Cytokinin signaling alters acid-fast staining of Mycobacterium tuberculosis

It was recently reported the human-exclusive pathogen Mycobacterium (M.) tuberculosis secretes cytokinins, which had only been known as plant hormones. While cytokinins are well-established, adenine-based signaling molecules in plants, they have never been shown to participate in signal transduction in other kingdoms of life. M. tuberculosis is not known to interact with plants. Therefore we tested the hypothesis that cytokinins trigger transcriptional changes within this bacterial species. Here, we show cytokinins induced the strong expression of the M. tuberculosis gene, Rv0077c. We found that Rv0077c expression is repressed by a TetR-like transcriptional repressor, Rv0078. Strikingly, cytokinin-induced expression of Rv0077c resulted in a loss of acid-fast staining of M. tuberculosis. While acid-fast staining is thought to be associated with changes in the bacterial cell envelope and virulence, Rv0077c-induced loss of acid-fastness did not affect antibiotic susceptibility or attenuate bacterial growth in mice, consistent with an unaltered mycolic acid profile of Rv0077c-expressing cells. Collectively, these findings show cytokinins signal transcriptional changes that affect M. tuberculosis acid-fastness, and that cytokinin signaling is no longer limited to the kingdom plantae.

Collectively, these findings show cytokinins signal transcriptional changes that affect M. tuberculosis acid-fastness, and that cytokinin signaling is no longer limited to the kingdom plantae.
M. tuberculosis is the causative agent of tuberculosis, one of the world's leading causes of mortality 1 . For this reason, researchers are eager to identify pathways that could be targeted for the development of new therapeutics to treat this devastating disease. Among the current prioritized targets is the mycobacterial proteasome. M. tuberculosis strains with defects in proteasome-dependent degradation are highly attenuated in mice, partly because they are sensitive to nitric oxide (NO) [2][3][4][5][6][7] . We recently reported that the NO-sensitive phenotype of a mutant defective for proteasomal degradation is due to an inability to degrade an enzyme called Log (Lonely guy), a homologue of plant enzymes involved in the biosynthesis of cytokinins, which are N 6substituted adenine-based molecules 8 . The accumulation of Log in M. tuberculosis results in a buildup of cytokinins, which break down into adenine and aldehydes that sensitize mycobacteria to NO 8 .
Cytokinins are hormones that regulate the growth and development of plants 9 . In addition, bacterial plant pathogens and symbionts use cytokinins to facilitate the parasitism of plants 10 . Our prior study was the first to identify a mammalian pathogen that secretes cytokinins. Outside of the laboratory, M. tuberculosis exclusively infects humans and is not known to have an environmental reservoir; therefore, it is unlikely M.  Supplementary Fig.   1a, b). Rv0077c is conserved among many mycobacterial species, while Rv0076c and Rv0078 are present only in several mycobacterial genomes ( Supplementary Fig. 1c) 11 .
M. smegmatis, a distant, non-pathogenic relative of M. tuberculosis, has a weak homologue of Rv0077c and no conspicuous Rv0078 homologue ( Supplementary Fig.   1c). mmpL6 is one of 13 mmpL (mycobacterial membrane protein large) genes in M.
tuberculosis. In strain H37Rv, mmpL6 is predicted to encode a 42 kD protein with five trans-membrane-domains and is truncated compared to the same gene in ancestral tuberculosis strains 12 . Thus, it is unclear if mmpL6 encodes a functional protein in strain H37Rv.
We raised polyclonal antibodies to recombinant Rv0077c protein and showed that protein levels were increased in M. tuberculosis treated with iP for 24 hours (Fig.   1a). Rv0077c was barely detectable in cell lysates of bacteria that had not been incubated with iP and was undetectable in a strain that has a transposon insertion mutation in Rv0077c (Fig. 1a). Rv0077c protein was restored to WT levels in the mutant upon complementation with an integrative plasmid encoding Rv0077c expressed from its native promoter (Fig. 1a). We also found a dose-dependent induction of Rv0077c production using iP concentrations from 1 nM to 100 µM (Fig. 1b).
We next synthesized and tested if the most abundantly produced cytokinin in M. tuberculosis, 2-methyl-thio-iP (2MeSiP) 8 , could also induce Rv0077c production.
The base structure of all cytokinins is adenine. We hypothesized while adenine could not induce Rv0077c expression, at high enough concentrations it could possibly inhibit Rv0077c induction by competing with iP for access to a transporter or receptor.
Indeed, adenine reduced the induction of Rv0077c by iP in a dose-dependent manner ( Fig. 1d).
