iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum
2016; Elsevier BV; Volume: 291; Issue: 38 Linguagem: Inglês
10.1074/jbc.m116.724088
ISSN1083-351X
AutoresMaikel Boot, Marion Sparrius, Kin Ki Jim, Susanna Commandeur, Alexander Speer, Robert van de Weerd, Wilbert Bitter,
Tópico(s)Mycobacterium research and diagnosis
ResumoTuberculosis can be treated with a 6-month regimen of antibiotics. Although the targets of most of the first-line antibiotics have been identified, less research has focused on the intrabacterial stress responses that follow upon treatment with antibiotics. Studying the roles of these stress genes may lead to the identification of crucial stress-coping mechanisms that can provide additional drug targets to increase treatment efficacy. A three-gene operon with unknown function that is strongly up-regulated upon treatment with isoniazid and ethambutol is the iniBAC operon. We have reproduced these findings and show that iniBAC genes are also induced in infected host cells, although with higher variability. Next, we set out to elucidate the genetic network that results in iniBAC induction in Mycobacterium marinum. By transposon mutagenesis, we identified that the operon is highly induced by mutations in genes encoding enzymes of the vitamin B12 biosynthesis pathway and the vitamin B12-dependent methylmalonyl-CoA-mutase MutAB. Lipid analysis showed that a mutA::tn mutant has decreased phthiocerol dimycocerosates levels, suggesting a link between iniBAC induction and the production of methyl-branched lipids. Moreover, a similar screen in Mycobacterium bovis BCG identified that phthiocerol dimycocerosate biosynthesis mutants cause the up-regulation of iniBAC genes. Based on these data, we propose that iniBAC is induced in response to mutations that cause defects in the biosynthesis of methyl-branched lipids. The resulting metabolic stress caused by these mutations or caused by ethambutol or isoniazid treatment may be relieved by iniBAC to increase the chance of bacterial survival. Tuberculosis can be treated with a 6-month regimen of antibiotics. Although the targets of most of the first-line antibiotics have been identified, less research has focused on the intrabacterial stress responses that follow upon treatment with antibiotics. Studying the roles of these stress genes may lead to the identification of crucial stress-coping mechanisms that can provide additional drug targets to increase treatment efficacy. A three-gene operon with unknown function that is strongly up-regulated upon treatment with isoniazid and ethambutol is the iniBAC operon. We have reproduced these findings and show that iniBAC genes are also induced in infected host cells, although with higher variability. Next, we set out to elucidate the genetic network that results in iniBAC induction in Mycobacterium marinum. By transposon mutagenesis, we identified that the operon is highly induced by mutations in genes encoding enzymes of the vitamin B12 biosynthesis pathway and the vitamin B12-dependent methylmalonyl-CoA-mutase MutAB. Lipid analysis showed that a mutA::tn mutant has decreased phthiocerol dimycocerosates levels, suggesting a link between iniBAC induction and the production of methyl-branched lipids. Moreover, a similar screen in Mycobacterium bovis BCG identified that phthiocerol dimycocerosate biosynthesis mutants cause the up-regulation of iniBAC genes. Based on these data, we propose that iniBAC is induced in response to mutations that cause defects in the biosynthesis of methyl-branched lipids. The resulting metabolic stress caused by these mutations or caused by ethambutol or isoniazid treatment may be relieved by iniBAC to increase the chance of bacterial survival. Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for 1.5 million deaths annually, making it the most deadly bacterial pathogen known to mankind (1..World Health Organization (2013) Global Tuberculosis Report 2013,Google Scholar). Although tuberculosis can be treated effectively with a long term multidrug regimen, failure to complete treatment has led to increasing occurrence of drug resistance, which in turn complicates treatment (1..World Health Organization (2013) Global Tuberculosis Report 2013,Google Scholar, 2Mitnick C.D. Shin S.S. Seung K.J. Rich M.L. Atwood S.S. Furin J.J. Fitzmaurice G.M. Alcantara Viru F.A. Appleton S.C. Bayona J.N. Bonilla C.A. Chalco K. Choi S. Franke M.F. Fraser H.S. Comprehensive treatment of extensively drug-resistant tuberculosis.N. Engl. J. Med. 2008; 359: 563-574Crossref PubMed Scopus (339) Google Scholar). Therefore, the need to develop new anti-mycobacterial compounds is evident. An attractive target for drug therapy is the mycobacterial cell wall, which is composed of non-canonical elements providing the opportunity to specifically target mycobacteria (3Brennan P.J. Nikaido H. The envelope of mycobacteria.Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1557) Google Scholar). In the past several antibiotics that target the mycobacterial cell wall have been identified and have proven to be effective in treatment of tuberculosis. Well known examples of specific anti-cell wall antibiotics for mycobacteria are ethambutol (EMB) 2The abbreviations used are: EMB, ethambutol; INH, isoniazid; CLC, carp leukocyte cell; PDIM, phthiocerol dimycocerosate; MIC, minimal inhibitory concentration; hpi, hours post infection; DLP, dynamin-like protein. 2The abbreviations used are: EMB, ethambutol; INH, isoniazid; CLC, carp leukocyte cell; PDIM, phthiocerol dimycocerosate; MIC, minimal inhibitory concentration; hpi, hours post infection; DLP, dynamin-like protein. and isoniazid (INH). Ethambutol targets the polymerization of arabinan, an essential component of the mycobacterial cell wall involved in covalently linking the peptidoglycan layer to the outer membrane (4Belanger A.E. Besra G.S. Ford M.E. Mikusová K. Belisle J.T. Brennan P.J. Inamine J.M. The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 11919-11924Crossref PubMed Scopus (398) Google Scholar). The prodrug isoniazid is activated by the peroxidase KatG and subsequently forms adducts with NAD+ to inhibit InhA, a NADH-dependent enoyl-ACP (acyl carrier protein) reductase (5Vilchèze C. Jacobs W.R. The mechanism of isoniazid killing: clarity through the scope of genetics.Annu. Rev. Microbiol. 2007; 61: 35-50Crossref PubMed Scopus (248) Google Scholar). As a consequence, biosynthesis of mycolic acids, the most abundant lipid of the mycobacterial outer membrane, is inhibited, causing an aberrant and lethal deformation of the cell wall (6Winder F.G. Collins P.B. Inhibition by isoniazid of synthesis of mycolic acids in Mycobacterium tuberculosis.J. Gen. Microbiol. 1970; 63: 41-48Crossref PubMed Scopus (174) Google Scholar). Recent research efforts have emphasized that also other essential steps in the biogenesis of the mycobacterial cell wall can serve as new targets for anti-tuberculosis interventions (7Kieser K.J. Baranowski C. Chao M.C. Long J.E. Sassetti C.M. Waldor M.K. Sacchettini J.C. Ioerger T.R. Rubin E.J. Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 13087-13092Crossref PubMed Scopus (63) Google Scholar, 8Stanley S.A. Kawate T. Iwase N. Shimizu M. Clatworthy A.E. Kazyanskaya E. Sacchettini J.C. Ioerger T.R. Siddiqi N.A. Minami S. Aquadro J.A. Grant S.S. Rubin E.J. Hung D.T. Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 11565-11570Crossref PubMed Scopus (80) Google Scholar). Although our knowledge on cell wall biosynthesis in mycobacteria has greatly increased over the last decades, the bacterial stress responses that follow after treatment with antibiotics targeting the mycobacterial cell wall have yet to be elucidated. By mapping the intra-bacterial responses upon treatment with antibiotics, key pathways that are necessary to cope with antibiotics or with antibiotic-induced stress can be identified. Moreover, mapping bacterial stress levels may help to explain the observed heterogeneity, displayed by bacterial subpopulations, to antibiotic treatment within patients (9Liu Q. Via L.E. Luo T. Liang L. Liu X. Wu S. Shen Q. Wei W. Ruan X. Yuan X. Zhang G. Barry 3rd, C.E. Gao Q. Within patient microevolution