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Journal of Bacteriology, September 2006, p. 6669-6679, Vol. 188, No. 18
0021-9193/06/$08.00+0     doi:10.1128/JB.00631-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Inactivation of Rv2525c, a Substrate of the Twin Arginine Translocation (Tat) System of Mycobacterium tuberculosis, Increases ß-Lactam Susceptibility and Virulence

Brigitte Saint-Joanis,1 Caroline Demangel,1 Mary Jackson,2 Priscille Brodin,1 Laurent Marsollier,1 Helena Boshoff,3 and Stewart T. Cole1*

Unité de Génétique Moléculaire Bactérienne,1 Unité de Génétique Mycobactérienne, Institut Pasteur, Paris, France,2 Tuberculosis Research Section, NIAID, National Institutes of Health, 12441 Parklawn Drive, Rockville, Maryland 208523

Received 4 May 2006/ Accepted 7 July 2006


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ABSTRACT
 
The twin arginine translocation (Tat) system is used by many bacteria to export fully folded proteins containing cofactors. Here, we show genetically that this system is essential for Mycobacterium tuberculosis, as the tatAC operon and tatB genes could be inactivated only in partially diploid strains. Using comparative genomics, the rv2525c gene of M. tuberculosis was identified as encoding a histidine-rich protein, with a twin arginine signal peptide, and orthologous genes were shown to be present in several but not all actinobacterial species. Conservation of this gene by Mycobacterium leprae, which has undergone reductive evolution, suggested an important role for rv2525c. An rv2525c knockout mutant was constructed, and biochemical analysis indicated that the mature Rv2525c protein is secreted. Upon exposure to antituberculous drugs, rv2525c expression is significantly up-regulated together with those of other genes involved in cell wall biogenesis. Phenotypic comparison of the mutant with the parental strain revealed an increase in susceptibility to some ß-lactam antibiotics and, despite slower growth in vitro, enhanced virulence in both cellular and murine models of tuberculosis. The Tat system thus contributes in multiple ways to survival of the tubercle bacillus.


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INTRODUCTION
 
Tuberculosis is a major public health problem worldwide, and in the last decade, great efforts have been made to develop better preventive and therapeutic measures. In particular, intensive research has been performed to clarify the molecular basis of the physiology and virulence mechanisms of the causative agent of the disease, Mycobacterium tuberculosis. This investigation was made easier thanks to the development of genetic tools for handling this organism (19) and the availability of the complete genome sequence (9). The protein secretion systems present in this bacterium have also been analyzed in some detail since secreted or exported proteins are central to the development of improved diagnostic tests and vaccines and also play a role in pathogenesis (1, 6).

In bacteria, two different types of signal peptide-dependent translocations have been described: the essential Sec-dependent pathway (23) and the more recently discovered Sec-independent pathway (3). This latter export system has been named Tat for twin arginine translocation because the precursor proteins engaged in this mechanism of export contain a conserved motif, S/T-R-R-X-F-L-K, with two contiguous arginine residues near the N terminus of the leader peptide. The Tat pathway, which has been identified in gram-positive as well as gram-negative bacteria and archaea, is related structurally and mechanistically to the {Delta}pH-dependent protein import pathway found in plant thylakoids. Some bacteria, such as Escherichia coli (17), preferentially use the Tat pathway for the export of redox enzymes involved in bacterial energy metabolism. However, it was shown that in certain organisms, the Tat system was responsible for the export of a wide variety of substrates and, in particular, virulence factors (11, 16, 22, 24). A most striking feature of Tat substrates is their adoption of a tertiary structure before crossing the cytoplasmic membrane, and it is the ability to transport folded proteins that distinguishes the Tat export system from the Sec-dependent, general secretory pathway (23). Many enzymes exported by the Tat system are synthesized as apoproteins requiring cofactors, such as iron-sulfur clusters and molybdopterin, and these are inserted in the cytoplasm prior to export (17).

The Tat pathway of E. coli is presently the best studied (3, 17, 27) and includes at least four components: TatA, TatB, and TatE, predicted to be anchored to the cytoplasmic membrane via an N-terminal hydrophobic alpha-helix, and TatC, with six predicted transmembrane helices. The tatE gene is cryptic and has arisen from a duplication of tatA. The tatA, tatB, and tatC genes comprise an operon, with tatD as the final cistron (Fig. 1), and the tatB and tatC genes were demonstrated to be essential pathway components. Interestingly, in some prokaryotes such as Bacillus subtilis, only tatA and tatC are present, suggesting that the minimal translocation system comprises TatA and TatC. The TatD protein, initially thought to be a component of the Tat machinery on the grounds of gene linkage, was recently shown to be a cytoplasmic protein exhibiting DNase activity and is not required for the Tat system to function (30). The genome sequences of M. tuberculosis (9) and Mycobacterium leprae (10) revealed that both mycobacteria contained clearly identifiable tatA, tatB, tatC, and tatD genes and should therefore produce a functional Tat system. However, the tatABCD gene cluster identified in E. coli was not found in M. tuberculosis, which resembles B. subtilis by having a tatAC operon, with tatB and tatD located elsewhere (Fig. 1).