In order to understand the consequences of cytokinin gene induction in M. tuberculosis, we sought to determine the function of Rv0077c. In the absence of cytokinin, Rv0077c is not expressed well under normal culture conditions; therefore, we sought to make a mutant that constitutively expressed Rv0077c in order to understand its function. Rv0077c is divergently expressed from Rv0078 ( Supplementary Fig. 1b), which encodes a putative TetR-like transcriptional regulator; the proposed translational start codons for these genes are separated by 61 base pairs therefore we hypothesized that Rv0078 encodes a repressor of Rv0077c expression. We identified the promoters for each gene by performing rapid amplification of 5' complementary DNA ends (5'RACE) analysis for Rv0077c and Rv0078 and determined the start of transcription for each gene is encoded within the 5' untranslated region of the other gene (Fig. 2a).
Furthermore, using an electrophoretic mobility shift assay (EMSA) we narrowed down the putative Rv0078 binding site to a palindrome overlapping the starts of transcription of both Rv0077c and Rv0078 (Fig. 2a, red box). Substitutions in the putative binding site prevented the binding of Rv0078 to the DNA probes (Fig. 2b). Notably, the addition of the cytokinin iP did not result in the release of Rv0078 from the probe (Supplementary  Table 3). Rv0078 formed a dimer with a fold highly similar to the TetR repressor and its family members 13 .
Helices α1 to α3 form the DNA binding domain (DBD), while helices α4 to α9 constitute an elongated ligand-binding domain (LBD) (Fig. 2d). The distance between the two DNA-binding α3 helices of the protomers is 42 Å, which is comparable to the distances of 35 -38 Å of other TetR family proteins 13,14 Interestingly, we found that W100, faces the LBD, which may explain why the Rv0078 W100R mutant allele was unable restore cytokinin-dependent regulation to the Rv0078 mutant strain (Fig. 2c, lane 8).
During our studies, a report was published on the identification of a small molecule of the spiroisoxazoline family, SMARt-420, which strongly induces the expression of the Rv0077c orthologue bcg_0108c in M. bovis Bacille Calmette-Guerin 15 .
Using x-ray crystallography and surface plasmon resonance techniques, the authors of this study found SMARt-420 binds to Rv0078 to derepress binding from the Rv0077c promoter 15,16 . SMARt-420 was identified in a search for compounds that could boost the efficacy of the second-line tuberculosis drug ethionamide (ETH). ETH is a pro-drug that is activated by the mono-oxygenase EthA, which transforms ETH into highly reactive intermediates. Activated ETH and nicotinamide adenine dinucleotide form a stable adduct, which binds to and inhibits InhA, an essential enzyme needed for mycolic acid synthesis in mycobacteria 17,18 . While spontaneous inactivating mutations in ethA can result in resistance to ETH, it was proposed that the induction of Rv0077c expression could bypass the need for EthA and transform ETH into its toxic form 15 . Based on this study, we predicted that an Rv0078 mutant of M. tuberculosis, which expresses high levels of Rv0077c (Fig. 2c, lanes 3 and 4), should be hypersensitive to ETH compared to the parental strain H37Rv. However, we observed either little to no significant change in the 50% minimum inhibitory concentration (MIC 50 ) of ETH between the WT and ΔRv0078::hyg strains (Supplementary Table 4). It is possible that the effects of SMARt-402 is growth-condition dependent, that this molecule affects another pathway to either increase the susceptibility of M. tuberculosis to ETH, or that another SMARt-420induced enzyme in addition to Rv0077c synergize together to activate ETH (A. Baulard, personal communication).
We also tested if the constitutive expression of Rv0077c changed the susceptibility of M. tuberculosis to other antibiotics, including two cell wall synthesis inhibitors. We observed no differences in the MIC 50 of these antibiotics between the WT and ΔRv0078::hyg strains (Supplementary Table 4).
To gain insight into the function of the most strongly cytokinin-induced gene Rv0077c, we prepared total cell lysate samples of WT and Rv0077c mutant strains treated with 100 µM iP for 24 hours for metabolomic analysis. From a total of 337 detectable metabolites, we observed a significant change in 24 molecules after the addition of iP to WT M. tuberculosis. Importantly, 17 metabolites showed a consistent difference between samples in which Rv0077c was produced and these changes disappeared in an Rv0077c-disrupted strain, suggesting the changes observed in iPtreated WT M. tuberculosis were specifically due to the presence of Rv0077c (Supplementary Table 5, highlighted in yellow). We observed an increased abundance of several phospholipids and a decrease in a major precursor of peptidoglycan, Nacetyl-glucosamine-1-phosphate (GlcNAc1P). We therefore hypothesized that Rv0077c modified one or more components of the cell envelope. Microscopic examination of Ziehl-Neelsen stained WT M. tuberculosis treated with iP showed a dramatic loss of acid-fast staining (Fig. 3, panel a compared to b), and this phenotype depended on the presence of Rv0077c (Fig. 3, panel c compared to d). Complementation of the mutation with Rv0077c alone restored the iP-induced loss of acid-fast staining (Fig. 3, panels e and f). Deletion of Rv0078 resulted in the constitutive loss of acid-fast staining, irrespective of the presence of iP (Fig. 3, panels g and h). Complementation of the Rv0078 deletion with the WT gene restored iP-control of loss acid-fast staining (Fig. 3, panels i and j) but complementation with Rv0078 W100R , which could not fully de-repress Rv0077c expression in the presence of iP (Fig. 2c), could not restore iP-induced loss of acid-fast staining (Fig. 3, panels k and l). Mixing and thus simultaneous staining of the Rv0077c and Rv0078 mutants further showed the staining differences were not a result of a technical artifact (Fig. 3m).