of Mycobacterium tuberculosis correlates with heterogeneous responses to treatment.Sci. Rep. 2015; 5: 17507Crossref PubMed Scopus (62) Google Scholar). Together, these findings might pave the way for novel drug targets that sensitize or enhance currently used antibiotics. Despite the availability of gene expression data after exposure of mycobacteria to anti-tuberculosis drugs, there is no comprehensive map of the stress response pathways that follow upon treatment. Moreover, antibiotic stress regulation pathways were not addressed in general. Insights into the pathways that mycobacteria deploy when confronted with antibiotic treatment will lead to a better understanding of the bacterial adaptive strategies and may consequently pinpoint drug-induced blind spots that can be targeted by novel compounds. Some individual genes or gene clusters that are regulated in response to antibiotic treatment have been well characterized. For instance, the acyl carrier protein kasA has been found to be up-regulated in response to isoniazid (10Wilson M. DeRisi J. Kristensen H.H. Imboden P. Rane S. Brown P.O. Schoolnik G.K. Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 12833-12838Crossref PubMed Scopus (495) Google Scholar). Another set of genes that was found to be highly induced by sublethal concentrations of ethambutol and isoniazid in M. tuberculosis and Mycobacterium bovis BCG is the iniBAC operon (11Alland D. Kramnik I. Weisbrod T.R. Otsubo L. Cerny R. Miller L.P. Jacobs Jr., W.R. Bloom B.R. Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13227-13232Crossref PubMed Scopus (94) Google Scholar, 12Alland D. Steyn A.J. Weisbrod T. Aldrich K. Jacobs Jr., W.R. Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition.J. Bacteriol. 2000; 182: 1802-1811Crossref PubMed Scopus (113) Google Scholar). This operon was found to be induced specifically by these two cell wall targeting antibiotics but not by general cell wall stress caused by disruption of the membrane integrity through the activity of granulysin or lysozyme (12Alland D. Steyn A.J. Weisbrod T. Aldrich K. Jacobs Jr., W.R. Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition.J. Bacteriol. 2000; 182: 1802-1811Crossref PubMed Scopus (113) Google Scholar). Further studies by the same group showed that overexpression of iniA seemed to confer tolerance to INH treatment in M. bovis BCG but not in M. tuberculosis (13Colangeli R. Helb D. Sridharan S. Sun J. Varma-Basil M. Hazbón M.H. Harbacheuski R. Megjugorac N.J. Jacobs Jr., W.R. Holzenburg A. Sacchettini J.C. Alland D. The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol.Mol. Microbiol. 2005; 55: 1829-1840Crossref PubMed Scopus (145) Google Scholar). This suggests a function for iniA in coping with the stress that is induced by INH or EMB treatment in M. bovis BCG. Despite these early studies, not much is known about the pathways that lead to induction of these three genes with unknown function. In Escherichia coli, disruption of cell wall biosynthesis can be sensed by extracytoplasmic function sigma factor E (14Erickson J.W. Gross C.A. Identification of the sigE subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression.Genes Dev. 1989; 3: 1462-1471Crossref PubMed Scopus (307) Google Scholar). Similarly, mycobacterial sigE has been shown to respond to heat-shock and oxidative stresses (15Manganelli R. Voskuil M.I. Schoolnik G.K. Smith I. The Mycobacterium tuberculosis ECF sigma factor sigE:role in global gene expression and survival in macrophages.Mol. Microbiol. 2001; 41: 423-437Crossref PubMed Scopus (350) Google Scholar). Although sigE is not a specific sensor for cell wall stress, other sigma factors may play a role in the stress network that is activated upon iniBAC induction. A few publications have addressed the regulation of the iniBAC operon. Colangeli et al. (16Colangeli R. Helb D. Vilchèze C. Hazbón M.H. Lee C.G. Safi H. Sayers B. Sardone I. Jones M.B. Fleischmann R.D. Peterson S.N. Jacobs Jr., W.R. Alland D. Transcriptional Regulation of multi-drug tolerance and antibiotic-induced responses by the histone-like protein Lsr2 in M. tuberculosis.Plos Pathog. 