical analysis by Mann–Whitney test. (G) Loss of pdtaR suppresses NO sensitivity of Δrip1. CFU counts of the indicated strains post treatment with vehicle or 200 µM DETA-NO for 3 days. Color coding of strain genotype is the same as in (E). n = 8 biological replicates for WT, Δrip1, Δrip1 ΔpdtaR; n = 6 biological replicates for Δrip1ΔpdtaR + pdtaR and Δrip1ΔpdtaR +pdtaR D65A; n = 4 biological replicates for ΔpdtaR. Statistical analysis by Mann–Whitney test. For all panels, statistical significance is represented as *p<0.05, **p<0.01, ***p<0.001.

To confirm that the PdtaS mutations are the functional variants reverting the Cu sensitivity, we performed complementation tests on the suppressor strains with PdtaS or PdtaS variants predicted to lack kinase activity (Figure 2C). Introduction of PdtaS into either Δrip1pdtaS(F37X) or Δrip1pdtaS(V54F) restored Cu sensitivity to both suppressor strains, indicating that the PdtaS single nucleotide polymorphisms (SNPs) are the functionally important suppressor mutations (Figure 2D). In addition, we constructed a chromosomal deletion of pdtaSrip1ΔpdtaS) and observed the same pattern of PdtaS-dependent Cu sensitivity (Figure 2D). To probe the requirement of PdtaS kinase activity, we mutated histidines 302/303 to glutamines, and glycines 443/445 to alanine, which are required for autophosphorylation and kinase activity respectively (Trajtenberg et al., 2010), and expressed these variants in the Δrip1pdtaS(F37X) strain. Although both kinase dead proteins were expressed to equivalent levels (Figure 2C), neither were able to restore Cu sensitivity (Figure 2D), indicating a requirement for PdtaS kinase activity in controlling Cu resistance.

PdtaS was reported to phosphorylate the RR PdtaR (Morth et al., 2005), which is itself an atypical RR in that it contains an ANTAR RNA binding domain (Ramesh et al., 2012) instead of the DNA binding domain present in many RRs. To validate the participation of PdtaR in the Δrip1 Cu sensitivity, we constructed Δrip1ΔpdtaR and tested Cu sensitivity. We observed that loss of pdtaR reverted the Cu sensitivity (Figure 2E) of Δrip1. Complementation of Δrip1ΔpdtaR with pdtaR restored copper sensitivity (Figure 2E). However, expression of a PdtaR-D65A, which lacks the receiver aspartate phosphorylated by PdtaS, was partially active, partially restoring Cu sensitivity to Δrip1ΔpdtaR at low-dose Cu and completely at higher-dose Cu (Figure 2E). These results suggest that phosphorylation of PdtaR is not absolutely required for its activity in the Cu sensitivity pathway.

The suppressor mutations in PdtaS were identified as suppressors of Cu sensitivity, but the NO sensitivity might be due to a different Rip1-dependent pathway. However, we observed that loss of pdtaS in Δrip1 also reverted the NO sensitivity nearly to the level of WT M. tuberculosis (Figure 2F), with the same requirement for PdtaS kinase activity. In addition, inactivation of pdtaS in wild-type cells conferred a hyperresistance to NO (Figure 2F). We next tested the involvement of PdtaR in the NO sensitivity phenotype of Δrip1 and found the loss of pdtaR reverted to the level of the wild-type strain (Figure 2G). We also observed a partial activity of PdtaR D65A, which partially complemented in comparison to WT PdtaR (Figure 2G). Loss of PdtaR in wild-type Mtb conferred hyperresistance to NO compared to the parental wild-type strain, again indicating that PdtaR suppresses NO resistance (Figure 2G). Taken together, these results demonstrate that the Cu and NO sensitivity conferred by loss of rip1 proceeds through an active PdtaS/R signaling system and that these two sensitivity phenotypes represent an integrated signaling pathway that responds to both stresses.

NO and Cu directly inhibit PdtaS kinase activity

The data above suggests that PdtaS/PdtaR acts as a negative regulator of Cu and NO resistance, and that this negative regulation is not relieved in the Δrip1 background. Genetic deletion of pdtaS/R relieves this inhibition and restores wild-type stress resistance. Although the PdtaS GAF domain was recently reported to bind cyclic di-GMP (Hariharan et al., 2021), the full set of ligands that interact with the PdtaS sensing domains are not known. To determine whether PdtaS/R directly senses cytosolic metals and/or nitric oxide, we reconstituted the PdtaS/R phosphotransfer reaction using purified proteins. The PdtaS kinase was active for autophosphorylation when assayed with radiolabeled ATP (Figure 3A), as previously reported (Morth et al., 2005), and was also active in phosphotransfer to PdtaR (Figure 3A). PdtaS autophosphorylation was not inhibited by calcium or iron at 1 mM (Figure 3—figure supplement 1A), but we observed strong dose-dependent inhibition of PdtaS autophosphorylation at 10–1000 μM Cu (Figure 3B) or similar zinc concentrations (Figure 3—figure supplement 1B,C). Titrations revealed a dose-dependent inhibition of PdtaS activity by these metals, with inhibition constants (Ki) of 34 μM for Cu and 26 μM for zinc (Figure 3B, F, Figure 3—figure supplement 1C). These data indicate that copper and zinc directly inhibit the kinase activity of PdtaS.

Figure 3 with 2 supplements see all
PdtaS is directly inhibited by copper and NO through a dicysteine motif in the N-terminal GAF domain.

(A) PdtaS phosphotransfer to PdtaR. Upper panel: phosphorscreen imaging of 32P incorporation; lower panel: Coomassie brilliant blue (CBB) staining to determine total protein. First reaction contains PdtaS alone, second and third lanes contain PdtaS/PdtaR incubated for 10 and 20 min, respectively. Fourth lane (M) is the molecular weight (MW) marker. (B) Cu++ inhibits PdtaS autophosphorylation. Autophosphorylation of PdtaS protein preincubated with increasing concentrations (5 µM to 104 µM) of CuCl2 or 1 mM of CaCl2 (Ca++) as a control. Upper panel: phosphorscreen imaging of 32P incorporation; lower panel: CBB staining to determine total protein. (C) NO inhibits PdtaS autophosphorylation. Autophosphorylation of PdtaS protein preincubated with increasing concentrations (5 µM to 104 µM) of spermine NONOate and 1 mM of spermine NONOate post NO release as control (designated by C). (D) PdtaS GAF domain cysteine 53 is required for copper inhibition. PdtaS-C53A protein preincubated with increasing concentrations (5 µM to 104 µM) of CuCl2 or 1 mM of CaCl2 was assayed for autophosphorylation as described in panel B . (E) PdtaS GAF domain cysteine 53 is required for NO inhibition. PdtaS-C53A protein preincubated with increasing concentrations (5 µM to 104 µM) of spermine NONOate and of 1 mM of spermine NONOate post NO release as control was followed by autophosphorylation. (F) Cu inhibition curve from replicate data represented by (B) and (D) for PdtaS (blue) and PdtaS-C53A (green) proteins yields Ki of (34 ± 8 µM) for PdtaS and (5540 ± 2381 µM) for PdtaS-C53A. Error bars are SEM for n = 3. (G) NO inhibition curve from replicate data represented by (C) and (E) and (Figure 3—figure supplement 2) for PdtaS (blue), PdtaS-C53A (green), or PdtaS-C57A (red) proteins yields Ki of (7 ± 2 µM) for PdtaS and (73 ± 34 µM) for PdtaS-C53A. Error bars are SEM for n = 3 for WT and C53A and n = 2 for C57A.

We next asked whether NO has a similar effect on PdtaS. We used spermine NONOate, an NO donor with a half-life of NO release of 40 min. NO had a dose-dependent inhibitory effect on PdtaS activity, but spermine NONOate, which had been exhausted for NO release, had no effect (Figure 3C). The Ki determined from dose titrations was 7 μM. There was no effect of hydrogen sulfide on PdtaS activity (Figure 3—figure supplement 1D). Consistent with the idea that these ligands inhibit signaling without affecting expression or stability of the signaling proteins, we observed no change in PdtaR protein expression or proteolysis in vivo upon treatment with Cu, Zn, or NO (Figure 2—figure supplement 1C). These data indicate that PdtaS directly senses metals and NO and implies that ligand sensing by the sensor kinase inhibits signaling through the PdtaS/R system.

PdtaS contains GAF and PAS domains N-terminal to the kinase domain. PAS domains bind a wide variety of small molecule ligands (Aravind and Ponting, 1997; Henry and Crosson, 2011). As noted above, the two suppressor mutations in PdtaS are in the GAF domain, one a frameshift that inactivates the protein and one (V54F) in close proximity to a dicysteine motif (53-CVAQC-57) (Figure 2B, Figure 2—figure supplement 1). To determine whether these domains are required for NO and Cu inhibition of PdtaS, we purified the PdtaS kinase domain, which was constitutively active (Figure 3—figure supplement 1E,F). However, neither Cu nor NO had a substantial inhibitory effect on the isolated kinase domain (Figure 3—figure supplement 1E,F), indicating that the GAF-PAS are required for the ligand-dependent inhibitory effect. We next mutated each cysteine in the PdtaS GAF in proximity to the V54F suppressor (C53A or C57A) and tested these kinases for Cu and NO inhibition. PdtaS-C53A was refractory to inhibition by both ligands (Figure 3D–G), directly implicating cysteine 53 in the GAF domain in Cu and NO sensing. PdtaS-C57A was also resistant to NO inhibition (Figures 3GFigure 3—figure supplement 2), but our attempts to determine Cu inhibition of this protein were unsuccessful due to protein aggregation, preventing accurate quantitation (data not shown). These results indicate that the PdtaS sensor kinase is a constitutively active, ligand-inhibited signaling protein that integrates Cu and NO sensing through a dicysteine motif in the GAF domain.

Rip1/PdtaR jointly control an NO-responsive regulon that includes chalkophore biosynthesis

The data above indicates that PdtaS/R is a negative regulator of Cu and NO resistance and that these ligands directly inhibit PdtaS signaling. The exact function of PdtaR in regulating gene expression is not known. PdtaR-type RRs contain an ANTAR domain in place of the more commonly encountered DNA binding domain of traditional RR. In the few examples that have been examined, ANTAR domains bind to dual hairpin structures at the 5′ ends of mRNA, and thereby influence transcriptional termination (Fox et al., 2009), but the full spectrum of gene regulation conferred by ANTAR domain RNA binding has yet to be elucidated. ANTAR-RRs in Listeria and Enterococcus are also regulated by trans acting small RNAs that compete for ANTAR domain binding and thereby titrate the RR away from other mRNA targets (DebRoy et al., 2014; Mellin et al., 2014). To understand the target genes controlled by Rip1/PdtaS/PdtaR, we performed RNA sequencing under NO stress. We focused on NO stress for several reasons, including (1) the strong phenotypic reversion of Δrip1 in iNOS-deficient mice (Figure 1G), (2) the strong phenotypic reversion of Rip1 NO sensitivity by the pdtaR mutation, and (3) the technical advantages of treating Mtb with carefully titrated NO donors in liquid culture.

RNA sequencing of WT, Δrip1, Δrip1ΔpdtaR, and ΔpdtaR treated with vehicle or DETA-NO revealed that the NO-induced DosR regulon was intact in the Δrip1 strain and unaffected by loss of PdtaR (Figure 4A). However, by clustering gene expression across all strains and conditions, we defined a cluster of NO-induced, Rip1-dependent genes for which the defective expression is restored to WT levels in the Δrip1ΔpdtaR strain, thereby matching the phenotypic pattern seen in our NO sensitivity tests (red box in Figure 4B). Consistent with these genes being negatively regulated by PdtaS/R in wild-type cells and the hyperresistance of the ΔpdtaR strain to NO (Figure 2G), this gene set was hyperinduced in the ΔpdtaR strain by NO (Figure 4). Importantly, there was no effect on basal gene expression with loss of pdtaR, indicating that inactivation of this TCS is not sufficient to activate gene expression in the absence of NO stress (Figure 4B).

NO-induced dual regulation of the chalkophore biosynthetic operon by Rip1/PdtaS/PdtaR.

(A) Unsupervised clustering of the diethylenetriamine nitric oxide (DETA-NO) (NO)-induced, DosR-regulated transcripts across the indicated genotypes. The scale bar represents the log2 of the normalized counts for each gene. (B) Unsupervised clustering of gene expression of M. tuberculosis WT, Δrip1, ΔpdtaR, and ΔrippdaR using the same color coding as in (A) treated with vehicle (V) or DETA-NO (NO) The highlighted cluster indicated by the red box on the left includes PPE1-5′, the PPE1 coding sequences after PPE1 (PPE1-after 5′), and the Rv0097-nrp cluster as independent elements. The scale bar represents the log2 of the scaled expression level for each row. The clustering of the genes and strains/conditions is done on the raw normalized counts. (C) Read coverage tracks across the ppe1-nrp cluster. Boxed areas highlight ppe1-5′ peak at the 5′ end of PPE1 and a region of potential termination at 5′ end of nrp. (D) DETA-NO-induced fold change values (DETA-NO/vehicle) across the same coverage region as in (C). 0097-nrp designates the entire Rv0097-nrp cluster.

Among the most strongly regulated genes controlled by Rip1/PdtaS/R under NO stress is a gene cluster from Rv0096-0101. This gene set encodes the PPE1 protein (rv0096) and five genes (rv0097-0101) that synthesize isonitrile lipopeptide chalkophores (Harris et al., 2017; Wang et al., 2017), which bind copper with high affinity (Wang et al., 2017; Xu and Tan, 2019). Alignment of RNA sequencing reads along the chalkophore cluster revealed low expression in basal conditions, but with a prominent small peak of 210 NT at the 5′ end of the nrp gene, indicating a site of potential termination (Figure 4C). With NO, chalkophore operon expression increased by three- to fivefold across the gene cluster in WT cells, but not in Δrip1 (Figure 4C, D). The defective chalkophore cluster expression is reversed in the Rip1/PdtaR double mutant. RNA reads mapping to the PPE1 locus, in contrast, mapped to a 336NT region that begins in the intergenic region at the reported transcription start site of the PPE1 mRNA and continues 277 nt beyond the PPE1 translational initiation codon (Figure 4C). We defined this element as ‘PPE1-5′’ and calculated its induction ratio with NO in comparison to the PPE1 coding sequence and the rest of the chalkophore operon. PPE1- 5′ is induced 33-fold by NO in wild-type cells, an induction that requires Rip1 (Figure 4D). PPE1-5′ is negatively regulated by PdtaR as evinced by hyperinduction in the ΔpdtaR strain. Loss of PdtaR restores the expression of PPE1-5′ in the Δrip1 background. Similar patterns were observed for each gene in the chalkophore operon and the chalkophore transcriptional unit as a whole (Figure 4D). These results identify a previously unappreciated NO-induced regulon that is subject to dual regulation by a positive transcriptional signal through the membrane-bound Rip1 protease and a negative signal from the cytoplasmic PdtaS/R TCS. Our data also identifies a previously unknown, NO-induced putative small RNA at the 5′ end of the chalkophore operon.

