INFECTION AND IMMUNITY, Apr. 2006, p. 2457–2461 0019-9567/06/$08.00⫹0 doi:10.1128/IAI.74.4.2457–2461.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 4
Posttranslational Regulation of Mycobacterium tuberculosis Extracytoplasmic-Function Sigma Factor L and Roles in Virulence and in Global Regulation of Gene Expression Elisa Dainese,1† Se´bastien Rodrigue,2† Giovanni Delogu,3 Roberta Provvedi,1 Liette Laflamme,2 Ryszard Brzezinski,4 Giovanni Fadda,3 Issar Smith,5 Luc Gaudreau,2 Giorgio Palu `,1 and Riccardo Manganelli1* Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy1; Centre de Recherche sur les Me´canismes du Fonctionnement Cellulaire, Universite´ de Sherbrooke, Sherbrooke, Que´bec, Canada2; Institute of Microbiology, Catholic University of the Sacred Hearth, Rome, Italy3; Centre de Valorisation de la Diversite´ Microbienne, De´partement de Biologie, Universite´ de Sherbrooke, Sherbrooke, Que´bec, Canada4; and TB Center, The Public Health Research Institute, Newark, New Jersey5 Received 9 November 2005/Returned for modification 12 December 2005/Accepted 23 January 2006
In this report, we demonstrate that SigL is posttranslationally regulated by a specific anti-sigma factor, RslA, and contributes to the expression of at least 28 genes. Several of these genes could mediate important cell enveloperelated processes. Importantly, a sigL-rslA mutant strain was significantly attenuated in a mouse model of infection. sigma factors (data not shown). Primer extension assays were performed according to standard procedures using the following oligonucleotide: Mtb sigB-EXT (5⬘-ATCCAGATCGCTG TCAACCCGG-3⬘). A putative promoter closely resembling
Sigma factors are interchangeable subunits that provide the promoter recognition function to bacterial RNA polymerase (RNAP) holoenzymes (10). Thirteen sigma factor genes were predicted from the genome sequence of Mycobacterium tuberculosis. Several of these were shown to be important for appropriate stress responses and virulence (9, 15–18, 20, 27, 29). Interestingly, the expression of the B-encoding gene (sigB) is induced in response to several stimuli (4, 14, 17, 18), suggesting a potential general role in stress responses (16, 24). Previous studies have shown that transcription of sigB can be initiated at the same nucleotide by RNAP holoenzymes that contain extracytoplasmic-function sigma factor E or H (17, 18, 21). It was also recently reported that the extracytoplasmic-function sigma factor L may also have a role in the expression of sigB (11, 16). Using an in vitro transcription assay, we have tested each M. tuberculosis sigma factor to determine whether additional sigma factors could elicit transcription of sigB. Reactions were performed as previously described (3) using purified Mycobacterium smegmatis RNAP, 150 fmol of sigB DNA template (encompassing base pairs ⫺361 to ⫹329 relative to the first translated nucleotide), and 3 pmol of recombinant sigma factors (13). As expected, primer extension products were detected for the E- and H-containing holoenzymes (Fig. 1A). Interestingly, the L-dependent transcription signal also originated from the same nucleotide (Fig. 1A). Importantly, the E-, H-, and L-dependent signals matched the sigB transcriptional start site identified in vivo (17, 21). In addition, an upstream F-dependent promoter with striking homology to the proposed F consensus was identified (Fig. 1A) (3, 9, 16). No signal was observed with the nine remaining M. tuberculosis
FIG. 1. Identification of M. tuberculosis (Mtb) sigma factors involved in the transcription of selected genes. (A) High-resolution mapping of transcription signals observed using an in vitro transcription screening of the sigB upstream region. E, H, and L initiate transcription at the same nucleotide, while F use an upstream promoter. (B) sigL is expressed from an autoregulated promoter. In vitro transcription with the corresponding sigma factors was followed by a primer extension procedure. Proposed promoter boxes are underlined. Arrows indicate transcriptional start sites.