2007; 3: e87Crossref PubMed Scopus (92) Google Scholar) have shown that lsr2, among regulating a large set of other genes, negatively regulates iniBAC expression, whereas recent studies have also implicated operon regulation by mtrA and the alleged INH binding regulation element inbR (17Li Y. Zeng J. Zhang H. He Z. The characterization of conserved binding motifs and potential target genes for M. tuberculosis MtrAB reveals a link between the two-component system and the drug resistance of M. smegmatis.BMC Microbiol. 2010; 10: 242Crossref PubMed Scopus (60) Google Scholar, 18Yang M. Gao C.H. Hu J. Zhao L. Huang Q. He Z.G. InbR, a TetR family regulator, binds with isoniazid and influences multidrug resistance in Mycobacterium bovis BCG.Sci. Rep. 2015; 5: 13969Crossref PubMed Scopus (13) Google Scholar). In this study we aimed to elucidate the genetic network that revolves around the induction of the iniBAC operon. We found that the operon can be induced by mutations in vitamin B12 biosynthesis or the vitamin B12-dependent methylmalonyl-CoA mutase MutAB in Mycobacterium marinum. Mutants in mutAB show decreased phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL) levels but no altered antibiotic susceptibility. To investigate the response of the iniBAC operon to stress, we generated a reporter gene construct by placing the gene encoding green fluorescent protein mEos3.1 under control of the M. marinum iniB promoter region, optimized as previously reported for M. tuberculosis (12Alland D. Steyn A.J. Weisbrod T. Aldrich K. Jacobs Jr., W.R. Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition.J. Bacteriol. 2000; 182: 1802-1811Crossref PubMed Scopus (113) Google Scholar). The reporter construct was transformed into M. marinum cells, and activity was assessed using the inducing antibiotics INH or EMB. The initial induction experiments were carried out by the addition of the antibiotics EMB or INH at their minimal inhibitory concentration (MIC) followed by flow cytometry analysis. Starting from day 1, we observed a strong induction of the iniBAC reporter construct after the addition of the antibiotic, as compared with the non-treated M. marinum cells; fluorescence was 114-fold (EMB) and 121-fold (INH) higher. The induction increased over time and reached a 357-fold difference by day 5 for EMB and 643-fold for INH (Fig. 1A). Next, we tested reporter activity in cell infection experiments. Carp leukocyte cells (CLC) and THP-1 macrophages were infected with M. marinum harboring the iniBAC reporter construct, and analysis of induction kinetics was tracked by flow cytometry. To differentiate between infected and non-infected macrophages and to assess which proportion of the infected cells expressed the iniBAC reporter construct, the red fluorescent protein crimson E2 was constitutively expressed on an episomal construct. We combined this with an integrative version of the stress marker containing the iniB promoter and mEos3.1. As such we could determine which proportion of the total pool of infected, crimson-positive cells was also iniBAC-induced and, thus, mEos3.1 positive. After infection, the phagocytized bacteria were challenged with 1× MIC of EMB or INH, and flow cytometry was used to measure induction of the iniBAC reporter on days 0, 1, 2, and 3. Fig. 1B shows a representative measurement of an experiment performed in triplicate. In THP-1 cells the population of macrophages that contained bacteria with induced iniBAC increased to 41.6% (S.D. 0.4%) on day 3 for ethambutol. Isoniazid showed a faster induction pattern in THP-1 cells with 13.4% (S.D. 0.4%) becoming mEos3.1-positive on day 1, increasing to 38.6% (S.D. 0.7%) on day 3 (Fig. 1, B and C). In CLC infection assays, similar patterns were observed (Fig. 1, B and C). In summary, the induction of the iniBAC reporter during intracellular infection seems to increase over time but does not seem to affect all bacteria, which was observed in culture. THP-1 cells were also analyzed by confocal imaging on days 1 and 2 (Fig. 2). Confocal imaging