PPE1-5′ binds directly to PdtaR

The data above indicate that Cu and NO resistance and PPE1-5′ expression in Mtb require a functional Rip1 and inhibition of the PdtaS/R system, which does not occur without Rip1. These cooperating systems jointly control chalkophore operon expression. One mechanism to unify these findings would be that the Rip1-controlled PPE1-5′ RNA binds directly to PdtaR to relieve the negative regulation of PdtaR on target genes such as nrp. As noted above, this mechanism of regulation by trans acting RNAs on ANTAR domain regulators has been reported previously (DebRoy et al., 2014; Mellin et al., 2014) and would explain why Rip1 is required to inactivate PdtaR activity. Inspection of the predicted RNA sequence of PPE-1-5′ for predicted RNA secondary structure that might bind PdtaR revealed three hairpins in the vicinity of the PPE-1 translational initiation codon (Figure 5A). To test whether this RNA may bind PdtaR, we produced a 284 nt PPE1-5′ RNA by in vitro transcription, along with positive (Mehta et al., 2020) and negative control RNAs (Figure 5—figure supplement 1A,B) and tested binding to PdtaR using microscale thermophoresis (MST). PdtaR was phosphorylated in vitro by PdtaS and binding was measured at varying concentrations of RNA. Negative control RNA derived from the dnaK gene did not bind PdtaR detectably (Figure 5BFigure 5—figure supplement 1C) and the previously reported PdtaR binding RNA from rv3864 (Mehta et al., 2020) bound with a Kd of 2 μM (Figure 5B). We detected strong binding between phosphorylated PdtaR and PPE1-5′ with a calculated Kd of 0.41 μM (Figure 5B). We tested the requirement for PdtaR phosphorylation in the PdtaR-PPE1-5′ interaction by omitting PdtaS. Surprisingly, given the usual requirement for phosphorylation in activating RR function, PdtaR binding to the rv3864 RNA was not affected by phosphorylation (Figure 5C). Unphosphorylated PdtaR still bound PPE1-5′, albeit with threefold lower affinity (Kd0.4 vs. 1.2 μM, Figure 5C), indicating that phosphorylation enhances binding but is not absolutely required.

Figure 5 with 2 supplements see all
The PPE1-5′ RNA is an effector of NO resistance through sequestration of PdtaR.

(A) Predicted structure of the RNA hairpins in PPE1-5′. The nucleotide numbering refers to distance from the first transcribed nucleotide of the ppe1 RNA. The PPE1 translational initiation codon is indicated within the first hairpin. (B) Phosphorylated PdtaR directly binds to ppe1-5′. Change in the thermophoretic movement of fluorescently labeled and phosphorylated PdtaR was measured as a function of titrant RNA concentration on the X-axis for ppe1-5′, rv3864, and dnaK (ranging from 0.31 nM to 10 µM) as described in Materials and methods yielding binding affinity constants (Kd) for ppe1-5′ = 410 ± 84 nM, rv3864 = 2117 ± 798 nM while dnaK shows no binding. Error bars are SEM for n = 3. (C) RNA binding by PdtaR is partially phosphorylation dependent. Identical assay as in (B) using PdtaR without phosphorylation by PdtaS. Changes in the thermophoretic movement of fluorescently labeled PdtaR were measured as a function of titrant RNA concentration: ppe1-5′ (0.15 nM to 10 µM), rv3864 (0.61 to 10 µM), and dnaK (0.15 nM to 10 µM). Binding affinities (Kd) are ppe1-5′ = 1281 ± 322 nM, rv3864 = 2592 ± 832 nM while dnaK shows no binding. Error bars represent SEM for n = 3. (D) Rip1/PdtaS/PdtaR control of PPE1-5′ and chalkophore biosynthesis controls NO resistance. Constitutive expression of annotated protein coding region of ppe1 alone (+ppe1 ORF hsp60), ppe1 coding region plus 222-nt 5′ of start codon (+ppe1-5′), both C-terminally fused to GFP, or the chalkophore cluster (+rv0097-nrp) in Δrip1 and testing for diethylenetriamine nitric oxide (DETA-NO) sensitivity. Relative survival of the indicated strains post treatment with vehicle or 200 µM DETA-NO. n = 8 biological replicates for WT, Δrip1, Δrip1: PPE1 ORFpHSP60; n = 10 biological replicates for Δrip1:PPE1 ORF +5'pHSP60 and Δrip1: Rv0097-nrppHSP60. Statistical analysis by Mann–Whitney test. (E) Rip1/PdtaS/PdtaR control of PPE1-5′ and chalkophore biosynthesis does not control copper resistance. Relative survival of the same strains as in (D) grown on agar plates supplemented with 0, 200, or 500 µM copper sulfate normalized as in Figure 1. Each value is the average of technical duplicate measurements for n = 3 biological replicates. For all panels, statistical significance by t-test analysis is represented as *p<0.05, **p<0.01, ***p<0.001.

Chalkophore operon expression is the functional target that mediates Rip1/PdtaS/R-controlled NO resistance

To determine whether Rip1/PdtaS/PdtaR regulation of PPE1-5′ or the chalkophore cluster controls NO sensitivity, we restored expression of either the PPE1 coding sequence lacking the PPE1-5′ (hsp60-PPE1), PPE1 including PPE1-5′, or the chalkophore biosynthetic machinery (hsp60-Rv0097-Rv0101) in the Δrip1 strain. Restored expression of chalkophore biosynthesis (rv0097-nrp) restored NO resistance to a level equivalent to WT cells (Figure 5D). Enforced expression of PPE1 without PPE1-5′ had a minimal effect on NO sensitivity, whereas expression of PPE1 with PPE1-5′ restored NO resistance to the level similar to that observed with chalkophore re-expression (Figure 5D), despite no difference in the protein levels of PPE1 expressed from these two promoter constructs (Figure 5—figure supplement 2A). Despite the strong copper avidity of chalkophores, which might suggest a role in ameliorating Cu toxicity by direct binding, as is the case for other Cu binding molecules such as MymT, restoration of chalkophore production or expression of either PPE-5′ or PPE1 did not rescue the copper sensitivity of the Δrip1 strain (Figure 5E). Deletion of the nrp gene alone did not alter NO resistance, indicating that there are multiple targets downstream of Rip1/PdtaS/R that contribute to NO resistance (Figure 5—figure supplement 2B). These results indicate that NO-induced expression of PPE1-5′ and chalkophore biosynthesis controlled by Rip1/PdtaS/PdtaR signaling are critical determinants of NO resistance in M. tuberculosis.

Rip1/PdtaS/R control of chalkophore biosynthesis mediates acute lung infection

The model that emerges from the data presented above is that M. tuberculosis utilizes a two-signal mechanism to respond to the combinatorial stresses of the host: Rip1-dependent inactivation of PdtaR through the PPE1 5′ RNA and direct inhibition of PdtaS through the cysteine motif in the PdtaS GAF domain. This combined inhibition of PdtaS/R signaling relieves the negative regulation of this system on chalkophore biosynthesis. This model predicts that the in vivo virulence defect conferred by loss of Rip1, which is reversed by ablation of host NO (Figure 1G), should also be (1) phenocopied by loss of chalkophore biosynthesis (2) reversed in the Δrip1pdtaR strain. We first tested the phenotype of M. tuberculosis lacking chalkophore biosynthesis (Δnrp). Loss of nrp conferred mild attenuation in the early stages of lung growth such that Δnrp titers were approximately 10-fold lower than wild type at 14 and 21 days after aerosol infection (Figure 6A). Loss of nrp also conferred more severe attenuation of extrapulmonary growth, which persisted during chronic infection (Figure 6B). We next infected mice by aerosol with Δrip1pdtaR along with WT, Δrip1, and ΔpdtaR. We observed that the severe attenuation of lung growth conferred by loss of Δrip1 was substantially reversed by loss of pdtaR such that bacterial lung titers of Δrip1pdtaR were 15- and 97-fold higher than Δrip1 at 7 and 14 days post aerosol deposition, respectively (Figure 6C). Although lung titers of Δrip1pdtaR were significantly lower than ΔpdtaR, which was attenuated compared to wild type, loss of pdtaR reversed the majority of the Rip1-dependent attenuation in the first two weeks of infection (Figure 6C). Importantly, the Rip1/PdtaR axis is only relevant to acute lung infection as loss of pdtaR did not reverse the severe attenuation of Δrip1 during chronic infection (Figure 6C) and had no effect on extrapulmonary tissues (Figure 6D), indicating that other pathways mediate these virulence phenotypes.

Rip1/PdtaS/PdtaR control of chalkophore biosynthesis controls early lung infection.

(A, B) Bacterial burden (colony-forming unit [CFU]) of the indicated strains in lungs (A) or spleens (B) of C57bl/6 mice infected with WT (blue circle), Δnrp (red square), or Δnrp +nrp (green triangle) at the indicated time points (days) post aerosol infection. For WT and Δnrp, n = 6 biological replicates on day 1 and n = 8 on all subsequent days. All p values calculated by unpaired t-test. (C, D) Bacterial burden (CFU) of the indicated strains in lungs (C) or spleens (D) of C57bl/6 mice infected with WT (blue circle), Δrip1 (red square), or Δrip1ΔpdtaR (purple inverted triangle) or ΔpdtaR (green triangle) at the indicated time points (days) post aerosol infection. For all strains, n = 5 biological replicates on day 1; n = 12 biological replicates on day 7; n = 8 biological replicates on days 15, 21, and 56; n = 4 biological replicates on days 105 and 156. For all panels, statistical significance by unpaired Welch’s t-test is represented as *p<0.05, **p<0.01.

Discussion

We have revealed a new branched pathway of signal transduction that integrates M. tuberculosis resistance to multiple host-derived stresses. M. tuberculosis is exposed to a mixture of toxic molecules within the host macrophage that include copper, zinc, and nitric oxide (Botella et al., 2011; Darwin, 2015; Darwin et al., 2003; Darwin and Nathan, 2005; Shi et al., 2014). Although signal transduction systems that respond to each of these molecules have been identified, the true in vivo determinants of M. tuberculosis resistance to these molecules are unknown. Additionally, although stresses are often assayed monolithically in vitro, this approach belies the situation in vivo, which the pathogen must sense and respond to simultaneous chemically distinct stresses that vary in intensity over time. Our studies identify an integrated sensing system that M. tuberculosis uses to directly sense combinations of stresses using a single sensing circuit.

The hub of this signaling system is the PdtaS/R TCS. This TCS is distinct from previously characterized TCS in M. tuberculosis in both structure and mechanism of regulation. The sensor kinase, PdtaS, is cytosolic rather than membrane bound. PdtaS is constitutively active in vitro (Mehta et al., 2020), and our data indicates the mechanism of signal transduction is ligand-induced inhibition of this constitutive activity. Rather than being activated by its ligands, PdtaS is inhibited by copper and NO through a dicysteine motif in the N-terminal GAF domain, defining an integrated mechanism by which two chemical effectors are sensed. The constitutively active PdtaS/R system negatively regulates a subset of genes required for NO resistance and relief of this inhibition by ligand-induced shutoff of PdtaS is necessary, but not sufficient, for expression of genes required NO resistance.

Our data also strongly demonstrates that full inhibition of PdtaS/R signaling requires the transduction of a cell surface signal by the intramembrane protease Rip1. Rip1 was originally identified as a S2P required for both acute growth in the mouse lung and persistence of the bacterium during chronic infection (Makinoshima and Glickman, 2005). Four anti-sigma factor substrates have been identified, but none of these previously identified pathways explain the virulence defects of M. tuberculosis lacking Rip1. The data presented here indicate that critical virulence function of Rip1 is to relieve PdtaR repression through control of a previously unrecognized small RNA at the 5′ end of the PPE1 gene. Rip1-controlled expression of PPE1 5′ is required for NO resistance as re-expression of this element is sufficient to reverse the NO sensitivity of Δrip1 and to re-express the chalkophore operon, itself sufficient to mediate NO resistance. The PPE1-5′ RNA binds directly to PdtaR in vitro, and this binding is enhanced, but does not absolutely require, PdtaR phosphorylation by PdtaS. This data, coupled with our genetic data, indicates that Rip1 control of PPE1-5′ directly inhibits PdtaR and de-represses PdtaR targets, which are ordinarily repressed by the PdtaS/R cascade. This mechanism of RNA-based titration of ANTAR domain RRs away from competitor target RNAs has been described as a mechanism of regulation in Enterococcus and Listeria (DebRoy et al., 2014; Mellin et al., 2014). This model suggests a branched logic of the Rip1/PdtaS/PdtaR system. Two signals are required to fully inactivate the PdtaS/R system and mediate NO resistance. A positive Rip1 transcriptional signal from the membrane to produce PPE1-5′ to sequester PdtaR, and a cytoplasmic inhibitory signal through the PdtaS kinase mediated by direct sensing of NO by the PdtaS GAF domain, which inhibits kinase activity. Our findings that PdtaR phosphorylation is not absolutely required for its function in NO resistance in vivo or RNA binding in vitro supports the model that two signals, PdtaS inhibition and PdtaR titration by the PPE1-5′ RNA, cooperate to titrate the activity of the PdtaS/R system. Our integrated model of this system is presented in Figure 7.

Model of the Rip1/PdtaS/PdtaR signaling circuit and its response to copper and nitric oxide.