Acid-fast staining is thought to be associated with mycolic acids; alterations in mycolic acid synthesis results in negative effects on cell growth in vitro and in vivo 19,20 .
We used thin layer and liquid or gas chromatography coupled to mass spectrometry to analyze the fatty acid, mycolic acid and lipid contents of the WT, Rv0077c and Rv0078 strains and observed no significant qualitative or quantitative differences between strains (Supplementary Figure 3 and Supplementary Table 6). In particular, the expression of Rv0077c did not conspicuously modify the chain length of mycolic acids, their cyclopropanation or the relative abundance of keto-to alpha-mycolates, and did not alter the wax ester and triglyceride content of the strains that have been previously been linked to acid-fast staining 20,21 .
Finally, we tested if either the Rv0077c or Rv0078 mutant had growth defects in vivo compared to the WT H37Rv strain. We infected C57BL/6J mice by a low-dose aerosol route with the parental, mutant, and complemented mutant strains, as well as with the Rv0078 mutant transformed with the Rv0078 W100R mutant allele. Interestingly, none of the strains revealed a difference in growth or survival compared to WT M. tuberculosis in mice as determined by the recovery of colony forming units (CFU) from the lungs and spleens (Fig. 4). While we did not observe any differences in bacterial burden in mice infected with the log 8 , Rv0077c or Rv0078 mutants, it is possible that

Methods
Bacterial strains, plasmids, primers, chemicals, and culture conditions. Bacterial strains, plasmids, and primer sequences used in this study are listed in Supplementary supplemented with 0.5% glycerol and BBL TM Middlebrook OADC enrichment (BD) was used for growth on solid medium ("7H11"). M. tuberculosis was transformed as described 27 . E. coli strains used for cloning and expression were grown in LB-Miller broth (Difco) at 37°C with aeration on a shaker or on LB agar. E. coli strains were chemically transformed as previously described 28 . The final concentrations of antibiotics used for M. tuberculosis growth: kanamycin, 50 µg/ml; hygromycin, 50 µg/ml; streptomycin, 25 µg/ml; and for E. coli: hygromycin, 150 µg/ml; kanamycin, 100 µg/ml; and streptomycin 50 µg/ml. AMP, adenine and iP were purchased from Sigma. iPR, 2MeSiP and 2MeSiPR were synthesized as previously described 29 .
The ΔRv0078::hyg mutant was made by deletion-disruption mutagenesis as described in detail elsewhere using pYUB854 30 . Cells were harvested and RNA was purified as described previously 30  5'RACE was performed as described by the manufacturer (Invitrogen). Briefly, 1 µg of RNA was used as template for cDNA production using a reverse primer 150-300 bp downstream of annotated translational start sites. A 3' poly-C tail was added to cDNA by recombinant Tdt. The cDNA was then amplified using a nested reverse primer and a primer that anneals to the poly-C tail. Products were cloned and sequenced.
Likely transcriptional start sites were selected based on clones that had the most nucleotide sequence upstream of the start codon. Total lipids extraction from bacterial cells and preparation of fatty acid and mycolic acid methyl esters from extractable lipids and delipidated cells followed earlier procedures 35 .

Acid
Total lipids and fatty acid/mycolic acid methyl esters were analyzed by one and twodimensional thin-layer chromatography (TLC) in a variety of solvent systems on aluminum-backed silica gel 60-precoated plates F 254 (E. Merck). TLC plates were revealed by spraying with cupric sulfate (10% in a 8% phosphoric acid solution) and heating. Alternatively, total lipids were run in both positive and negative mode and the released fatty acids/mycolic acids in negative mode only, on a high resolution Agilent 6220 TOF mass spectrometer interfaced to a LC as described 36,37 . Data files were analyzed with Agilent's Mass hunter work station software and most compounds were identified using a database of M. tuberculosis lipids developed in-house 36 Table S1). c, Deletion and disruption of Rv0078 results in the constitutive expression of Rv0077c. Total cell lysates were prepared and separated on a 10% SDS-PAGE gel. IB = immunoblot. The membrane was stripped and incubated them with antibodies to DlaT to confirm equal loading of samples. Molecular weight standards are indicated to the left of the blots and are in kilodaltons (kD). d, A cartoon view of the crystal structure of the apo Rv0078 dimer at 1.85 Å resolution (PDB pending). LBD, ligand binding domain. DBD, DNA binding domain. e, The purple mesh shows a putative ligand binding pocket surrounded by helices α4 to α8 in the LBD of one Rv0078 monomer. Trp100, which was required for cytokinin-dependent induction, is labeled.