confirmed the observations of flow cytometry assays. Moreover, the EMB- and INH-treated macrophages showed a clear pattern of heterogeneity from day 2 onward, coinciding with the significant increase in induction seen by flow cytometry for both antibiotics. In conclusion, we have confirmed that M. marinum expresses iniBAC upon treatment with EMB and INH. Furthermore, this induction is observed in culture and in two different cell types using a cell infection assay. However, induction is more variable during cell infection as compared with culture. Next we set out to probe which genes and regulation networks play a role in iniBAC induction. For this, a wild type M. marinum MUSA containing an exosomal variant of the iniBAC stress marker was used as a parent strain to create a transposon library. The resulting mutants were plated out and selected for high fluorescence. Analysis of the transposon insertion site was performed for a total of 50 positive mutants. Strikingly, 32 of the 50 transposons insertions were identified in genes that are responsible for cobalamin synthesis, the so-called cob family genes (Fig. 3A). These genes are part of the set of 25 genes that are required for vitamin B12 biosynthesis in mycobacteria (19Gopinath K. Moosa A. Mizrahi V. Warner D.F. Vitamin B12 metabolism in Mycobacterium tuberculosis.Future Microbiol. 2013; 8: 1405-1418Crossref PubMed Scopus (46) Google Scholar). Furthermore, 12 mutants were identified in methylmalonyl-CoA-mutase encoding genes MutAB or in the gene coding for its alleged foldase, meaB (20Savvi S. Warner D.F. Kana B.D. McKinney J.D. Mizrahi V. Dawes S.S. Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growthon fatty acids.J. Bacteriol. 2008; 190: 3886-3895Crossref PubMed Scopus (178) Google Scholar). The MutAB enzyme plays a role in propionate metabolism, converting (S)-methylmalonyl-CoA, a precursor for methyl-branched lipids, to (R)-methylmalonyl-CoA, which is further processed to succinyl-CoA to enter the tricarboxylic acid or glyoxylate cycle (Fig. 3B). The methyl-branched lipids synthesized from (S)-methylmalonyl-CoA include PDIM, diacyl trehalose/penta-acyl trehalose, and SL-1 (21Minnikin D.E. Kremer L. Dover L.G. Besra G.S. The methyl-branched fortifications of Mycobacterium tuberculosis.Chem. Biol. 2002; 9: 545-553Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). MutAB is vitamin B12-dependent, suggesting that the mutants affected in vitamin B12 biosynthesis were most likely identified because of their downstream effect on MutAB. Besides identification of most known cobalamin biosynthesis genes, this screen also confirms that MMAR_4563, is probably coding for the vitamin B12 biosynthesis enzyme CobF (data not shown), and MaeB is indeed functionally linked to MutAB. Other mutants that showed induction, albeit to a lesser extent, include mutations in pks16, mshD, or mas, genes that are all involved in cell wall lipid biosynthesis (Table 1).TABLE 1List of M. marinum mutants identified in transposon mutagenesis screenGene IDGene productH37Rv orthologueGene lengthTransposon position(s) within gene (bp from 5' of gene)MMAR_0989PE_PGRS31532149MMAR_1350tetR repressorRv3208684421MMAR_1768masRv2940c56161173MMAR_1885cobBRv28481377148, 194, 416, 429, 597, 610, 905, 1271, 1290, 1293, 1324MMAR_2302mutARv1492187814, 225, 427, 1836MMAR_2303mutBRv149322626, 41, 521, 1095, 1724, 2179MMAR_2304meaBRv14961011204, 379MMAR_3037cobNRv2062c357376, 129, 311, 1486, 3374, 3394MMAR_3045cobHRv2065627123MMAR_3046cobIRv20661470256MMAR_3052cobKRv2070c72911MMAR_3053cobMRv2071c740715MMAR_3054cobLRv2072c11731093MMAR_3251cobURv0254c56175MMAR_3252cobTRv22071077525MMAR_3307cobCRv2231c1053273MMAR_3311cobDRv2236c942792MMAR_4476pks16Rv10131635652MMAR_4563cobFnone74430, 134, 155, 232, 610MMAR_4863mshDRv0819963469, 526 Open table in a new tab To confirm that the effects seen on the induction of the iniBAC stress marker were due to abrogated vitamin B12 biosynthesis, we compared the induction of three mutants, mutA::tn, mshD::tn, and cobN::tn. Without the addition of vitamin B12, both mutA::tn and cobN::tn showed clear