NO and copper resistance is mediated by cell surface and cytoplasmic convergent signals that inactivate PdtaS/PdtaR. In basal conditions, PdtaS/R is constitutively active, and that activity holds virulence genes, including chalkophore biosynthesis, inactive. NO or copper directly inhibit PdtaS activity through direct sensing by PdtaS N-terminal GAF domain. NO shutoff of PdtaS kinase activity requires C53 and 57, whereas Cu shutoff requires C53 with the role of C57 unknown. Sensing of NO at the cell surface contributes a second signal to relief of PdtaS/R by inducing Rip1-dependent expression of a hairpin containing small RNA at the 5′ end of the PPE1 gene (PPE1-5′), which binds directly to the ANTAR domain containing PdtaR. These signals relieve PdtaS/R repression of virulence gene expression, including chalkophore biosynthesis, to activate NO resistance. Although our data clearly implicates the upstream Rip1/PdtaS/PdtaR cascade in Cu resistance, the downstream targets that mediate Cu resistance are not known (indicated by ?). Despite the high Cu affinity of the isonitriles produced by the nrp locus, these molecules are involved in the NO resistance arm of the pathway rather than Cu. Figure constructed with BioRender.

Our findings also clearly indicate that the coupled Rip1/PdtaS/R circuit controls Cu and NO resistance, but the gene sets downstream of this cascade that mediate resistance to each ligand are distinct. The NO resistance is due to regulation of isonitrile chalkophore biosynthesis. These lipopeptide molecules were recently defined in Streptomyces and Mycobacterium marinum as the products of the Nrp nonribosomal peptide synthase (Harris et al., 2017; Wang et al., 2017). The nrp gene has also been identified in multiple studies as a determinant of virulence (Bhatt et al., 2018), although the mechanism was not defined. The structure of the TB chalkophore is not known, but the Streptomyces molecule binds copper with extremely high affinity (Xu and Tan, 2019). Despite this Cu binding and an expectation that this property would predict a role in Cu resistance, our data instead indicate that these molecules are not acting to diminish copper toxicity and rather that their function is to ameliorate or prevent NO toxicity. The exact mechanisms by which chalkophores contribute to NO resistance in M. tuberculosis are not defined here, but in Methanotrophs chalkophores help maintain the activity of Cu-containing enzymes in the membrane (Kenney and Rosenzweig, 2018). An analogous function in mycobacteria might imply that NO stress displaces Cu from thiol metal centers, an activity that has been shown with MymT (Gold et al., 2008), and that chalkophores may compensate for this toxicity. This hypothesis and others will require further characterization of chalkophore functions in mycobacteria.

Although the Rip1/PdtaS/PdtaR cascade jointly controls NO and Cu resistance, we did not identify the chromosomal targets of PdtaS/R that mediate the Cu arm of the pathway. The Cu resistance controlled by this signaling cascade is distinct from known mechanisms of Cu binding or efflux based on our genetic data. Our data showing that NO-induced transcription of the PPE1-5′ RNA is the second signal in the NO resistance pathway, but not Cu resistance, leads us to hypothesize that as yet unidentified Rip1-controlled small RNA titrates away PdtaR from targets involved in Cu resistance. This model (Figure 7) implies that the target RNAs controlled by PdtaR may be modified based on the activating stress, a hypothesis that will be pursued in future work to determine the full complement of RNAs binding to PdtaR under Cu and NO stress.