* Corresponding author. Mailing address: Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Via Gabelli 63, 35100 Padova, Italy. Phone: 39 49 827 2366. Fax: 39 49 827 2355. E-mail:
[email protected]. † E.D. and S.R. contributed equally to this work. 2457
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FIG. 3. Time-to-death analysis in DBA/2 mice after aerosol infection with M. tuberculosis H37Rv (squares) and with the sigL mutant strain (circles). FIG. 2. RslA is a L-specific anti-sigma factor. (A) GST-RslA interacts with L. Results of GST pull-down assays between GST-RslA and hexahistidine-tagged H or L revealed by immunoblotting using an anti-His antibody are shown. L, loading control; S, supernatant; P, pellet. (B) RslA inhibits L activity in a dose-dependent manner. In vitro transcription inhibition assays at the sigB promoter were carried out in the absence (⫺) or presence (⫹) of L and increasing amounts (triangle) of hexahistidine-tagged RslA cytoplasmic moiety.
the E-H-L-dependent sigB promoter was also found upstream of sigL. In vitro transcription was thus performed as in Fig. 1A using a sigL DNA template (base pairs ⫺345 to ⫹138 relative to the translation start site) and oligonucleotide Mtb sigL-EXT (5⬘-CTCTTGGACGACGTCTTC-3⬘). Interestingly, L was the only sigma factor that allowed transcription from this promoter, suggesting that minor divergences in DNA sequences may account for promoter discrimination by E, H, and L (Fig. 1B). Hahn and colleagues recently proposed that RslA could be a L anti-sigma factor and detected an interaction between these proteins in a bacterial two-hybrid assay (11). Since RslA is a predicted transmembrane protein, we have fused its cytoplasmic moiety (amino acids 1 to 114) to glutathione S-transferase (GST) and performed direct interaction assays with hexahistidine-tagged E, F, H, and L. Assays were performed as previously described (3) and analyzed by immunoblotting using anti-His antibody. Figure 2A shows a direct
interaction between L and GST-RslA. No interaction was detected between GST-RslA and E, F, and H or between GST and any sigma factor (Fig. 2A and data not shown). In vitro transcription assays next demonstrated that RslA inhibits L-dependent transcription of the sigB gene in a dose-dependent manner (Fig. 2B). In this latter experiment, increasing concentrations (1.5, 3.0, 6.0, and 12 pmol) of the N-terminally hexahistidine-tagged RslA cytoplasmic moiety were added to a fixed amount of L-containing holoenzyme and sigB promoter template as described above for Fig. 1. Importantly, the Ldependent signal was not affected by the addition of RshA or Rv1222, a H (26) and a E anti-sigma factor, respectively (data not shown). Taken together, these results demonstrate that RslA is a L-specific anti-sigma factor. The DNA region including sigL and rslA was next replaced in the genome of M. tuberculosis H37Rv by an hygromycin resistance gene using specialized transduction (2). The growth curve and colony morphology of the resulting strain (TB1) was indistinguishable from the wild-type (wt) strain (data not shown). In agreement with the results obtained by Hahn et al. using a similar sigL mutant (11), we have not observed any
TABLE 1. Bacterial counts in organs of C57BL/6 mice after aerosol infection Log CFUa (mean ⫾ SD) on:
Strain and organ
Day 1
Day 28
Day 60
H37Rv Lung Spleen
2.04 ⫾ 0.11 ND
5.06 ⫾ 0.10 3.75 ⫾ 0.17
4.77 ⫾ 0.18 3.77 ⫾ 0.26
TB1 Lung Spleen
2.12 ⫾ 0.15 ND
4.92 ⫾ 0.15 3.51 ⫾ 0.18
4.85 ⫾ 0.13 3.63 ⫾ 0.15
a
Data from at least five mice. ND, not determined.
FIG. 4. Measurement of sigL expression. M. tuberculosis H37Rv harboring a reporter plasmid carrying the sigL upstream region fused to lacZ was grown in Middlebrook 7H9 medium, and -galactosidase activity was measured at different points of the growth curve. The bars show -galactosidase activity (in Miller units), and the line shows the optical density at 540 nm (OD 540).