expression of mCherry (Fig. 4A). As expected, the cobN transposon mutant clearly reverts from high iniBAC induction to a near background expression level by the addition of 10 μg/ml vitamin B12 (Fig. 4B). We also tested which concentration of vitamin B12 was required to phenotypically complement the cob mutants. Lowering the concentration to <100 ng/ml vitamin B12 abrogated complementation, which was also confirmed by flow cytometry (data not shown). Because usually only trace amounts of vitamin B12 are required, this means that M. marinum does not efficiently transport vitamin B12 under these conditions. The other two mutants, mutA::tn and mshD::tn, were unaffected by the addition of vitamin B12, as confirmed by fluorescence microscopy (data not shown). Besides complementation with vitamin B12, a genetic complementation was also performed for three mutants, i.e. cobB::tn, mshD::tn, and mutA::tn. The resulting complementing strains were analyzed by flow cytometry for fluorescence induction of the stress marker as compared with their mutant parent strain. This confirmed that complementation of cobB::tn and mshD::tn was achieved by introducing an intact copy of the respective gene (Fig. 4C). The mutA::tn mutant did not complement when only mutA was reintroduced in the mutant strain, suggesting that the transposon mutant we obtained in mutA likely also disturbs expression of the downstream genes mutB and MMAR_2304 (meaB orthologue). Complementation was achieved when we introduced mutA, mutB, and MMAR_2304, as fluorescence induction was restored to wild type levels. To assess whether we could find the metabolic cue that induces iniBAC, we performed an experiment on HdB minimal medium plates containing only acetic acid, propionic acid, or valeric acid as carbon source. Because acetic acid and propionic acid are both major players in the pathways in which MutAB operates, we suspected that the addition of these fatty acids could influence either bacterial growth of some of our mutants or iniBAC induction. Glucose and glycerol served as a growth control. However, we did not observe any significant growth differences upon the addition of 0.1% acetic acid, propionic acid, or valeric acid when comparing a cobB::tn and mutA::tn mutant versus a WT M. marinum (Fig. 5A). We also analyzed the iniBAC induction of the bacteria that grew on these plates by flow cytometry. Also in this experiment, we did not observe any significant changes in induction (Fig. 5B). These results suggest that the induction of the iniBAC operon is probably not dependent directly on the level of metabolic intermediates or fluxes in the routes outlined in Fig. 3. To assess whether vitamin B12 availability can complement the cob mutants in vivo, three different mutants (cobN::tn, mutA::tn, and mshD::tn) were selected for analysis in a zebrafish embryo hindbrain infection model using WT as a control. All four strains contained a constitutive, integrated hsp60-mEos3.1 for visualization of infection and an episomal version of the mCherry iniB stress marker. After 24 h of infection, a homogeneous induction of iniBAC was seen for all three mutants, indicating that the induction has not changed in this period of time (Fig. 6), which is not unexpected because the bacteria were already highly expressing mCherry and the protein is relatively stable. However, after 120 h post infection (hpi), a clear reduction of mCherry was observed in all three mutants, whereas the green fluorescent signal, i.e. mEos3.1, was still present or even increased. In summary, we show that iniBAC induction is declining in vivo, with virtually no induction after 120 h. The decrease in induction was seen for all tested mutants and, therefore, seems to be independent of vitamin B12 availability. Because MutAB mediates the isomerization of (R)-methyl-malonyl-CoA to succinyl-CoA, we hypothesized that a mutA transposon mutant could have an excess amount of (R)-methylmalonyl-CoA. Consequently, this could influence the amount of methyl-branched lipids that are synthesized, such as PGLs/PDIMs (Fig. 3B). To assess whether this was the case, we performed TLC analysis to detect both PDIM and PGL variants for WT MUSA and transposon mutants in genes mas, mutA, and cobB. Strikingly, when comparing the amount of PDIMs of the mutA and cobB transposon mutants, a significant reduction in PDIM types A, B, and C can be distinguished (Fig. 7A). This reduction is phenotypically similar to a mas transposon mutant, which is known to be strongly affected in PDIM production (22Azad A.K. Sirakova T.D. Rogers L.M. Kolattukudy P.E. Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 4787-4792Crossref PubMed Scopus (86) Google Scholar). Moreover, PGL production was also severely decreased in mutA::tn and cobB::tn compared with WT, whereas the mas mutant, as expected, showed a null phenotype for this lipid (Fig. 7B). In conclusion, abrogation of vitamin B12 biosynthesis or mutation of mutAB caused a decrease in methyl-branched lipids PGL and PDIM in M. marinum. To see whether the mutations that induce iniBAC are also changed in antibiotic susceptibility, we determined the MIC values of mutA::tn, mutB::tn, mshD::tn, and cobB::tn upon exposure to five first-line antibiotics. There were no differences observed for the four mutants tested, indicating that, although differences in PDIM and PGL can be seen for mutA::tn and cobB::tn, this does not seem to affect antibiotic susceptibility (Table 2). This finding seems to contradict the previously published results by Colangeli et al. (13Colangeli R. Helb D. Sridharan S. Sun J. Varma-Basil M. Hazbón M.H. Harbacheuski R. Megjugorac N.J. Jacobs Jr., W.R. Holzenburg A. Sacchettini J.C. Alland D. The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol.Mol. Microbiol. 2005; 55: 1829-1840Crossref PubMed Scopus (145) Google Scholar), where they stated that up-regulation of iniA in M. tuberculosis caused a moderate but significantly increased resistance phenotype against ethambutol.TABLE 2MIC determination on M. marinum mutants shows no change in antibiotic susceptibilityStrainRifampicinEthambutolIsoniazidStreptomycinCiprofloxacinμg/mlμg/mlμg/mlμg/mlμg/mlWT0.321.63244mutA::tn0.320.83244mutB::tn0.320.83244cobB::tn0.320.83244mshD::tn0.320.83244 Open table in a new tab The array of mutants that show an increased induction of the iniBAC operon seem to revolve around methyl-branched lipid biosynthesis. To see whether a defect in lipid biosynthesis and cell envelope integrity in general would also cause induction of the operon, we transformed a kasB::tn mutant, a mutant known to produce truncated mycolic acids, with the iniBAC stress marker (23Bhatt A. Fujiwara N. Bhatt K. Gurcha S.S. Kremer L. Chen B. Chan J. Porcelli S.A. Kobayashi K. Besra G.S. Jacobs W.R. Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5157-5162Crossref PubMed Scopus (149) Google Scholar). This mutant shows reduced growth and is attenuated in zebrafish embryos. 3E. Stoop, G. van den Brink, A. van der Sar, and W. Bitter, unpublished results. Comparison of induction on plate to a mutA::tn and mshD::tn mutant revealed that the kasB::tn mutant does not seem to be highly induced on plate (data not shown). Subsequent analysis by flow cytometry revealed that the iniBAC operon is indeed only 1.4-fold induced in this mutant, comparable with a mshD::tn mutant, whereas a mutA transposon mutant showed a 14.2-fold induction compared with a WT expressing the stress marker (Fig. 8). Therefore, not all genes that cause cell wall alterations contribute equally to the induction of the iniBAC operon. Although most genes coding for vitamin B12 biosynthesis enzymes are present in species belonging to the M. tuberculosis complex, research by Warner et al. (24Warner D.F. Savvi S. Mizrahi V. Dawes S.S. A riboswitch regulates expression of the coenzyme B12-independent methionine synthase in Mycobacterium tuberculosis: implications for differential methionine synthase function in strains H37Rv and CDC1551.J. Bacteriol. 2007; 189: 3655-3659Crossref PubMed Scopus (64) Google Scholar) has shown that M. tuberculosis does not seem produce vitamin
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