Figure 1
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  		FIG. 1. Organization of Mycobacterium tuberculosis tat genes. The genetic organization of M. tuberculosis tat genes is compared with that of E. coli. The percentage of identity between the corresponding homologous Tat proteins is indicated. In M. tuberculosis, tatA and tatC are separated by an intergenic region of 16 bp.

Bioinformatics has been used to predict potential substrates for the Tat system in the proteome of M. tuberculosis, and in an early analysis using very stringent criteria (8), 11 candidates were recognized with appropriately positioned signal peptides harboring the distinctive twin arginine motif (Table 1), while the less restrictive TATFIND algorithm predicts 31 (11). Four of the 11 Tat substrates are phospholipases C and may serve as virulence factors (24), whereas no functional information is available for 5 others. During the extensive reductive evolution of the genome of M. leprae, only one of the corresponding genes, ml1190, escaped inactivation; it is orthologous to the rv2525c gene of M. tuberculosis. The conservation of this coding sequence by M. leprae, despite genome downsizing and gene decay (10), was a strong indication that rv2525c/ml1190 might play an important biological role. Here, we have performed detailed genetic and biochemical analysis of the rv2525c gene and its product and also demonstrated that three tat genes are apparently essential for the growth of M. tuberculosis.


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  		TABLE 1. Possible twin arginine substrates


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MATERIALS AND METHODS
 
Bacterial strains, growth conditions, and plasmids. Mycobacterium tuberculosis H37Rv was used as the reference strain and for the construction of knockout mutants. Mycobacteria were grown at 32°C, 37°C, or 39°C in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween 80 and albumin-dextrose-catalase (ADC) in Sauton medium (preparation of samples for protein chip array experiments) or on solid Middlebrook 7H11 medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase. When required, the media were supplemented with 2% sucrose or the following concentrations of antibiotics: 20 µg/ml of kanamycin, 200 µg/ml of hygromycin for Escherichia coli or 50 µg/ml for mycobacteria, and 25 µg/ml gentamicin. Plasmid pPR27 is a thermosensitive vector, carrying the sacB counterselectable marker (19); pYUB412 is an integrating cosmid vector (2), and pYUB412-IE214 and pYUB412-IE254 contain the tatAC (kb 2330 to 2373) and tatB (kb 1356 to 1396) regions, respectively, from the genome of the Erdman strain.

Cloning procedures. E. coli XL2-Blue electrocompetent cells from Stratagene were used for cloning. M. tuberculosis electrocompetent cells were generated from 400 ml of exponential-phase cultures; bacilli were harvested by centrifugation at 3,000 x g for 20 min at 15°C, washed with H2O, and resuspended in 2 ml of 10% glycerol after recentrifugation. Bacilli (250 µl) and approximately 5 µg of purified plasmids were mixed and electroporated with a Bio-Rad Gene Pulser (2.5 kV, 25 µF, 1,000 {Omega}). After electroporation, bacilli were resuspended in 2 ml of culture medium and left overnight at the appropriate temperature. Transformants were selected on Middlebrook 7H11 agar supplemented with the appropriate antibiotics.

Construction of recombinant plasmids. PCR fragments bearing the genes of interest and ~300 bp of flanking regions were synthesized and introduced in the pGEM-T Easy vector (Promega). See Table 2 for primer details. Then, a unique restriction site (HindIII or BglII) was created inside the genes using the QuikChange site-directed mutagenesis kit from Stratagene and the appropriate primer pairs (Table 2). Disrupted alleles rv2525c::Km, tatAC::Km, tatB::Km, and tatD::Km were then constructed by cloning the kanamycin resistance cassette from pUC4K (Amersham Biosciences), carried on a PstI-cut, blunt-ended restriction fragment, into the unique, blunt-ended HindIII or BglII sites. The resulting plasmids were then digested by EcoRI; the restriction fragments containing the disrupted alleles rv2525c::Km, tatA,C::Km, tatB::Km, and tatD::Km were blunt ended and inserted into SmaI-cut pXYL4 (19), yielding the plasmids prv2525c::Km-XYL, ptatA::Km-XYL, ptatB::Km-XYL, and ptatD::Km-XYL. Finally, BamHI fragments carrying the disrupted alleles and a copy of xylE were extracted from the plasmids prv2525c::Km-XYL, ptatA::Km-XYL, ptatB::Km-XYL, and ptatD::Km-XYL and inserted into the BamHI site of the vector pPR27. This last step yielded the plasmids pPR27-rv2525c, pPR27-tatAC, pPR27-tatB, and pPR27-tatD, the constructs used for allelic replacement. For complementation purposes, rv2525c was cloned in the integrative vector pRBexint-rv2525c, where it was expressed from the dnaK promoter. All constructs were checked by DNA sequencing.