In summary, our results identify a previously unrecognized mechanism of bacterial signal transduction that allows Mtb to rapidly adapt to the toxic environment of the host lung. This study opens a window into several important future questions, including the full spectrum of RNAs bound by the PdtaR RR, the ultimate mechanism by which this sensing circuit controls NO resistance, and the mechanisms of PdtaS sensing of ligand. These future questions will add additional detail to the critical mechanisms of TB pathogenesis identified here and potentially identify a critical pathway for therapeutic development against Mtb.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Mycobacterium tuberculosis)M.tb Erdman (WT, EG2)Lab StockATCC 35801Animal passaged
Strain, strain background (Escherichia coli)DH5αLab StockATCC SCC2197Plasmid maintenance strain
Strain, strain background (Escherichia coli)EL350/ phAE87Lab StockPhage packaging strain
Strain, strain background (Escherichia coli)Rosetta 2 (DE3)Millipore SigmaCat# 71397Recombinant protein expression strain
Strain, strain background (Mus musculus) femaleC57BL/6JJackson LaboratoryStock no: 000664; RRID:IMSR_JAX:000664
Strain, strain background (Mus musculus) femaleB6;129P2-Nos2tm1Lau/JJackson LaboratoryStock no: 002596; RRID:IMSR_JAX:002596
Gene (M. tuberculosis)rip1ATCC 35801Erdman_3146
Gene (M. tuberculosis)sigKATCC 35801Erdman_0488
Gene (M. tuberculosis)sigLATCC 35801Erdman_0808
Gene (M. tuberculosis)sigMATCC 35801Erdman_4291
Gene (M. tuberculosis)sigDATCC 35801Erdman_3735
Gene (M. tuberculosis)mymTATCC 35801Rv0186Ant217703-217864 Erdman Genome
Gene (M. tuberculosis)ctpVATCC 35801Erdman_1076
Gene (M. tuberculosis)csoRATCC 35801Erdman_1074
Gene (M. tuberculosis)nrpATCC 35801Erdman_0118
Gene (M. tuberculosis)ppe1ATCC 35801Erdman_0113
Gene (M. tuberculosis)rv0097ATCC 35801Erdman_0114
Gene (M. tuberculosis)fcoTATCC 35801Erdman_0115
Gene (M. tuberculosis)fadD10ATCC 35801Erdman_0116
Gene (M. tuberculosis)rv0100ATCC 35801Erdman_0117
Gene (M. tuberculosis)pdtaSATCC 35801Erdman_3533
Gene (M. tuberculosis)pdtaRATCC 35801Erdman_1787
Genetic reagent (M. tuberculosis)Δrip1Sklar et al., 2010MGM3206; rip1 KOChromosomal deletion of Erdman_3146 nt5-1212 by double crossover recombination followed by marker excision by LoxP recombination
Genetic reagent (M. tuberculosis)ΔsigDSchneider et al., 2014JSS0005; sigD KOChromosomal deletion of Erdman_3735 nt6-622 by double crossover recombination
Genetic reagent (M. tuberculosis)ΔsigKSklar et al., 2010MGM3259; sigK KO
Genetic reagent (M. tuberculosis)ΔsigLSklar et al., 2010MGM3254; sigL KO
Genetic reagent (M. tuberculosis)ΔsigMSklar et al., 2010MGM3260, sigM KO
Genetic reagent (M. tuberculosis) sigL sigM KOΔsigLΔsigMSklar et al., 2010MGM3255; sigL sigM KO
Genetic reagent (M. tuberculosis)ΔsigKΔsigLΔsigMSchneider et al., 2014MGM3256; sigK sigL sigM KO
Genetic Reagent (M. tuberculosis)ΔsigKΔsigLSklar et al., 2010MGM3261; sigK sigL KO
Genetic reagent (M. tuberculosis)ΔsigKΔsigMSklar et al., 2010MGM3283; sigK sigM KO
Genetic reagent (M. tuberculosis)ΔsigKΔsigDThis study, available from corresponding authorsigK::loxP sigD::hygR; MGM3288Chromosomal deletion of Erdman_3735 nt6-622 by double crossover recombination MGM3259 background
Genetic reagent (M. tuberculosis)ΔsigLΔsigDThis study, available from corresponding authorsigL::loxP sigD::hygR; MGM3289Chromosomal deletion of Erdman_3735 nt6-622 by double crossover recombination MGM3254 background
Genetic reagent (M. tuberculosis)ΔsigMΔsigDThis study, available from corresponding authorsigM::loxP sigD::hygR; MGM3290Chromosomal deletion of Erdman_3735 nt6-622 by double crossover recombination MGM3260 background
Genetic reagent (M. tuberculosis)ΔctpVThis study, available from corresponding authorctpV::hygRChromosomal deletion of nt 175-2136 of Erdman_1076 by double crossover recombination
Genetic reagent (M. tuberculosis)ΔcsoRThis study, available from corresponding authorcsoR::hygRChromosomal deletion of nt 1-360 of Erdman_1074 by double crossover recombination
Genetic reagent (M. tuberculosis)ΔmymTThis study, available from corresponding authormymT::hygRChromosomal deletion of nt 1-162 of mymT by double crossover recombination
Genetic reagent (M. tuberculosis)Δrip1ΔctpVThis study, available from corresponding authorrip1::loxP ctpV::hygRChromosomal deletion of nt 175-2136 of Erdman_1076 by double crossover recombination MGM3206 background
Genetic reagent (M. tuberculosis)Δrip1ΔcsoRThis study, available from corresponding authorrip1::loxP csoR::hygRChromosomal deletion of nt 1-360 of Erdman_1074 by double crossover recombination MGM3206 background
Genetic reagent (M. tuberculosis)Δrip1ΔmymTThis study, available from corresponding authorrip1::loxP mymT::hygRChromosomal deletion of nt 1-162 of mymT by double crossover recombination MGM 3206 background
Genetic reagent (M. tuberculosis)ΔpdtaSThis study, available from corresponding authorpdtaS::hygRChromosomal deletion of nt1-1506 of Erdman_3533 by double crossover recombination
Genetic reagent (M. tuberculosis)ΔpdtaS + VectorThis study, available from corresponding authorpdtaS::hygR:pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (M. tuberculosis)ΔpdtaRThis study, available from corresponding authorpdtaR::hygRChromosomal deletion of nt 1-576 of Erdman_1787 by double crossover recombination
Genetic reagent (M. tuberculosis)ΔpdtaR + vectorThis study, available from corresponding authorpdtaR::hygR:pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (M. tuberculosis)ΔnrpThis study, available from corresponding authornrp::hygRChromosomal deletion of nt 21-7515 of Edman_0118 by double crossover recombination
Genetic reagent (M. tuberculosis)Δnrp + vectorThis study, available from corresponding authornrp::hygR: pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (M. tuberculosis)Δnrp + WT nrpThis study, available from corresponding authornrp::hygR: WT nrpWT copy of nrp integrated at AttB, Erdman_0116 promoter
Genetic reagent (M. tuberculosis)Δrip1ΔpdtaSThis study, available from corresponding authorrip1::loxP pdtaS::hygRChromosomal deletion of nt1-1506 of Erdman_3533 by double crossover recombination MGM3206 background
Genetic reagent (M. tuberculosis)Δrip1ΔpdtaS + vectorThis study, available from corresponding authorrip1::loxP pdtaS::hygR: pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1ΔpdtaRThis study, available from corresponding authorrip1::loxP pdtaR::hygRChromosomal deletion of nt 1-576 of Erdman_1787 by double crossover recombination MGM3206 background
Genetic reagent (M. tuberculosis)Δrip1ΔpdtaR + vectorThis study, available from corresponding authorrip1::loxP pdtaR::hygR: pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1ΔpdtaR + WT pdtaRThis study, available from corresponding authorrip1::loxP pdtaR::hygR: WT pdtaR HAWT copy of pdtaR integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1ΔpdtaR + D65A pdtaRThis study, available from corresponding authorrip1::loxP pdtaR::hygR: D65A pdtaR HAD65A variant of pdtaR integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1ΔnrpThis study, available from corresponding authorrip1::loxP nrp::hygRChromosomal deletion of nt 21-7515 of Edman_0118 by double crossover recombination MGM3206 background
Genetic reagent (M. tuberculosis)WT + vectorMakinoshima and Glickman, 2005EG2: pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (M. tuberculosis)WT + vectorMakinoshima and Glickman, 2005EG2:pMV261Transformed with empty pMV261K episomal plasmid
Genetic reagent (M. tuberculosis)Δrip1 + vectorSklar et al., 2010Δrip1: pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1 + vectorSklar et al., 2010rip1::loxP: pMV261Transformed with empty pMV261K episomal plasmid
Genetic reagent (M. tuberculosis)Δrip1 + WT Rip1Sklar et al., 2010 Makinoshima and Glickman, 2005rip1::loxP: WT rip1Transformed with WT rip1 pMV261K episomal plasmid
Genetic reagent (M. tuberculosis)Δrip1 + H21A Rip1Makinoshima and Glickman, 2005rip1::loxP: H21A rip1Transformed with H21A variant rip1 pMV261K episomal plasmid
Genetic reagent (M. tuberculosis)Δrip1: PPE1 ORF pHSP60This study, available from corresponding authorrip1::loxP: PPE1 GFP ORF onlyTransformed with PPE1 GFP ORF only pMV261K episomal plasmid
Genetic reagent (M. tuberculosis)Δrip1: PPE1 ORF + 5' pHSP60This study, available from corresponding authorrip1::loxP: PPE1 GFP ORF + 5' UTR pHSP60Transformed with PPE1 GFP + 5' UTR pMV261K episomal plasmid
Genetic reagent (M. tuberculosis)Δrip1: Rv0097-nrp pHSP60This study, available from corresponding authorrip1::loxP: Rv0097-nrpTransformed with Rv0097-nrp pMV261K episomal plasmid
Genetic reagent (M. tuberculosis)Δrip1 pdtaS F37XThis study, available from corresponding authorrip1::loxP pdtaS F37XIsolated spontaneous Erdman_3533 variant in MGM3206 background
Genetic reagent (M. tuberculosis)Δrip1 pdtaS F37X + vectorThis study, available from corresponding authorrip1::loxP pdtaS F37X: pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1 pdtaS F37X + pdtaSThis study, available from corresponding authorrip1::loxP pdtaS F37X: WT pdtaS 10xHisWT pdtaS 10xHis integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1 pdtaS F37X + pdtaS V54FThis study, available from corresponding authorrip1::loxP pdtaS F37X: V54F pdtaS 10xHisV54F variant of Erdman_3533 10xHis integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1 pdtaS F37X + pdtaS H302Q/H303QThis study, available from corresponding authorrip1::loxP pdtaS F37X: H302Q/H303Q pdtaS 10xHisH302Q/H303Q variant of Erdman_3533 10xHis integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1 pdtaS F37X + pdtaS G443A/G445AThis study, available from corresponding authorrip1::loxP pdtaS F37X: G443A/G445A pdtaS 10xHisG443A/G445A variant of Erdman_3533 10xHis integrated at AttB
Genetic reagent (M. tuberculosis)Δrip1 pdtaS V54FThis study, available from corresponding authorrip1::loxP pdtaS V54FIsolated spontaneous Erdman_3533 variant in MGM3206 background
Genetic reagent (M. tuberculosis)Δrip1 pdtaS V54F + vectorThis study, available from corresponding authorrip1::loxP pdtaS V54F: pMV306KEmpty pMV306K integrated at AttB
Genetic reagent (E. coli)Rosetta 2 DE3: pET WT PdtaSThis study, available from corresponding authorRosetta 2 DE3: pET WT PdtaST7lac recombinant expression of C-terminally 10xHis tagged protein
Genetic reagent (E. coli)Rosetta 2 DE3: pET WT PdtaS kinase domainThis study, available from corresponding authorRosetta 2 DE3: pET WT PdtaS kinase domainT7lac recombinant expression of C-terminally 10xHis tagged protein
Genetic reagent (E. coli)Rosetta 2 DE3: pET C53A PdtaSThis study, available from corresponding authorRosetta 2 DE3: pET C53A PdtaST7lac recombinant expression of C-terminally 10xHis tagged protein
Genetic reagent (E. coli)Rosetta 2 DE3: pET C57A PdtaSThis study, available from corresponding authorRosetta 2 DE3: pET C57A PdtaST7lac recombinant expression of C-terminally 10xHis tagged protein
Genetic reagent (E. coli)Rosetta 2 DE3: pET WT PdtaRThis study, available from corresponding authorRosetta 2 DE3: pET WT PdtaRT7lac recombinant expression of C-terminally 10xHis tagged protein
Genetic reagent (E. coli)Rosetta 2 DE3: pET D65A PdtaRThis study, available from corresponding authorRosetta 2 DE3: pET D65A PdtaRT7lac recombinant expression of C-terminally 10xHis tagged protein
Recombinant DNA reagentpET SUMO (plasmid)Reverter and Lima, 2009pET SUMOT7lac promoter E. coli expression vector
N-terminal 10xHIS Sumo Fusion Protein
Recombinant DNA reagentpET (plasmid)This study, available from corresponding authorpETT7lac promoter E. coli expression vector
Recombinant DNA reagentPdtaS (plasmid)This study, available from corresponding authorpET WT PdtaS 10XHISnt 1-1503 of Erdman_3533 fused N-terminal to Enterkinase cleavage site followed by 10xHis tag
Recombinant DNA reagentPdtaS C53A (plasmid)This study, available from corresponding authorpET C53A PdtaS 10XHISnt 1-1503 of Erdman_3533 C53A variant fused N-terminal to Enterkinase cleavage site followed by 10xHis tag
Recombinant DNA reagentPdtaS C57A (plasmid)This study, available from corresponding authorpET C57A PdtaS 10XHISnt 1-1503 of Erdman_3533 C57A variant fused N-terminal to Enterkinase cleavage site followed by 10xHis tag
Recombinant DNA reagentPdtaS kinase domain (plasmid)This study, available from corresponding authorpET WT PdtaS kinase domain 10XHISnt 678-1503 of Erdman_3533 variant fused N-terminal to Enterkinase cleavage site followed by 10xHis tag
Recombinant DNA reagentPdtaR D65A (plasmid)This study, available from corresponding authorpET Sumo WT PdtaRN-terminal 10xHIS Smt3 fused to nt 1-615 of Erdman_1787
Recombinant DNA reagentPdtaR D65A (plasmid)This study, available from corresponding authorpET Sumo D65A PdtaRN-terminal 10xHIS Smt3 fused to nt 1-615 of Erdman_1787 D65A variant
Recombinant DNA reagentVector (plasmid)Sklar et al., 2010 Makinoshima and Glickman, 2005pMV261KEpisomal M.tb plasmid
Recombinant DNA reagentVector (plasmid)Sklar et al., 2010 Makinoshima and Glickman, 2005pMV306KAttB integrating M.tb plasmid
Recombinant DNA reagentpMV261K GFP (plasmid)This study, available from corresponding authorpMV261K GFPEpisomal M.tb plasmid for C-terminal GFP fusion constructs
Recombinant DNA reagentWT rip1 (plasmid)Makinoshima and Glickman, 2005pHMG121
Recombinant DNA reagentH21A rip1 (plasmid)Makinoshima and Glickman, 2005pHMG141
Recombinant DNA reagentpdtaS (plasmid)This study, available from corresponding authorpMV306K WT PdtaS 10xHisnt 1-1503 of Erdman_3533 tagged at C-term with 10x His
Recombinant DNA reagentpdtaS V54F (plasmid)This study, available from corresponding authorpMV306K V54F PdtaS 10xHisnt 1-1503 of Erdman_3533 tagged at C-term with 10x His V54F variant
Recombinant DNA reagentpdtaS H302Q/H303Q (plasmid)This study, available from corresponding authorpMV306K H302Q/H303Q PdtaS 10xHisnt 1-1503 of Erdman_3533 tagged at C-term with 10x His H302Q/H303Q variant
Recombinant DNA reagentpdtaS G443A/G445A (plasmid)This study, available from corresponding authorpMV306K G443A/H445A PdtaS 10xHIsnt 1-1503 of Erdman_3533 tagged at C-term with 10x His G443A/G445A variant
Recombinant DNA reagentpdtaR (plasmid)This study, available from corresponding authorpMV306K WT PdtaR HAnt 1-615 of Edman_1787 tagged at C-term with HA epitope
Recombinant DNA reagentpdtaR D65A (plasmid)This study, available from corresponding authorpMV306K D65A PdtaR HAnt 1-615 of Edman_1787 tagged at C-term with HA epitope D65A variant
Recombinant DNA reagentPPE1 ORF pHSP60 (plasmid)This study, available from corresponding authorpMV261K ORF PPE1 GFPnt 1-1389 of Erdman_0113 tagged at C-term with GFP
Recombinant DNA reagentPPE1 ORF + 5' pHSP60 (plasmid)This study, available from corresponding authorpMV261K ORF PPE1 GFP + 5'PPE1nt -222-1389 of Erdman_0113 tagged at C-term with GFP
Recombinant DNA reagentRv0097-nrp pHRP60 (plasmid)This study, available from corresponding authorpMV261K Rv0097-nrpnt 1 of Erdman_0114 to nt 7539 of Erdman_0118
Recombinant DNA reagentWT nrp (plasmid)This study, available from corresponding authorpMV306 WT nrpnt 1-96 of Erdman_0116 (predicted TSS Wadsworth) + nt 1-7539 of Erdman_0118
Recombinant DNA reagentpmsg360Hyg (plasmid)Sklar et al., 2010 Makinoshima and Glickman, 2005pmsg360HygPhage packaging vector for hygR allelic exchange
Recombinant DNA reagentpmsg360Zeo (plasmid)Sklar et al., 2010 Makinoshima and Glickman, 2005pmsg360ZeoPhage packaging vector for zeoR allelic exchange
Recombinant DNA reagentpmsg318-2 (plasmid)Sklar et al., 2010 Makinoshima and Glickman, 2005pmsg318-2M.tb Cre recombinase expression vector
Recombinant DNA reagentpmsg360Hyg CtpV flanks (plasmid)This study, available from corresponding authorΔctpV targeting vectorHyg cassette flanked by 616 bp 5' CtpV nt 175 + 600 bp 3' CtpV nt 2136
Recombinant DNA reagentpmsg360 Hyg CsoR flanks (plasmid)This study, available from corresponding authorΔcsoR targeting vectorHyg cassette flanked by 600 bp 5' CsoR start codon + 600 bp 3' of CsoR stop codon
Recombinant DNA reagentpmsg360 Hyg MymT flanks (plasmid)This study, available from corresponding authorΔmymT targeting vectorHyg cassette flanked by 600 bp 5' MymT start codon + 600 bp 3' of MymT stop codon
Recombinant DNA reagentpmsg360 Hyg pdtaS flanks (plasmid)This study, available from corresponding authorΔpdtaS targeting vectorHyg cassette flanked by 600 bp 5' PdtaS start codon + 579 bp 3' PdtaS stop codon
Recombinant DNA reagentpmsg360 Hyg pdtaR flanks (plasmid)This study, available from corresponding authorΔpdtaR targeting vectorHyg cassette flanked by 600 bp 5' PdtaR start codon + 647 bp 3' PdtaR nt 576
Recombinant DNA reagentpmsg360 Hyg nrp Flanks (plasmid)This study, available from corresponding authorΔnrp targeting vectorHyg cassette flanked by 621 bp 5' nrp nt 21+ 621 bp 3' nrp nt 7515
Sequence-based reagentoSigA-1Integrated DNA TechnologiesqPCR primercgtcttcatcccagacgaaat
Sequence-based reagentoSigA-2Integrated DNA TechnologiesqPCR primercgacgaagaccacgaagac
Sequence-based reagentoCtpV-1Integrated DNA TechnologiesqPCR primergtgtcccatgttcgaggtcaa
Sequence-based reagentoCtpV-2Integrated DNA TechnologiesqPCR primergtcaatgttcttcggtgcttac
Sequence-based reagentoLpqS-1Integrated DNA TechnologiesqPCR primergcatcgagttgtccaccag
Sequence-based reagentoLpqS-2Integrated DNA TechnologiesqPCR primertcaatgtggctcacccaaac
Sequence-based reagentoMymT-1Integrated DNA TechnologiesqPCR primergggtgatacgaatgacgaacta
Sequence-based reagentoMymT-2Integrated DNA TechnologiesqPCR primeracagtggcatgggacttc
Sequence-based reagento2625c-FIntegrated DNA TechnologiesqPCR primertcttgatcgcgttgggattg
Sequence-based reagento2625c-RIntegrated DNA TechnologiesqPCR primercccggcgaattgatgtagag
Sequence-based reagentoDesR-FIntegrated DNA TechnologiesqPCR primertctgatcctcacgtcctacac
Sequence-based reagentoDesR-RIntegrated DNA TechnologiesqPCR primeragcgcccacatctttgac
Sequence-based reagentoHspX-FIntegrated DNA TechnologiesqPCR primergaattcgcgtacggttccttc
Sequence-based reagentoHspX-RIntegrated DNA TechnologiesqPCR primergccaccgacacagtaagaatg
Sequence-based reagentoPdtaS_C53A-FIntegrated DNA TechnologiesPCR primergcgacgacggtgtcctggtggcggttgcg
Sequence-based reagentoPdtaS_C53A-RIntegrated DNA TechnologiesPCR primercgcaaccgccaccaggacaccgtcgtcgc
Sequence-based reagentoPdtaS_C57A-FIntegrated DNA TechnologiesPCR primergcgcaagcccggccgaacaccgggccgacg
Sequence-based reagentoPdtaS_C57A-RIntegrated DNA TechnologiesPCR primercgtcggcccggtgttcggccgggcttgcgc
Sequence-based reagentoPdtaR_D65A-FIntegrated DNA TechnologiesPCR primergtgatcatggccgtgaaga
Sequence-based reagentoPdtaR_D65A-RIntegrated DNA TechnologiesPCR primertcttcacggccatgatcac
Sequence-based reagentoPdtaS_H302Q/H303Q-FIntegrated DNA TechnologiesPCR primergggaaatccagcagcgggtt
Sequence-based reagentoPdtaS_H302Q/H303Q-RIntegrated DNA TechnologiesPCR primeraacccgctgctggatttccc
Sequence-based reagentoPdtaS_G443A/G445A-FIntegrated DNA TechnologiesPCR primeracgacgcgcttgctctgccg
Sequence-based reagentoPdtaS_G443A/G445A-FIntegrated DNA TechnologiesPCR primercggcagagcaagcgcgtcgt
Sequence-based reagentSense5'PPE1-FIntegrated DNA TechnologiesPCR primer; in vitro transcriptiontaatacgactcactatagggggccgactaacaccgcgg
Sequence-based reagentSense5'PPE1-RIntegrated DNA TechnologiesPCR primer; in vitro transcriptionggtttgctcaagccaggc
Sequence-based reagentRv3864-FIntegrated DNA TechnologiesPCR primer; in vitro transcriptiontaatacgactcactataggcaaaaaattcgtgcaccaacc
Sequence-based reagentRv3864-RIntegrated DNA TechnologiesPCR primer; in vitro transcriptiontttccttacgctcgccgt
Sequence-based reagentAntiDNAK-FIntegrated DNA TechnologiesPCR primer; in vitro transcriptiongatccacctagttctagaatggctcgtgcggtcg
Sequence-based reagentAntiDNAK-RIntegrated DNA TechnologiesPCR primer; in vitro transcriptiontaatacgactcactatagggggcgtcattgaagtaggcg
AntibodyAnti E. coli RNA polymerase B antibody (α-Rpoβ)BioLegend663903 (RRID:AB_2564414)1:10,000 dilution
AntibodyAnti-HA.11 epitope tag antibody (α-HA)BioLegend901513 (RRID:AB_2565335)1:1000 dilution
Antibodyα-CarD (Rabbit)Pocono Rabbit FarmStallings et al., 20091:10,000 dilution
Antibodyα-MymT (Rabbit)Gold et al., 20081:1000 dilution
AntibodyAnti-GFP(Rabbit) Antibody (α-GFP)Rockland600-401-215L (RRID:AB_2612813)1:1000 dilution
Chemical compound, drugSpermine NONOateMillipore Sigma567703
Chemical compound, drugDiethylenetriamine/nitric oxide adduct (DETA-NO)Millipore SigmaD185
Chemical compound, drugHydrogen peroxide, 30%Fisher ScientificH325-100
Chemical compound, drugSodium hydrosulfide hydrateFisher ScientificAC296200250
Commercial assay, kitIn-Fusion HD Cloning KitTakara Bio USA639650
Commercial assay, kitMaxima H Minus cDNA Synthesis Master Mix, with dsDNaseThermo Fisher ScientificM1681
Commercial assay, kitDyNAmo Flash SYBR Green qPCR KitThermo Fisher ScientificF415F
Commercial assay, kitHiScribe T7 High Yield RNA Synthesis KitNew England BiolabsE2040S
Commercial assay, kitRibo-Zero Magnetic Bacterial KitEpicentreMRZB12424
Commercial assay, kitTruSeq Stranded Total RNA kitIllumina20020599
Commercial assay, kitKAPA Hyper Prep KitRoche7962312001
Commercial assay, kitTruSeq SBS Kit v3IlluminaFC-401-3002
Commercial assay, kitGeneJET RNA Purification KitFisher ScientificFERK0731
Commercial assay, kitTURBO DNA-free kitFisher ScientificAM1907
Peptide, recombinant proteinPhusion High Fidelity PolymeraseFisher ScientificF530L
Commercial assay, kitGeneJET Plasmid Miniprep KitFisher ScientificFERK0503
Commercial assay, kitNanoTemper Technologies Inc PROTEIN LABELING KIT RED-NHS (MOL011)Fisher ScientificNC1491187
Commercial assay, kitNanoTemper Technologies Inc
Standard capillaries
Fisher ScientificNC1408770
Software, algorithmViennaRNA Web Serviceshttp://www.viennarna.at/forna/; RRID:SCR_008550
Software, algorithmfastqchttp://www.bioinformatics.babraham.ac.uk/projects/fastqcRRID:SCR_014583
Software, algorithmbwa memLi et al., 2009RRID:SCR_010910
Software, algorithmsamtoolsLi et al., 2009RRID:SCR_002105
Software, algorithmBioconductor Rsubread packageLiao et al., 2019RRID:SCR_016945
Software, algorithmDESeq2 R packageLove et al., 2014RRID:SCR_015687
Software, algorithmRThe R Project for Statistical Computing; https://www.R-project.org/RRID:SCR_001905
Software, algorithmGATKBroad Institute; https://gatk.broadinstitute.org/hc/en-usRRID:SCR_001876
Software, algorithmpheatmaphttps://www.rdocumentation.org/packages/pheatmap/versions/0.2/topics/pheatmapRRID:SCR_016418
OtherNUPAGE 4-12% BT GelFisher ScientificNPO312BOX/NPO336BOX
OtherProtran Nitrocellulose Hybridization Transfer MembranePerkin ElmerNBA08C001EA
OtherImmun-Blot PVDF MembraneBio-Rad1620177
OtherNanoTemper Technologies Premium CapillariesFisher ScientificNC1408772
OtherHisProbe-HRP ConjugateThermo Fisher Scientific15165