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TABLE 2. Genes under L controla Locusb
Geneb
q value (%)c
Fold inductiond
Rv0593
lpr
0
Rv0594
mce2F
0
3.4 (3.5) 关1.0兴
0 0 0
18.2 12.6 12.8 (61.7) 关0.7兴
Rv1137c Rv1138c Rv1139c
20.3
Rv1145
mmpL13a
0
8.3 (16.0) 关1.1兴
Rv1146
mmpL13b
5.2
6.5
Rv1147 Rv1509 Rv1510 Rv1660 Rv1661 Rv1662 Rv1663 Rv1664 Rv1665 Rv1727 Rv2710e Rv2877c Rv2878c Rv2931 Rv3161c Rv3163c Rv3164c Rv3166c Rv3242c Rv3518c
6.5 5.2 0 0 0 0 0 6.7 7.3 0 10.7 0
2.1 2.5 1.5 17.1 (102.5) 关1.4兴 8.1 7.1 5.1 2.4 2.9 10.1 1.7 (1.6) 关1.1兴 13.5
mpt53 ppsA
0 5.0
3.4 (8.2) 关2.2兴 1.8
moxR3
7.0 0 14.2
1.7 4.7 5.5
cyp142
14.1 0 4.5
4.5 9.6 4.3
pks10 pks7 pks8 pks17 pks9 pks11 sigB
Gene product/function
Unknown; thought to be involved in host cell invasion (lipoprotein) Unknown; thought to be involved in host cell invasion (secreted protein) Unknown Oxidoreductase Isoprenylcysteine carboxymethyltransferase (ICMT) family (transmembrane protein) Unknown; thought to be involved in fatty acid transport (transmembrane protein) Unknown; thought to be involved in fatty acid transport (transmembrane protein) Unknown Unknown Unknown (integral membrane protein) Polyketide synthase Polyketide synthase Polyketide synthase Polyketide synthase Polyketide synthase Polyketide synthase Unknown Primary-like sigma factor Unknown; contains CcdA domain, similar to cytochrome c biogenesis protein (integral membrane protein) Unknown; similar to DsbE (secreted protein) Phenolpthiocerol synthesis type I polyketide synthase; involved in dimycocerosyl phthiocerol biosynthesis Dioxigenase, contains a Rieske iron-sulfur domain Unknown (secreted protein) Unknown, AAA ATPase domain, similar to Mg chelatase Unknown (transmembrane protein) Unknown; possible ATPase precursor Probable cytocrome P450 monooxygenase
Since it is absent from M. tuberculosis TB1, sigL was not considered in this table. Genes were included in the table if their q value was ⱕ5 and fold induction was ⱖ1.7. Genes that showed a q value of ⱕ15 that are part of a putative operon in which at least one gene fit the previous criteria were also included in the table. Genes with an upstream putative sigL consensus sequence are shown in bold type. b Genes are annotated as described by the Pasteur Institute on TUBERCULIST (http://genolist.pasteur.fr/TubercuList/). c The q value is the estimated false discovery rate calculated by significance analysis of microarray. d mRNA levels in strain TB3 divided by those in strain TB1. Values in parentheses represent the fold induction obtained by qRT-PCR (TB3/TB1). Values in brackets represent the fold induction obtained by qRT-PCR (H37Rv/TB1). e sigB was added into the table even though it did not fit the q value criteria, since its overexpression in the “partially complemented strain” was confirmed by quantitative RT-PCR. a
difference in the sensitivity of the TB1 and wt strains to diamide, cumene hydroperoxide, H2O2, and sodium nitroprusside (data not shown) using a previously described disk diffusion assay methodology (18). Moreover, the TB1 and wt strains were not differentially affected by EDTA, vancomycin, tetracycline, heat shock (45°C), acidic pH, hyperosmolarity, and dithiothreitol. However, in contrast to the findings of Hahn and colleagues, we have noticed a small but reproducible sensitivity of TB1 to the superoxide generator plumbagine and to the detergent sodium dodecyl sulfate relative to the wt strain in disk diffusion assays performed as previously described (18); these phenotypes were fully complemented by the introduction of the sigL-rslA locus (or by the sigL gene alone, see TB3 strain described below) at the L5 integration site (data not shown). However, no significant increase in sigL expression was monitored by quantitative reverse transcription-PCR (qRT-PCR)