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  		TABLE 2. Primers used in this study

Construction of the knockout mutants, merodiploids, and complemented strains. Knockout mutants were constructed by allelic exchange using the Ts/sacB method described by Pelicic et al. (19). The essentiality of the tatAC and tatB genes was investigated using the same method, with M. tuberculosis H37Rv merodiploid strains as the starting material. The integrating cosmid pYUB412-IE57 (containing the region from kb 2833 to 2870 of the genome of the strain Erdman) was transformed in the rv2525c knockout mutant to yield the complemented strain carrying both the wild and inactivated rv2525c alleles. Plasmids pYUB412-IE214 and pYUB412-IE254 were used to obtain tatAC and tatB merodiploid strains, respectively.

DNA extraction and Southern analysis. Mycobacterial genomic DNA was extracted using standard protocols (19). To confirm successful allelic exchange in M. tuberculosis H37Rv or in merodiploid strains, genomic DNA was cleaved with the appropriate restriction enzymes, subjected to 1% agarose gel electrophoresis before capillary blotting to a Hybond-C nitrocellulose membrane (Amersham) and hybridization with {alpha}-32P-labeled probes. The different probes used were the BamHI fragments carrying the disrupted alleles previously cloned in pPR27 vector. Labeling of DNA probes was performed with the Prime-It II kit (Stratagene).

Transcriptomics, reverse transcription (RT)-PCR, and microarray hybridizations. M. tuberculosis was grown to an optical density at 600 nm of 0.5, and cells were harvested by centrifugation. The pellet was resuspended in 1 ml of GTC (5 M guanidium thiocyanate, 0.5% N-lauryl sarcosine, 0.5% Tween 80, 0.1 M ß-mercaptoethanol) for every 50 mg (wet weight) of bacteria. These bacteria were then broken in 500 µl of TRIzol with mini glass beads using a TissueLyser apparatus (QIAGEN) at maximum speed. RNA was extracted with 500 µl of chloroform-isoamyl alcohol. The aqueous phase was precipitated overnight at 4°C by adding 500 µl isopropanol and 50 µl 3 M sodium acetate, pH 6.0. After precipitation, the pellet was rinsed with 500 µl of a 70% ethanol solution before resuspension in diethyl pyrocarbonate-treated water. This solution was digested by DNase I (Ambion) for 2 h at 37°C and finally purified using a QIAGEN kit.

RT-PCR experiments were carried out according to the manufacturer's instructions (Superscript one-step RT-PCR kit from Invitrogen). When the cotranscription of tatA and tatC was tested, 1 µg of target RNA from H37Rv and specific primers (Table 2) flanking either the tatA gene (primers 17 and 18) or the tatA and tatC genes (primers 19 and 20) were used in the RT-PCRs. To verify the inactivation of transcription of rv2525c in the knockout mutant, 1 µg of target RNA from either H37Rv or the rv2525c knockout mutant and specific primers (Table 2) flanking the gene rv2525c (primers 21 and 22) were used in the RT-PCRs. Hybridization experiments involving microarrays and mRNA isolated from M. tuberculosis in the presence or absence of various drugs and inhibitors were done exactly as described previously (5).