Reagents

Media and salts were purchased from Thermo Fisher, USA. Glycine, imidazole, and ATP from Merck Sigma-Aldrich, USA. Protein marker from Thermo Scientific, USA. Antibiotics, isopropyl β-D-1-thiogalactopyranoside (IPTG) and dithiothreitol (DTT) from GoldBio Inc, USA. Protease inhibitor cocktail from Amresco, USA. Ni2+-NTA resin from Qiagen, GmBH. γ32P-labeled ATP (>3000 Ci/mmol) from Perkin Elmer, USA.

General growth conditions, strains, and DNA manipulations

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M. tuberculosis (Erdman) and derivatives were grown and maintained in 7H9 media (broth) or on 7H10 (agar) supplemented with 10% Oleic Acid-Albumin-Dextrose-Catalase supplement (OADC), 05% glycerol, and 0.05% Tween-80(broth only) (7H9 OADC/7H10 OADC) at 37°C unless otherwise noted below. The wild-type strain used in this study is animal passaged, minimally passaged in vitro, and confirmed to be phthicerol dimycocerosates (PDIM) positive both by metabolic labeling and without mutations in PDIM biosynthesis by whole genome sequencing. Deletion mutations were generated by specialized transduction utilizing the temperature-sensitive phage phAE87. Mutant strains were confirmed by Southern blot or PCR using primers in the Hyg cassette and outside the cloned region, followed by sequencing of the amplified PCR product to confirm the location of chromosomal insertion. For a complete strain list with relevant features, see Supplementary file 1. Plasmids utilized in this study were generated using standard molecular techniques and are listed with their features in Supplementary file 2.

Metal sensitivity assays

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For agar-based assays, duplicate logarithmically growing (OD600 of 0.4–0.6) cultures were centrifuged and washed twice with room temperature phosphate buffered saline (PBS) containing 0.05% Tween-80 (PBS Tween). Resulting washed suspensions were all adjusted to OD600 of 0.2 and serial diluted in PBS Tween. Dilutions were then plated onto control unsupplemented 7H10 OADC agar media, as well as media supplemented with the indicated concentration of copper (II) sulfate or iron (III) chloride. Colony-forming units (CFUs) were enumerated after 28 days of growth at 37°C, 5% CO2. Relative survival was calculated by dividing the average number of CFU on treatment plates by the average number of CFU on control plates. Experiments were repeated a minimum of two biological replicates defined as individual samples treated independently throughout. Where applicable, technical replicate refers to repeated quantification of identical biological samples to account for error during dilutions for agar plate cultures.

Liquid growth curve assays were pre-grown and washed as above. Growth curves were started at an initial OD600 of 0.005 by adding 1 mL of washed culture, at an OD600 of 0.05, to 9 mL of 7H9 OADC supplemented with 100 μM zinc (II) sulfate. Biological replicates defined as individual samples treated independently throughout.

For copper liquid sensitivity CFU assays, 25 mL cultures were pre-grown to an OD600 of 0.8–1.0 in 7H9 OADC media. Cultures were washed twice in 7H9 supplemented with albumin dextrose saline, 05% glycerol, 02% Tween-80 (7H9 ADS), and normalized to an OD600 of 0.1 in four-replicate 10 mL cultures. After equilibration at 37°C for 2 hr, duplicate cultures were treated with 200 µM copper sulfate (Sigma-Aldrich), while control cultures were left untreated. On days 0 and 3 post treatment, aliquots of each culture were removed, washed, and serial diluted in PBS Tween. Dilutions were then plated on 7H10 OADC plates for CFU determination. CFUs were enumerated after 28 days of growth at 37°C, 5% CO2. Relative survival was calculated by dividing the average CFU day 3 by the average number of CFU on day 0. Biological replicates defined as individual samples treated independently throughout.

Nitric oxide sensitivity assays

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Duplicate 25 mL cultures were pre-grown to an OD600 between 0.8 and 1.0 in 7H9 OADC media. Cultures were washed twice in 7H9 ADS and normalized to an OD600 of 1.0 in 10 mL of 7H9 ADS. Four-replicate, 9 mL 7H9 ADS cultures were then inoculated with 1 mL of this suspension for a starting OD600 of 0.1 in 30 mL inkwell bottles (Fisher Scientific Cat# 03-313-89A) with ~2 in. of headspace. After equilibration at 37°C for 2 hr, duplicate cultures were treated with 100 μL of freshly prepared 20 mM DETA-NO, or 0.1 M sodium hydroxide (final concentration 1mM, vehicle for DETA-NO). Treatment was repeated every 24 hr for 3 days. Days 0 and 3 post treatment, aliquots of each culture were removed, washed, and serial diluted in PBS Tween. Dilutions were then plated on 7H10 OADC plates for CFU determination. CFUs were enumerated after 28 days of growth at 37°C, 5% CO2. Relative survival was calculated by dividing the average CFU day 3 by the average number of CFU on day 0. Experiments were repeated a minimum of two times. Biological replicates defined as individual samples treated independently throughout.

PBS starvation

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Cultures of the indicated strains were pre-grown in 7H9 OADC media to an OD of between 0.5 and 0.7. Cells were then collected by centrifugation and washed twice with PBS pH 7.0. Replicate flasks containing 30 mL of PBS were then inoculated to an OD of 0.1. Flasks were incubated standing in an incubator at 37°C with 5% CO2. At the indicated times, aliquots were removed, serially diluted in PBS, and plated onto 7H10 OADC agar plates. CFUs were enumerated after 28 days of incubation at 37°C. Biological replicates defined as individual samples treated independently throughout.

Lysozyme, hydrogen peroxide, and SDS sensitivity

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Cultures of the indicated strains were pre-grown in 7H9 OADC to an OD of between 0.5 and 0.7. Cells were then washed twice with 7H9 ADS media and diluted to an OD of 0.1 in the same media. 1 mL of washed culture was then inoculated into replicate 9 mL bottles of fresh 7H9 ADS media (untreated control) or 7H9 ADS media containing 2.5 mg/mL lysozyme, 10 mM hydrogen peroxide, or 0.05% (v/v) sodium dodecyl sulfate (SDS). Following 3 hr of treatment at 37°C, aliquots from each culture were removed, serial diluted in 7H9 ADS media, and then spread onto 7H910 OADC plates. CFUs were enumerated following 28 days of incubation at 37°C. Biological replicates defined as individual samples treated independently throughout.

Survival at pH 4.5

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Cultures of the indicated strains were pre-grown in 7H9 OADC to an OD of between 0.5 and 0.7. Cultures were then divided in half and washed with either 7H9 ADS-pH 7.0 or 7H9 ADS-pH 4.5. Replicate 10 mL bottles of pH 4.5 or pH 7.0 media were then inoculated with washed culture at on OD of 0.01. Aliquots of each culture were removed immediately after inoculation, and after 7 days of growth at 37°C, serial diluted, and then plated onto 7H10 OADC plates. CFUs were enumerated following 28 days of incubation at 37°C. Biological replicates defined as individual samples treated independently throughout.

Copper-induced MymT expression

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MymT expression was induced as follows: cultures grown in 7H9 OADC to an OD600 of ~0.5 were divided into 20 mL replicate aliquots. Copper sulfate (0–500 µM) was then added to individual replicates followed by 2 hr incubation at 37°C. Following incubation, cells were processed for immunoblot as described in Materials and methods, excepting that the PVDF membrane was used for blotting.

RNA extraction and RT-qPCR mRNA levels were quantified as follows. For copper-stimulated transcripts, triplicate 40 mL cultures grown to an OD600 of 0.4 in 7H9 OADC were divided into two sets of 3, 20 mL cultures. Half were left untreated for quantification of basal transcript level, while the other half were treated with 200 μM copper (II) sulfate for 2 hr.

For nitric oxide-stimulated transcripts, triplicate 40 mL culture grown to an OD600 of 0.4 in 7H9 ADS were divided into two sets of 3X20 mL cultures. Half were then treated with 200 μL of 20 mM DETA-NO dissolved in 0.1 M sodium hydroxide for 3 hr or with vehicle (0.1 M sodium hydroxide)-alone control. Cells were collected by centrifugation, washed once in PBS Tween, and then suspended in 1 mL of Trizol (Invitrogen). Cells were mechanically disrupted with zirconia bead via 3 × 30 s pulses in a BioSpec Mini24 beadbeater. Total RNA was then extracted according to the manufacturer’s instructions. Contaminating genomic DNA was removed using the Turbo DNA-free kit (Invitrogen), and RNA was then further purified utilizing RNeasy Mini spin columns (Qiagen). 1 μg of resulting total RNA was used to synthesize cDNA via random priming utilizing the Maxima H-Minus cDNA Synthesis kit (Thermo Fisher). Real-time qPCR was performed on a 7500 real-time PCR system (Applied Biosystems). Amplification product was detected by SYBR green using the Dynamo Flash qPCR kit (Thermo Fisher). For each gene of interest (GOI)-normalized cycle threshold, C(t) was determined relative to the housekeeping gene sigA. Relative expression level was calculated using the formula 2^-(C(t)GOI-C(t)sigA). Primer sets used to amplify individual GOI are listed in Supplementary file 3.

Aerosol infection of mice

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Mice used in this study were purchased from The Jackson Laboratory. 8–10-week-old C57Bl/6J (stock number 00064) were used. iNOS ko mice were purchased from Jackson, stock number 002609. All purchased mice were rested within our animal facility to normalize microbiota for 2 weeks. Care, housing, and experimentation on laboratory mice were performed in accordance with the National Institute of Heath guidelines and the approval of the Memorial Sloan-Kettering Institutional Animal Care and Use Committee (IACUC). Protocol approval number is 01-11-030.

Strains for infection were grown to an OD600 of 0.5–0.7 in 7H9 OADC. They were then washed twice with PBS Tween followed by brief sonication to disrupt aggregates. Final inoculums were prepared by suspending 8 × 107 CFU in 10 mL of sterile water. Appropriate mouse strains were then exposed to 4 × 107 CFU in a Glas-Col aerosol exposure unit. At the indicated time point post infection, 4–8 individual mice were humanely euthanized and both lungs and spleens were harvested for CFU determination. Organ homogenates were cultured on 7H10 OADC plates, and CFU was enumerated after 28 days of incubation at 37°C, 5% CO2.

Suppressor screen and whole genome sequencing

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Spontaneous copper suppressor mutants were identified by selecting ~3 × 105 CFU of Δrip1 bacteria on each 7H10 OADC agar dish supplemented with 500 μM copper sulfate. A total of approximately 5x107 bacteria were selected. Plates were then incubated at 37°C, 5% CO2 for 28 days. Individual genetic suppressor candidates were picked and expanded to an OD600 of 0.5 in 40 mL of 7H9 OADC without addition of copper followed by confirmation of Cu resistance. Whole genome sequencing was performed acoustically shearing genomic DNA, and HiSeq sequencing libraries were prepared using the KAPA Hyper Prep Kit (Roche). PCR amplification of the libraries was carried out for 10 cycles. 5−10 × 106 50 bp paired-end reads were obtained for each sample on an Illumina HiSeq 2500 using the TruSeq SBS Kit v3 (Illumina). Post-run demultiplexing and adapter removal were performed and fastq files were inspected using fastqc (Andrews S. (2010). FastQC: a quality control tool for high-throughput sequence data; available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Trimmed fastq files were then aligned to the reference genome (M. tuberculosis str. Erdman; GenBank: AP012340.1) using bwa mem (Li and Durbin, 2009). Bam files were sorted and merged using Samtools (Li et al., 2009). Read groups were added and bam files de-duplicated using Picard tools, and GATK best practices were followed for SNP and indel detection (DePristo et al., 2011).