after exposure to plumbagine or sodium dodecyl sulfate (data not shown). Hahn and colleagues have shown that a sigL mutant of M. tuberculosis is attenuated in BALB/c mice in a high-dose intravenous infection model. We have performed a similar analysis using a low-dose aerosol infection model for two different mouse strains with different natural resistances to infection. C57BL/6 and DBA/2 mice were challenged, with ⬃100 and 200 CFU/mouse of the TB1 or H37Rv strain (6). No major differences were observed in the abilities of the two strains to colonize lung and spleen tissues or in the size and extent of lesions as assessed by histopathology analysis at 28 and 60 days postinfection (Table 1 and data not shown). DBA/2 mice were also held for observation and determination of the time to death. A significant extension of the median survival time was observed for mice infected with the TB1 mutant compared to the pa-
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rental strain, thus indicating that sigL is important for virulence (Fig. 3). Using qRT-PCR and primer extension assays, Hahn and colleagues have concluded that sigL is constitutively expressed through mid-exponential and stationary phase by a L-independent promoter located 130 bases upstream of sigL (11). However, by using a plasmid containing a fusion of the first 564 nucleotides upstream of sigL to lacZ, -galactosidase activity in the TB1 and wt strains (measured by the method of Miller [19]) tended to increase with culture density, especially in the transition from lag to exponential phase (Fig. 4). In order to study the role of L by mimicking naturally inducing conditions (release of L by RslA), we have complemented the TB1 mutant strain by the introduction of sigL and its upstream region at the L5 integration site (12). The resulting “partially complemented” strain (TB3) thus contains sigL but not rslA, which should allow a constitutive activity of L. A 25-fold induction of sigL was indeed measured by qRT-PCR in this strain with respect to the H37Rv strain (data not shown). The TB1 and TB3 strains were grown to mid-exponential phase, and the RNA expression profiles were compared using oligonucleotide microarrays as previously described (18). The resulting data were analyzed using the significance analysis of microarray method (28). A total of 27 genes from 12 putative transcription units were up-regulated in TB3 relative to TB1 (Table 2). No repressed genes were identified. Six induced genes were chosen, and their induction levels were confirmed by qRT-PCR (Table 2). The mRNA levels of the selected genes in H37Rv and TB1 mutant strains were also compared. All genes were expressed at similar levels in both strains, suggesting that inhibition of L activity by RslA is virtually complete under these conditions (Table 2). Hahn and colleagues have recently identified 19 genes induced by the overexpression of L from an acetamide-inducible promoter (11). Some genes (pks10, pks7, Rv1138c, mpt53, and Rv2877c) were identified by both approaches. However, the remaining four genes from the pks10 operon, three genes from the putative mmpL13 operon, four genes from the putative operon starting with Rv3166c, mce2F and its upstream gene, sigB, and others, were identified only in this work. Putative promoter boxes similar to those of in vitro-identified Ldependent promoters (Fig. 1) and compatible with the proposed L consensus sequence (11) were found upstream of eight of these genes (Table 2). Rapid amplification of cDNA end (5⬘-RACE) experiments, performed as previously described (17), confirmed that the transcriptional start sites of sigL, mmpL13A, and mpt53 were suitably located to allow L to recognize these promoter elements (data not shown). Interestingly, although E, H, and L all recognize the same promoter upstream of sigB, no other genes seem to be regulated by any combination of these sigma factors (17, 18). This suggests that although the consensus sequences recognized by these sigma factors are similar, few differences are sufficient for promoter discrimination. Many of the genes regulated by L could be involved in processes related to the cell envelope (Table 2). For example, the pks10 operon and ppsA gene products are likely to be involved in the biosynthesis of dimycocerosyl phthiocerol, an important component of the mycobacterial cell wall (1, 5, 8, 22, 23). Moreover, the mmpL13a and mmpL13b gene products