Immunoblotting

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Lysates for immunoblotting were prepared from 40 mL cultures grown in 7H9 OADC to an OD600 of between 0.5 and 0.8. Cells were cooled to 4°C on ice, centrifuged, and washed 1× with PBS. Washed pellets were then suspended in 0.4 mL of PBS, and ~100 μL of zirconia beads were added. Lysis was performed by 3, 45 s pulses in a BioSpec Mini24 beadbeater with 5 min intervening rest periods on ice. Beads were then removed by centrifugation at 1000×g for 5 min, and the resulting supernatant was mixed 1:1 with 2× Laemmli sample buffer supplemented with .1 M DTT. 20 μL of each sample, heated for 10 min at 100°C, was then separated on 4–12% NuPAGE bis-tris poly acrylamide gels. Separated proteins were then transferred to nitrocellulose and probed with the appropriate antibodies. Antibodies used in this study are monoclonal anti-HA.11 (BioLegend), monoclonal anti-E. coli RNA-polymerase β (BioLegend), rabbit polyclonal anti-GFP (Rockland), rabbit polyclonal anti-CarD, and rabbit polyclonal anti-MymT (a kind gift of Dr. Carl Nathan, Gold et al., 2008). His tag fusion proteins were detected using HisProbe-HRP (Thermo Fisher).

Transcriptional profiling

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RNA samples for comparative nitric oxide transcriptional profiling were isolated from cells grown and treated as described above, except that nitric oxide treatment was carried out for 5 hr instead of 3 hr. RNA sequencing was performed as previously reported (Hubin et al., 2017). Full RNAseq dataset is given in Supplementary file 4 and deposited under NCBI BioProject Accession Number PRJNA719428 (https://www.ncbi.nlm.nih.gov/sra/PRJNA719428).

Recombinant PdtaS/PdtaR purification

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Full-length C-terminal 10x His tagged PdtaS and N-terminal 10xHisSmt3 PdtaR fusions were both expressed and purified from Rosetta 2 (DE3) E. coli (Millipore Sigma). Cultures, 2–4 L, grown in Luria–Bertani (LB) media to an OD600 of 1.0 at 37°C were equilibrated to 30°C for 30 min prior to induction. Protein expression was then induced with 1 mM IPTG, and the cultures were allowed to grow for 3–4 additional hours at 30°C. Cells were then collected by centrifugation, resuspended in lysis/wash buffer (350 mM sodium chloride, 20 mM Tris-HCl pH 8.0, 20 mM imidazole, 1 mM β-mercaptoethanol) at a ratio of 2 mL of buffer to 1 g of wet pellet weight, and stored at −80°C. The following day frozen cell pellets were thawed and disrupted by two passes through a French pressure cell at an internal pressure of 14,000 psi (Sim-Aminco, 40,000 psi cell). Crude lysates were clarified by centrifugation at 20,000×g for 30 min in an SS-34 Rotor (Sorvall), and then passed over TALON metal affinity resin (Takara) equilibrated in wash buffer. Columns were then washed with 20 column volumes of Wash buffer. Bound proteins were eluted with two-column volumes of Elution Buffer (Wash Buffer + 250 mM imidazole).

After elution from the TALON column, PdtaS 10XHis was dialyzed and concentrated to ~2 mg/mL in a dialysis buffer containing 50 mM Tris-HCl pH 8.0, 50 mM sodium chloride, 100 µM DTT with 40% (v/v) glycerol. Eluted 10xHis-Smt3-PdtaR was cleaved from PdtaR by overnight treatment with Ulp1 protease at 4°C. Free PdtaR was then recovered in the flow-through fraction of a second round of TALON resin purification. PdtaR was then dialyzed and stored in the dialysis buffer.

Histidine kinase autophosphorylation and phosphotransfer activity

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For autophosphorylation assay, 5 μM of the purified histidine kinase PdtaS (HK) was incubated for 20 min in autophosphorylation buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 10 mM MgCl2) containing 50 μM ATP and 1 μCi of γ32P-labeled ATP at 30°C. In the phosphotransfer assay, 10 μM of RR PdtaR was added to the autophosphorylated HK and incubated for time points described in the figure legends. The reaction was terminated by adding 1× SDS-PAGE sample buffer (2% w/v SDS, 50 mM Tris.HCl, pH 6.8, 0.02% w/v bromophenol blue, 1% v/v β-mercaptoethanol, 10% v/v glycerol). The samples were resolved on 15% v/v SDS-PAGE. After electrophoresis, the gel was washed and exposed to phosphor screen (Fujifilm Bas cassette2, Japan) for 4 hr followed by imaging with Typhoon 9210 phosphorimager (GE Healthcare, USA). Quantitative densitometric analysis was performed with ImageJ.

PdtaS autophosphorylation inhibition assays

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5 μM of the purified HK PdtaS, PdtaSC53A mutant and PdtaSkinase domain (amino acids 277–501) proteins were preincubated for 10 min in the autophosphorylation buffer with copper chloride and zinc sulfate at various concentrations mentioned in the figure legends and with 1 mM of calcium chloride as a negative control. Similarly, the effect of NO and H2S was studied by preincubating 5 μM of PdtaS, PdtaSC53APdtaSC57A, or PdtaSkinase domain protein with spermine NONOate (Cayman Chemical Company, USA) for 40 min and sodium hydrosulfide hydrate (Acros Organics, Belgium) as spontaneous H2S donor for 30 min at various concentrations specified in the figure legends. The negative control (C) for use of spermine NONOate as NO donor was 1 mM of spermine NONOate allowed to exhaust NO release by overnight incubation at room temperature in Tris.Cl buffer at pH = 6.8. The autophosphorylation reaction was then initiated by adding 50 μM ATP along with 1 μCi of γ32P-labeled ATP at 30°C for 20 min. The reaction was terminated by adding 1× SDS-PAGE sample buffer. The samples were resolved on 15% v/v SDS-PAGE and analyzed as above. The dose-response curves were plotted and inhibition constant (Ki) was obtained by fitting the percentage inhibition to concentration plot using one site-specific binding fit using GraphPad Prism software.

MST determination of binding affinity

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Purified HK PdtaS protein (5 μM) was autophosphorylated in autophosphorylation buffer for 20 min at 30°C using 50 μM of ATP to generate phosphorylated HK PdtaS (PdtaS~P). Phosphotransfer reaction was carried out at 30°C for 10 min from HK PdtaS~P to fluorescently labeled RR PdtaR protein (50 nM) prelabeled using amine coupling Monolith Protein Labeling kit RED-NHS 2nd generation (Cat# MO-L011, NanoTemper Technologies GmbH) as prescribed in the manufacturer’s protocol. Binding assays were performed immediately with in vitro transcribed RNAs ppe1-5′, rv3864, and dnaK at concentrations from 0.31 nM to 10 µM diluted with autophosphorylation buffer having 0.025% Tween-20. The sample was then loaded into standard treated capillaries and analyzed using a Monolith NT. 115 (NanoTemper Technologies GmbH). The red laser was used for a duration of 35 s for excitation (MST power = 40%, LED power 100%). The data were analyzed using MO Control software (NanoTemper Technologies GmbH) to determine the apparent KD values represented as fraction bound.

For assays using unphosphorylated PdtaR, 50 nM of the fluorescently labeled PdtaR protein was incubated with 50 μM of ATP for 20 min at 30°C in an autophosphorylation buffer. MST assay was immediately performed post addition with ppe1-5′ (concentration range from 0.15 nM to 10 µM), rv3864 (concentration range from 0.61 nM to 10 µM), and dnaK (concentration range from 0.15 nM to 10 µM) diluted with autophosphorylation buffer having 0.025% Tween-20, and apparent KD values represented as fraction bound were similarly obtained.

Data availability

Raw RNA sequencing data has been deposited into SRA, accession number PRJNA719428. The processed data is included in the supplementary data.

The following data sets were generated
    1. Buglino J
    2. Sankhe G
    3. Lazar N
    4. Bean JM
    5. Glickman MS
    (2021) NCBI BioProject
    ID PRJNA719428. Rip1 and PdtaR mutant transcriptional responses to Nitric oxide.

References

Decision letter

  1. Bavesh D Kana
    Senior and Reviewing Editor; University of the Witwatersrand, South Africa
  2. Martin Voskuil
    Reviewer
  3. Jeffery S Cox
    Reviewer; University of California, Berkeley, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

In this study the authors identify several components of a regulatory circuit in Mycobacterium tuberculosis, the causative agent of tuberculosis, that allows this important pathogen to respond in a novel manner to nitric oxide and copper ions. The experiments demonstrate that a two-component system, together with a protease, sense and integrate NO stress to control expression of biosynthetic genes required for synthesis of high affinity copper-binding molecules. The authors thus demonstrate a complex set of mechanisms for response to important stress determinants that prevail during colonization of macrophages. The study will be of interest to bacterial physiologists, infectious disease researchers and microbiologists.

Decision letter after peer review:

Thank you for submitting your article "Integrated sensing of host stresses by inhibition of a cytoplasmic two component system controls Mtb infection" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Bavesh Kana as the Senior and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Martin Voskuil (Reviewer #2); Jeffery S Cox (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

In this submission, Buglino and colleagues investigate a regulatory circuit in Mycobacterium tuberculosis that facilitates the response to nitric oxide and copper ions, both of which are components of antibacterial response in macrophages. They build on previous work demonstrating that the membrane-embedded site-2 protease (S2P), Rip1 is required for virulence in the mouse model of tuberculosis infection, an effect that is related to activity of σ factors. In this work, the authors further explore the role of this protease in regulating the response to stress conditions.

Key findings:

1. In M. tuberculosis, Rip1 is required for resistance to copper ions, nitric oxide, and zinc, with deletion of rip1 resulting in a 10 000 fold increase in sensitivity to copper, which did not appear to result from the activity of any known copper pathways in this organism.

2. In the mouse model of tuberculosis infection, Rip1 appears to defend against the antimicrobial effect of NO, as attenuate virulence of the mutant was reversed in infections of NOS2 deficient mice.

3. Using a screen for suppression of the copper sensitivity, two mutations were detected in PdtaS, suggesting that the PdtaS/R system was involved in the observed response. The authors confirm this by complementing the mutant with variants of PdtaS that lack kinase activity and by creating a deletion mutant of the two-component regulator.

4. The authors then reconstitute the Pdta phosphotransfer reaction and show that this two-component system is able to sense copper, zinc and nitric oxide. Binding of these ligands inhibits signalling through the system. Ligand binding requires the GAF domain of the sensor and required a cysteine residue.

5. Using RNA sequencing of their mutant panel, the authors define the set of genes regulated by the PdtaS/R system, including a gene cluster responsible for the synthesis of isonitrile lipopeptide chalkophores (which bind copper with high affinity) and PPE1-5.

6. To further investigate the link with PPE1-5, the authors demonstrate that PPE1-5 RNA binds to PdtaR and these combined effects are critical for chalkophore biosynthesis and NO resistance in vitro and during infection.

Conclusion: The study shows that the PdtaS/R two-component system and the Rip1 protease sense and integrate NO stress to control expression of biosynthetic genes required for synthesis of high affinity copper-binding molecules.

Essential revisions:

All reviewers commented on the high quality and rigour of the data presented in the manuscript but also raised concerns that there was insufficient evidence presented to support the conclusion that the circuit is important for copper stress resistance. It is our assessment that your work still has significant value without a strong focus on copper stress resistance. I suggest you modify the manuscript to keep the focus on NO stress resistance and indicate the role for copper in regulation of the circuit is not firmly delineated. In a separate section below, I have provided the reviewer suggestions for additional copper experiments to give you a sense of the concerns and how best to address them. You can address these by modifying the manuscript or adding any new supporting data, if these are available. Extensive new experimentation on this aspect is not expected.

Concerns regards copper sensing/resistance:

It seems counterintuitive that a copper acquisition system would be activated during copper stress and it appears that it is probably not from the data presented, although chalkophore gene expression during copper stress was not investigated. If copper is blocking PdtaS/R signaling as is indicated by the in vitro biochemistry, then the unknown Rip1 mediator may be preventing the downstream activation of the chalkophore biosynthesis genes. Although this would be an opposite role for Rip1 than that convincingly demonstrated with NO. Figure 5D shows overexpression of the chalkophore biosynthetic operon instead of increasing copper resistance, as is the case with NO, results in a modest increase in sensitivity to copper stress on plates. However, deletion of the nrp gene, which is part of the chalkophore biosynthetic operon, appears to also modestly increase sensitivity to copper stress on plates (Figure S7B and C). These two findings provide the phenotypic data that suggest a role for the system in copper stress resistance, but they also appear to be contradictory findings. In addition, restoration of chalkophore gene expression or expression of PPE5' or PPE1 did not rescue copper sensitivity of the rip1 mutant strains demonstrating that chalkophore regulation is not likely the mechanism in which loss of Rip1 causes copper sensitivity. Since the PdtaS/R system controls chalkophore synthesis it seems unlikely that the in vitro finding that copper blocks the system like NO occurs in the cell during copper stress. Alternatively, if copper mediate blockage of activation does occur in the bacilli, it may not result in de-repression of chalkophore biosynthesis genes as occurs with NO. As gene expression was not determined during copper stress, the impact of copper on gene expression cannot be assessed. While it appears that copper stress is in some manner integrated into the sensory inputs of the overall circuit, the above findings make the role of copper sensing vague. The authors indicate that the PdtaS/R system is not involved in copper resistance, but it is not clear why copper would not trigger de-repression of chalkophore genes as it appears to block the system like NO. To establish the role of copper in the system several experiments would be helpful. These include expression profiling during copper stress to determine if genes such as the chalkophore operon are regulated during copper stress as they are induced by NO.

Copper stress experiments could also be performed in liquid culture to determine true lethality upon plating instead of copper exposure only on plates. As copper challenge experiments were done with copper added to plates not in liquid culture, as was the case for NO and zinc experiments, it is difficult to compare NO and copper experiments. As solid agar copper toxicity experiments cannot differentiate lethal and static effects of copper, the copper experiments should be labeled as "relative inhibition" instead of "relative survival."

The Ki for copper binding PdtaS was found to be 34 microM, since PdtaS is cytosolic and free intracellular copper is known to be highly toxic, this level of intracellular copper should be put into context with potential intracellular levels.

A mechanism should be discussed to incorporate the contradictory findings that copper should activate chalkophore synthesis genes through its interaction with PdtaS, even though this interaction seems to not have a role. It may be the impact on PdtaS autophosphorylation by copper is unavoidable due to the chemistry therefor the Rip1 system is employed to short circuit the activation of chalkophore synthesis from copper stress to prevent increased copper accumulation.

The paragraph starting on line 103: The logical basis for the epistasis tests is not entirely clear. The first idea makes sense – Rip1 mutants may be sensitive to high copper levels because they are unable to derepress the RicR and CsoR regulons which function to export copper, and thus removal of the repressors would suppress the sensitivity phenotype of the rip1 mutant. While the rip1 csoR double mutant is sensitive to high copper, isn't it possible that RicR is still repressing something that is required for copper resistance. That is, wouldn't it be more definitive to remove both ricR and csoR? This needs some clarification. Secondly, the transition from the two copper regulators (RicR and CsoR) to the two gene targets is not intuitive, it would help the reader if the functions of MymT and CtpV are explained. This part is a bit confusing as the mymT and ctpV mutants aren't sensitive to copper concentrations that are toxic to rip1 mutant cells – isn't that the main evidence that the Rip1 pathway for copper resistance is mediated through mechanisms other than MymT or CtpV mediated copper efflux (or perhaps not due to efflux at all)? Or are the mymT and ctpV mutants still able to efflux copper? Here the logic for the epistasis test isn't clear – it would seem that if the Rip1 pathway for copper resistance is independent of the MymT/CtpV pathway, the main expectation of double mutants would be that there would be ultra-sensitivity, i.e. synergy or "negative epistasis". It seems that there is a fairly potent synergistic effect of ctpV and csoR in combination with the rip1 mutation in Figure S3C, especially at medium [Cu] (2 and 3 logs). This is not evident with mymT. Finally, the term "copper handling" is vague (line 116).

Major concerns that must be addressed:

1. In order for the authors to claim that NO and copper modulates the PtdaS/R/Rip1 signal transduction system via direct interaction with a thiol switch, they need to demonstrate a direct effect of copper or NO on the Cys residue. For example, evidence of S-nitrosylation, the covalent addition of NO moiety to the sulfur atom of Cys, would provide conclusive evidence of a thiol switch. Also, the authors have not demonstrated whether copper could modify the Cys resideu. Standard spectrophotometry, mass spectrometry or other assays (e.g., biotin switch) could demonstrate these modifications. Also, if the Cys residue plays a key role, why was not it identified in the suppressor screen? What about the 2nd Cys residue (C57)? In fact, other residues were identified strongly suggesting that Cys is not central to a thiol switch as other residues (e.g., V) are also involved. As indicated above, rephrasing these conclusions and removing the emphasis on copper should assist in addressing these issues.

2. Did the authors consider that PdtaS may bind other cofactors (e.g. c/gAMP) or prosthetic groups such as heme? Although doi.org/10.1016/j.jsb.2011.11.012 reported that there is no evidence of a bound metabolite they did not comment on the presence of heme in the GAF domain and stated that the internal cavity is too small to bind heme in the PAS domain. Although not required, since the authors have purified PdtaS, a simple UV-vis scan will reveal whether this protein binds heme (potentially loosely as it was not identified in the crystal structure), which will provide a clear mechanism of responding to NO and potentially copper.

3. The rational for performing RNA seq experiments using NO and not also copper is unclear. Can you explain this reasoning? The main reason for this request is you identified a putative copper binding operon under NO stress. Please also expand on your statement that isonitrile binds copper with high affinity as this binding data is not provided in the references (at least not in the PNAS paper), which suggests it is involved in copper transport and not binding. Strong copper binding is also mentioned in lines 303 without any reference; please provide.

4. In figure 6, the authors examined 4 mutants (some of them are double knockouts) in vivo, but no complementation experiments were performed. We understand the challenges and complexities involved in re-doing these time-consuming experiments however, genetic complementation is the standard in the field. Can the authors provide a compelling reason for the lack of complementation?

5. Although the authors predict in lines 323-326 that the in vivo virulence defect conferred by loss of rip1 should be reversed in a rip1/pdtaR knockout, this is not entirely true. Although the authors attempt to explain this by separating the response during acute infection versus chronic infection, this is not entirely consistent with their prediction in lines 323 – 326 and the data points in figure 6C and D. This will become more evident if the authors provide the statistics for not only the wild-type and rip1 comparison, but also for the wild-type and rip1/pdtaR and wild-type and pdtaR comparisons. As it stands, it is incomplete.

6. Lines 133-134. this conclusion is too strong – it seems that increased growth of an Mtb mutant in an iNOS knockout mouse does not establish that the gene is directly involved in defending against the antimicrobial effect of NO. Certainly this is consistent with the direct NO sensitivity of the mutant in culture, but there are many effects of NO during anti-TB immune responses.

7. Figure 3. There is a bit of a disconnect between the gel in panel B and the quantification in panel F. Perhaps it is the pdf but while there is a noticeable decrease in the band intensity in the two-fold drop from 5 to 10 uM, but the 10-100 μm bands all look the same to me (unlike the Zn inhibition data in Figure S5, which looks really good). Also, a -Cu control is missing – how was the % inhibition calculated without this?

8. Isn't the simple expectation that chalkophore synthesis would lead to Cu resistance? It seems unexpected that it would only affect NO and not Cu sensitivity. That is mentioned in the Discussion section but it seems confusing in the results without any explanation.

9. The role of PPE1-5' RNA could be more thoroughly explored. First, the expression of the PPE1-5' RNA is in the context of expression of the entire PPE1 gene. Thus, it is not clear that expression of PPE1-5' is sufficient to suppress the NO sensitivity phenotype of the Rip1 mutant. Can just the RNA be expressed?

https://doi.org/10.7554/eLife.65351.sa1

Author response

Essential revisions:

All reviewers commented on the high quality and rigour of the data presented in the manuscript but also raised concerns that there was insufficient evidence presented to support the conclusion that the circuit is important for copper stress resistance. It is our assessment that your work still has significant value without a strong focus on copper stress resistance. I suggest you modify the manuscript to keep the focus on NO stress resistance and indicate the role for copper in regulation of the circuit is not firmly delineated. In a separate section below, I have provided the reviewer suggestions for additional copper experiments to give you a sense of the concerns and how best to address them. You can address these by modifying the manuscript or adding any new supporting data, if these are available. Extensive new experimentation on this aspect is not expected.

Concerns regards copper sensing/resistance:

It seems counterintuitive that a copper acquisition system would be activated during copper stress and it appears that it is probably not from the data presented, although chalkophore gene expression during copper stress was not investigated. If copper is blocking PdtaS/R signaling as is indicated by the in vitro biochemistry, then the unknown Rip1 mediator may be preventing the downstream activation of the chalkophore biosynthesis genes. Although this would be an opposite role for Rip1 than that convincingly demonstrated with NO. Figure 5D shows overexpression of the chalkophore biosynthetic operon instead of increasing copper resistance, as is the case with NO, results in a modest increase in sensitivity to copper stress on plates. However, deletion of the nrp gene, which is part of the chalkophore biosynthetic operon, appears to also modestly increase sensitivity to copper stress on plates (Figure S7B and C). These two findings provide the phenotypic data that suggest a role for the system in copper stress resistance, but they also appear to be contradictory findings. In addition, restoration of chalkophore gene expression or expression of PPE5' or PPE1 did not rescue copper sensitivity of the rip1 mutant strains demonstrating that chalkophore regulation is not likely the mechanism in which loss of Rip1 causes copper sensitivity. To establish the role of copper in the system several experiments would be helpful. These include expression profiling during copper stress to determine if genes such as the chalkophore operon are regulated during copper stress as they are induced by NO.

We agree with the reviewers that some further explanation is required, and that our study does not delineate the full mechanisms of Cu resistance downstream of Rip1. However, we disagree that the findings are contradictory. The statement that “ a copper acquisition system would be activated during copper stress” seems to assume that the nrp operon is involved in copper acquisition because the natural product produced by these enzymes binds copper. The molecular function of the molecules produced by the nrp operon encoded proteins is not known. The reviewers seem to assume that any molecule that binds copper is acting to modulate copper toxicity via binding, as is the case for copper binding proteins such as MymT. However, in the case of the chalkophores of Methanococcus, the function of the chalkophore is to supply copper to a membrane bound copper enzyme, not to directly ameliorate toxicity. Although we agree that it is surprising that the chromosomal target of the cascade that mediates NO resistance, but not copper resistance, is a copper binding molecule, the experiments in the paper were executed to definitively demonstrate this point.

Therefore, we agree that our study does not fully define the Cu arm of the pathway. However, what is definitive in our data, and acknowledged in the review, is: (1) Rip1 is required for resistance to both stresses (2) loss of PdtaS/R function is a suppressor of BOTH the NO and Cu sensitivity of the Drip1 strain, and (3) Cu and NO both inhibit the signaling system biochemically. The fact that the chromosomal targets that mediate resistance to each of these stresses are distinct indicates that there is some mechanism that specifies PdtaR function to specific gene sets under specific stress conditions. Our data identifies the Ppe1-5’ RNA as that factor under NO stress and we hypothesize, but do not identify, a Cu induced similar factor. The fact that Cu and NO both inhibit PdtaS must be understood in the context of our two signal model, in which inhibition of the kinase is not sufficient to depress gene expression without a second signal (PPE1-5’ in the case of NO) and therefore the assertion in the review that inhibition of PdtaS by copper would stimulate nrp expression is not correct. The RNAseq data in the PdtaR ko in the absence of any stress demonstrates this unambiguously, there is no induction of the nrp operon in the PdtaR ko in basal conditions.

In addition, the review points to several pieces of data for example “Figure 5D shows…” and “appears to also modestly increase sensitivity to copper stress.” that are nonsignificant differences and therefore were not highlighted in our analysis or integrated into the model. They should not be invoked as supporting the assertion that our data is “contradictory”.

In summary, although we agree that our study does not resolve the ultimate downstream chromosomal target of the Rip1/PdtaS/PdtaR cascade that controls Cu resistance, we do definitively implicate this cascade in Cu resistance. The fact that a Cu binding molecule mediates NO, but not Cu resistance, may be surprising based on the notion that any Cu binding molecule must be ameliorating toxicity, but this is a presumption that is not borne out in the data. Multiple other hypotheses can be advanced but must be explored by further experimentation. For example, NO may induce a Cu deficient state in membrane bound enzymes which is ameliorated by a restorative function of chalkophore-bound Cu through a loading function.

In response to these points raised by the reviewers, we have clarified the discussion to better incorporate these concerns and make clear that our study does not elucidate the downstream mechanisms of Cu resistance controlled by the Rip1/PdtaS/R system. We have focused the data presentation more on the NO phenotypes. We have maintained the model figure to emphasize this point because this visual representation clearly indicates a “?” on the chromosomal target of Cu arm of the pathway.

Copper stress experiments could also be performed in liquid culture to determine true lethality upon plating instead of copper exposure only on plates. As copper challenge experiments were done with copper added to plates not in liquid culture, as was the case for NO and zinc experiments, it is difficult to compare NO and copper experiments. As solid agar copper toxicity experiments cannot differentiate lethal and static effects of copper, the copper experiments should be labeled as "relative inhibition" instead of "relative survival."

We thank the reviewer for this suggestion. We have added data to Figure 1-Figure Supplement 1D showing that the copper sensitivity of the Δrip1 strain is present when cells are exposed to Cu in liquid media.

The Ki for copper binding PdtaS was found to be 34 microM, since PdtaS is cytosolic and free intracellular copper is known to be highly toxic, this level of intracellular copper should be put into context with potential intracellular levels.

Thank you for this question. The intracellular copper concentration noted in phagosomes is in the range of 20 – 40 μm (Wagner D, et al. J Immunol 2005;174:1491–1500). It is difficult extrapolate these levels to the physiologic relevance to PdtaS particularly because of the equilibrium between Cu+1 and Cu+2. Although we apply Cu+2 in these assays, we cannot control the conversion to Cu+1. As a point of reference, the biochemical data in PMID 21166899 indicates that dissociation of RicR from its operator requires 15-20 μM copper, suggesting that the cytosolic concentration reaches this level under copper stress given the robust induction of RicR targets under the conditions we test.

Since the PdtaS/R system controls chalkophore synthesis it seems unlikely that the in vitro finding that copper blocks the system like NO occurs in the cell during copper stress. Alternatively, if copper mediate blockage of activation does occur in the bacilli, it may not result in de-repression of chalkophore biosynthesis genes as occurs with NO. As gene expression was not determined during copper stress, the impact of copper on gene expression cannot be assessed. While it appears that copper stress is in some manner integrated into the sensory inputs of the overall circuit, the above findings make the role of copper sensing vague. The authors indicate that the PdtaS/R system is not involved in copper resistance, but it is not clear why copper would not trigger de-repression of chalkophore genes as it appears to block the system like NO.

A mechanism should be discussed to incorporate the contradictory findings that copper should activate chalkophore synthesis genes through its interaction with PdtaS, even though this interaction seems to not have a role. It may be the impact on PdtaS autophosphorylation by copper is unavoidable due to the chemistry therefor the Rip1 system is employed to short circuit the activation of chalkophore synthesis from copper stress to prevent increased copper accumulation.

As noted above, this comment presumes the inhibition of PdtaS/R alone is sufficient to activate all downstream gene expression, which is not correct. We have performed RNA sequencing of the PdtaR mutant without stress and find very few differences in gene expression (see figure 4B,C) and specifically that the chalkophore operon is not induced. This piece of data is part of the support for our two-signal model in which inhibition of PdtaS signaling is necessary but not sufficient for activation of gene expression. Our data indicate that a second signal to modify PdtaR binding is required, and, in the case of NO, we identify this signal as the NO induced transcription of PPE1-5’. The reviewer is correct that we have not identified this second signal for Cu, nor identified the chromosomal target(s) it regulates, but the data does not support the assertion that Cu inhibition of PdtaS is sufficient to activate chalkophore biosynthesis and that, based this is assumption, our findings are contradictory.

The paragraph starting on line 103: The logical basis for the epistasis tests is not entirely clear. The first idea makes sense – Rip1 mutants may be sensitive to high copper levels because they are unable to derepress the RicR and CsoR regulons which function to export copper, and thus removal of the repressors would suppress the sensitivity phenotype of the rip1 mutant. While the rip1 csoR double mutant is sensitive to high copper, isn't it possible that RicR is still repressing something that is required for copper resistance. That is, wouldn't it be more definitive to remove both ricR and csoR? This needs some clarification.