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belong to a family of proteins involved in lipid transport (5), sulfolipid biosynthesis (7), and peptidoglycolipid biogenesis (25). From this perspective, it is noteworthy that RslA is a predicted transmembrane protein that could possibly sense the condition of the cell envelope or an external signal. The absence of L could thus result in a weakening the cell surface or in an inappropriate modulation of the host immune response by the bacterium, which could at least partially explain the attenuation of the sigL mutant reported in this paper and by others (11). This work was supported by grants from ISS (PN-AIDS 50F.24 [R.M.] and 50F.13 [G.F.]), MIUR (PRIN 2003 grant no. 2003059340 [R.M.]), and NIH (grant no. AI-44856 [I.S.]) and from the NSERC “Genomics projects” (R.B. and L.G.). L.G. holds a Canada Research Chair on mechanisms of gene transcription. S.R. is the recipient of fellowships from NSERC and FRSQ. REFERENCES 1. Azad, A. K., T. D. Sirakova, N. D. Fernandes, and P. E. Kolattukudy. 1997. Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria. J. Biol. Chem. 272:16741– 16745. 2. Bardarov, S., S. Bardarov, Jr., M. S. Pavelka, Jr., V. Sambandamurthy, M. Larsen, J. Tufariello, J. Chan, G. Hatfull, and W. R. Jacobs, Jr. 2002. Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148:3007–3017. 3. Beaucher, J., S. Rodrigue, P. E. Jacques, I. Smith, R. Brzezinski, and L. Gaudreau. 2002. Novel Mycobacterium tuberculosis anti-sigma factor antagonists control SigF activity by distinct mechanisms. Mol. Microbiol. 45:1527– 1540. 4. Betts, J. C., P. T. Lukey, L. C. Robb, R. A. McAdam, and K. Duncan. 2002. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 43: 717–731. 5. Camacho, L. R., P. Constant, C. Raynaud, M. A. Laneelle, J. A. Triccas, B. Gicquel, M. Daffe, and C. Guilhot. 2001. Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier. J. Biol. Chem. 276:19845– 19854. 6. Collins, F. M. 1985. Protection to mice afforded by BCG vaccines against an aerogenic challenge by three mycobacteria of decreasing virulence. Tubercle 66:267–276. 7. Converse, S. E., and J. S. Cox. 2005. A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis. J. Bacteriol. 187:1238–1245. 8. Dubey, V. S., T. D. Sirakova, M. H. Cynamon, and P. E. Kolattukudy. 2003. Biochemical function of msl5 (pks8 plus pks17) in Mycobacterium tuberculosis H37Rv: biosynthesis of monomethyl branched unsaturated fatty acids. J. Bacteriol. 185:4620–4625. 9. Geiman, D. E., D. Kaushal, C. Ko, S. Tyagi, Y. C. Manabe, B. G. Schroeder, R. D. Fleischmann, N. E. Morrison, P. J. Converse, P. Chen, and W. R. Bishai. 2004. Attenuation of late-stage disease in mice infected by the Mycobacterium tuberculosis mutant lacking the SigF alternate sigma factor and identification of SigF-dependent genes by microarray analysis. Infect. Immun. 72:1733–1745. 10. Gruber, T. M., and C. A. Gross. 2003. Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol. 57:441– 466. 11. Hahn, M. Y., S. Raman, M. Anaya, and R. N. Husson. 2005. The Mycobacterium tuberculosis extracytoplasmic-function sigma factor SigL regulates polyketide synthases and secreted or membrane proteins and is required for virulence. J. Bacteriol. 187:7062–7071. 12. Hatfull, G. F., and G. J. Sarkis. 1993. DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics. Mol. Microbiol. 7:395–405. 13. Jacques, J.-F., S. Rodrigue, R. Brzezinski, and L. Gaudreau. 2006. A recombinant Mycobacterium tuberculosis in vitro transcription system. FEMS Microbiol. Lett. 255:140–147. 14. Manganelli, R., E. Dubnau, S. Tyagi, F. R. Kramer, and I. Smith. 1999. Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis. Mol. Microbiol. 31:715–724. 15. Manganelli, R., L. Fattorini, D. Tan, E. Iona, G. Orefici, G. Altavilla, P. Cusatelli, and I. Smith. 2004. The extra cytoplasmic function sigma factor SigE is essential for Mycobacterium tuberculosis virulence in mice. Infect. Immun. 72:3038–3041.
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