The RicR and CsoR regulons are nonoverlapping according to prior literature. It is possible there is redundancy in function, but the experiment requested would require creation of triple mutant and based on the data that all RicR targets measured are intact, in addition to the lack of epistasis with RicR targets (i.e. CtpV and MymT, see below), we have not created this mutant.

Secondly, the transition from the two copper regulators (RicR and CsoR) to the two gene targets is not intuitive, it would help the reader if the functions of MymT and CtpV are explained. This part is a bit confusing as the mymT and ctpV mutants aren't sensitive to copper concentrations that are toxic to rip1 mutant cells – isn't that the main evidence that the Rip1 pathway for copper resistance is mediated through mechanisms other than MymT or CtpV mediated copper efflux (or perhaps not due to efflux at all)? Or are the mymT and ctpV mutants still able to efflux copper? Here the logic for the epistasis test isn't clear – it would seem that if the Rip1 pathway for copper resistance is independent of the MymT/CtpV pathway, the main expectation of double mutants would be that there would be ultra-sensitivity, i.e. synergy or "negative epistasis". It seems that there is a fairly potent synergistic effect of ctpV and csoR in combination with the rip1 mutation in Figure S3C, especially at medium [Cu] (2 and 3 logs). This is not evident with mymT.

The reviewer is correct, the data shows that the Cu resistance phenotype of the Rip1 strain is independent of MymT and CtpV. The confusion may arise because the MymT and CtpV mutants are not themselves sensitive to Cu, but this result is actually consistent with the published literature, which shows variable copper sensitivity in M. tuberculosis strains. For example, CDC1551 (more similar to Erdman than H37Rv) is 100 fold more resistant to Cu than H37Rv, and loss of mmcO, which confers severe sensitivity to Cu in H37Rv, has no phenotype in CDC1551 (PMID 24549843). With regard to epistasis, if the CtpV or MymT mutants are not sensitive to Cu, but the rip1 mutant is, then it is not plausible that the phenotype of the Rip1 strain is due to MymT or CtpV dysfunction. If the CtpV or MymT mutants were indeed sensitive, then the predicted result would have been additive sensitivity, i.e. lack of epistasis.

Finally, the term "copper handling" is vague (line 116).

We have modified this wording.

Major concerns that must be addressed:

1. In order for the authors to claim that NO and copper modulates the PtdaS/R/Rip1 signal transduction system via direct interaction with a thiol switch, they need to demonstrate a direct effect of copper or NO on the Cys residue. For example, evidence of S-nitrosylation, the covalent addition of NO moiety to the sulfur atom of Cys, would provide conclusive evidence of a thiol switch. Also, the authors have not demonstrated whether copper could modify the Cys resideu. Standard spectrophotometry, mass spectrometry or other assays (e.g., biotin switch) could demonstrate these modifications.

Our apologies, our intention in using the term “thiol switch” was not to imply that the PdtaS cysteine is directly modified by either Cu or NO, which we did not show, and we were unaware that the term “thiol switch” implicitly indicates covalent modification. Our intended meaning was that the thiol (originally C53 but see below for our new data also implicating C57) is required for the NO and Cu inhibition of PdtaS kinase activity. The structural and biochemical mechanism of this thiol dependent inhibition will require further study, but our data does demonstrate that PdtaS lacking either thiol cannot be inhibited by NO or Cu. We have altered our terminology to eliminate the term “thiol switch.”

Also, if the Cys residue plays a key role, why was not it identified in the suppressor screen?

There could be multiple explanations for this finding such as non-saturation of the screen, some maladaptive property of the Cysteine mutation in vivo that prevents its isolation, or the fact that both thiols seem to be involved in sensing and therefore a mutation that lies in proximity to both may uniquely disrupt their geometry in a way that is maximally suppressive.

What about the 2nd Cys residue (C57)? In fact, other residues were identified strongly suggesting that Cys is not central to a thiol switch as other residues (e.g., V) are also involved. As indicated above, rephrasing these conclusions and removing the emphasis on copper should assist in addressing these issues.

We thank the reviewer for this question. We agree that C57 is plausibly involved due to its proximity to C53 and to the suppressor mutation that was identified. To investigate this possibility, we purified PdtaS C57A and assayed this protein for kinase activity and inhibition by Cu and NO. We found that the C57A mutation did not modify the basal activity of the kinase but did confer resistance to inhibition by the NO (see revised Figure 3G and Figure 3-Figure Supplement 2). We were unable to assay this protein for Cu inhibition due to solubility problems in the assay with high Cu concentrations and therefore the role of this residue in Cu inhibition remains unresolved. Figure 7 has been updated to incorporate C57.

2. Did the authors consider that PdtaS may bind other cofactors (e.g. c/gAMP) or prosthetic groups such as heme? Although doi.org/10.1016/j.jsb.2011.11.012 reported that there is no evidence of a bound metabolite they did not comment on the presence of heme in the GAF domain and stated that the internal cavity is too small to bind heme in the PAS domain. Although not required, since the authors have purified PdtaS, a simple UV-vis scan will reveal whether this protein binds heme (potentially loosely as it was not identified in the crystal structure), which will provide a clear mechanism of responding to NO and potentially copper.

We thank the reviewer for this question, which we have also considered. We are aware of reports that the PdtaS binds cyclic-di-GMP (https://www.biorxiv.org/content/10.1101/615575v1.abstract) at the GAF domain of the sensor. The interaction of this metabolite with Cu and NO sensing documented here is an interesting subject for further study but beyond the scope of this study. It does suggest that PdtaS integrates multiple inputs and we have added this point to the discussion.

With regard to heme binding by PdtaS, we have performed UV-vis spectroscopy on our purified protein in comparison to BSA as a negative control and cytochrome C as a positive control. We see clear absorbance for CytC, but no signal for either BSA or PdtaS, suggesting that PdtaS does not contain heme, as previously reported. The graph is shown in Author response image 1.

Author response image 1

3. The rational for performing RNA seq experiments using NO and not also copper is unclear. Can you explain this reasoning?

We have added this rationale to the text:

“We focused on NO stress for several reasons, including: (1) the strong phenotypic reversion of Drip1 in iNOS deficient mice (2) the strong phenotypic reversion of Rip1 by the pdtaR mutation and (3) the technical advantages of treating Mtb with carefully titrated NO donors in liquid culture. “

The main reason for this request is you identified a putative copper binding operon under NO stress. Please also expand on your statement that isonitrile binds copper with high affinity as this binding data is not provided in the references (at least not in the PNAS paper), which suggests it is involved in copper transport and not binding. Strong copper binding is also mentioned in lines 303 without any reference; please provide.

The copper binding properties of the isonitrile chalkophores is reported in reference 42 of the manuscript (Wang et al. ACS Chemical biology) and in reference 46 (Xu et al). Reference 46 uses synthetic molecules of defined structure from Streptomyces. Neither the PNAS paper, nor the ACS chemical biology paper (reference 42) define the physiologic role of the molecules, only that they bind Cu.

4. In figure 6, the authors examined 4 mutants (some of them are double knockouts) in vivo, but no complementation experiments were performed. We understand the challenges and complexities involved in re-doing these time-consuming experiments however, genetic complementation is the standard in the field. Can the authors provide a compelling reason for the lack of complementation?

We agree with the reviewer that complementation is an essential genetic control. We have included complementation in Figure 6A,B for the Δnrp strain. For the Δrip1 strain in figure 6C,D, the complemented strain has been previously reported in mice and is therefore not repeated here (although it is included in all in vitro assays, see all prior figures). For the rip1/pdtaR double mutant, we are interrogating reversion of the Rip1 in vivo phenotype by the PdtaR null mutation. The complementation control we assume the reviewer is asking about is testing is the animal phenotype of Δrip1ΔpdtaR+pdtaR to show that the reversion we observe is due to the pdtaR mutation and not a spontaneous suppressor. We acknowledge that we have not done this infection due to the large number of strains, time, and expense, but note that this strain is included in every in vitro assay in Figure 2, alongside analogous complemented strains for the PdtaS suppressors. Both the PdtaS SNP, the ΔpdtaS::hyg mutation, and the ΔpdtaR::hyg mutation all share the same reversion phenotype, strongly arguing against a spontaneous mutation. In addition, in all assays in Figure 2, these suppressors are complemented, and, in all cases, the suppressor effect is reverted by complementation.

5. Although the authors predict in lines 323-326 that the in vivo virulence defect conferred by loss of rip1 should be reversed in a rip1/pdtaR knockout, this is not entirely true. Although the authors attempt to explain this by separating the response during acute infection versus chronic infection, this is not entirely consistent with their prediction in lines 323 – 326 and the data points in figure 6C and D. This will become more evident if the authors provide the statistics for not only the wild-type and rip1 comparison, but also for the wild-type and rip1/pdtaR and wild-type and pdtaR comparisons. As it stands, it is incomplete.

We apologize if there was some display problem with the figure, since the statistics requested are mostly displayed on the figure. We have added additional statistics. We agree with the reviewer that the suppression of the Rip1 virulence phenotype is limited to early infection in the lung, but in this period the suppression is very strong, reversing the Rip1 CFU decrement by approximately 100 fold. The PdtaR single mutant has a virulence phenotype alone, although this was not our focus. However, this result indicates that the suppression of the Rip1 phenotype by PdtaR would not be expected to replicate WT titers since the Rip1 independent virulence function of PdtaR, which we do not define, is still defective in the Rip1 PdtaR double mutant. Indeed, there is a significant difference between WT and PdtaR and WT vs Rip1/PdtaR for this reason (see added stats in Figure 6). We do not believe the stage specific reversion of the Rip1 phenotype in the lung is a weakness; it shows that the signal transduction cascade we identify is only relevant in early infection.

6. Lines 133-134. this conclusion is too strong – it seems that increased growth of an Mtb mutant in an iNOS knockout mouse does not establish that the gene is directly involved in defending against the antimicrobial effect of NO. Certainly this is consistent with the direct NO sensitivity of the mutant in culture, but there are many effects of NO during anti-TB immune responses.

We have modified this wording to accommodate this possibility.

7. Figure 3. There is a bit of a disconnect between the gel in panel B and the quantification in panel F. Perhaps it is the pdf but while there is a noticeable decrease in the band intensity in the two-fold drop from 5 to 10 uM, but the 10-100 μm bands all look the same to me (unlike the Zn inhibition data in Figure S5, which looks really good). Also, a -Cu control is missing – how was the % inhibition calculated without this?

The densitometric analysis of the band displayed in percentage inhibition dose response plots are mean of replicates while the image is representative of it. Instead of a no copper control, we have used an unrelated divalent cation, Ca2+, as a negative control to control for charge. Calculations are made in relation to this control.

8. Isn't the simple expectation that chalkophore synthesis would lead to Cu resistance? It seems unexpected that it would only affect NO and not Cu sensitivity. That is mentioned in the Discussion section but it seems confusing in the results without any explanation.

See above. It was our expectation as well, but the data does not support this idea. In this case, the simplest model, one of copper chelation by copper binding, is not the correct model. We have modified the discussion to expand on this point.

9. The role of PPE1-5' RNA could be more thoroughly explored. First, the expression of the PPE1-5' RNA is in the context of expression of the entire PPE1 gene. Thus, it is not clear that expression of PPE1-5' is sufficient to suppress the NO sensitivity phenotype of the Rip1 mutant. Can just the RNA be expressed?

We have not yet been able to define the minimal PPE-5’ element that can reproduce the restoration of NO resistance in vivo. The in vitro biochemistry showing that PdtaR binds directly to the PPE1 5’ RNA uses the RNA (shown in Figure S6A) derived from the PPE1 5’ peak, strengthening our assertion that this RNA is the functionally important element, but we have not yet been able to reproduce this effect in vivo and it will require further experimentation.

https://doi.org/10.7554/eLife.65351.sa2

Article and author information

Author details

  1. John A Buglino

    Immunology Program Sloan Kettering Institute, New York City, United States
    Contribution
    Conceptualization, Investigation, Visualization, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8961-8672
  2. Gaurav D Sankhe

    Immunology Program Sloan Kettering Institute, New York City, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5627-2879
  3. Nathaniel Lazar

    Immunology and Microbial Pathogenesis Graduate Program, Weill Cornell Graduate School, New York City, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. James M Bean

    Immunology Program Sloan Kettering Institute, New York City, United States
    Contribution
    Formal analysis, Visualization, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6733-5794
  5. Michael S Glickman

    1. Immunology Program Sloan Kettering Institute, New York City, United States
    2. Immunology and Microbial Pathogenesis Graduate Program, Weill Cornell Graduate School, New York City, United States
    3. Division of Infectious Diseases, Memorial Sloan Kettering Cancer Center, New York City, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    glickmam@mskcc.org
    Competing interests
    MSG is a consultant for Fimbrion Therapeutics, serves on the SAB of Vedanta Biosciences and receives consulting fees and equity, and serves of the SAB of PRL NYC.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7918-5164

Funding

National Institute of Allergy and Infectious Diseases (R01AI138446)

  • John A Buglino
  • Gaurav D Sankhe
  • Michael S Glickman

National Cancer Institute (P30 CA008748)

  • John A Buglino
  • Gaurav D Sankhe
  • Nathaniel Lazar
  • James M Bean
  • Michael S Glickman

National Institute of Allergy and Infectious Diseases (U19AI11143)

  • John A Buglino
  • James M Bean
  • Michael S Glickman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was funded by NIH grants R01AI138446, U19AI11143, and P30 CA008748. The authors thank Carl Nathan and Ben Gold for providing the anti-MymT antibody.

Ethics

Animal experimentation: Care, housing, and experimentation on laboratory mice were performed in accordance with the National Institute of Heath guidelines, and the approval of the Memorial Sloan-Kettering Institutional Animal Care and Use Committee (IACUC).Protocol approval number is 01-11-030.

Senior and Reviewing Editor

  1. Bavesh D Kana, University of the Witwatersrand, South Africa

Reviewers

  1. Martin Voskuil
  2. Jeffery S Cox, University of California, Berkeley, United States

Publication history

  1. Received: December 1, 2020
  2. Accepted: April 25, 2021
  3. Version of Record published: May 18, 2021 (version 1)

Copyright

© 2021, Buglino et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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