Protein chip analysis. M. tuberculosis H37Rv and the rv2525c knockout mutant were grown in Sauton medium (Difco) until late log phase and then centrifuged at 3,000 rpm; supernatants were collected and concentrated using a Millipore filter with a 3-kDa cutoff (Bedford, Massachusetts). The surface-enhanced laser desorption/ionization (SELDI) analysis was performed using 10 µg of culture supernatant proteins of each strain and a CM10 ProteinChip array (Ciphergen Biosystems). Protein samples were detected using a PBSII reader (Ciphergen Biosystems Inc.). Before the supernatants were loaded, the CM10 arrays were equilibrated first with buffer 1 (250 µl 50 mM Na acetate, pH 4.0) for 5 min and then with buffer 2 (250 µl 50 mM Na acetate, pH 4.0, 0.1% Triton X-100) for 5 min. After the equilibration buffer was removed, 10 µg of each protein sample was diluted in 80 µl of buffer 2 per spot; samples were incubated for 45 min on a rocker at room temperature. Samples were removed, and the surface was washed once with buffer 2 for 5 min and twice with buffer 1 for 5 min. A quick final rinse with water was performed. The surface was allowed to dry, followed by two additions of 0.8 µl saturated sinapinic acid (Ciphergen Biosystems) in 50% acetonitrile and 0.5% trifluoroacetic acid.

Immunoblotting. Immunoblotting was carried out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer of proteins to a nitrocellulose membrane (Hybond-C Extra; Amersham), followed by incubation with an anti-Rv2525c polyclonal antibody (diluted 1 in 1,000). Detection of bound immunoglobulin was achieved with an enhanced chemiluminescence peroxidase system (Amersham). The anti-Rv2525c antibody was raised in rabbits following immunization with two doses of 50 µg of nickel affinity column-purified His-tagged protein produced from recombinant E. coli.

MIC determinations. MICs for antituberculosis drugs and ß-lactam antibiotics were determined using the Bactec TB system (Becton Dickinson Diagnostic Instrumentation Systems, Sparks, Md.). Briefly, the MIC was determined in 7H12B (Becton Dickinson) containing twofold dilutions of antibiotic from 64 to 0.125 µg/ml. The MIC was defined as the lowest concentration that inhibited more than 99% of the bacteria. MICs were also determined by the resazurin test (18).

Briefly, twofold serial dilutions of each drug were prepared in 100 µl of medium directly in 96-well plates, and after 6 days of incubation at 37°C, 30 µl of resazurin solution was added and incubation continued overnight before color development was assessed. A change from blue to pink indicates reduction of resazurin and therefore bacterial growth. The MIC was defined as the lowest drug concentration that prevented this color change.

Whole-cell radiolabeling experiments. Radiolabeling of whole M. tuberculosis cells with [1,2-14C]acetic acid (specific activity, 113 Ci/mol; MP Biomedicals Inc.) and [1-14C]propionate (specific activity, 56.7 Ci/mol; MP Biomedicals Inc.) was performed in 7H9 broth supplemented with ADC and 0.05% Tween 80. Radiolabeled precursors (0.5 µCi/ml) were added to mid-log cultures, and cultures were incubated at 37°C for a further 18 h with shaking.

Preparation and analysis of lipids and fatty acids. Fatty acid and mycolic acid methyl esters were prepared from extractable lipids and from delipidated cells as described previously (20). Total lipids from cold or radiolabeled bacterial cells were extracted with CHCl3/CH3OH (1:2) for one night, followed by two overnight extractions with CHCl3/CH3OH (2:1). Culture filtrates were also collected and filtered through 0.2-µm-pore-size sterile filters to yield sterile extracellular materials. Exocellular lipids were extracted by adding 2 volumes of CH3OH and 1 volume of CHCl3 to 0.8 volumes of extracellular materials to yield a homogeneous single-phase mixture. The mixture was incubated at room temperature for 2 h and then partitioned into two phases by adding 1 volume of H2O/CHCl3 (1:1, vol/vol). The organic phase was recovered, washed with 0.9% NaCl, and dried to yield the secreted lipid extracts.

Lipids and fatty acid methyl esters were analyzed by thin-layer chromatography (TLC) on Silica Gel 60 F254 precoated plates (E. Merck, Darmstadt, Germany) in solvent systems of various polarities as described previously (26). Radiolabeled lipids and fatty acids were visualized by exposure of TLC to Kodak BioMax MR films at –70°C. Nonradiolabeled short-chain fatty acid methyl esters (up to C26) derived from wild-type and mutant strains were analyzed by gas chromatography on a Shimadzu GC-14A chromatograph using a methyl-5% phenyl silicone column operating at a temperature of 175°C for 2 min, followed by a programmed increase of 8°C per minute to 300°C.

Infection of bone marrow macrophages. Bone marrow macrophages were obtained from the femurs of 6-week-old C57BL/6 mice. Cells were seeded in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and 10% L929 cell-conditioned medium at a density of 105 cells/ml. Culture medium was replaced after 4 and 7 days, and infection was performed on differentiated day 7 macrophages. Activated macrophages were obtained by incubating the cells with 100 U/ml gamma interferon and 10 ng/ml lipopolysaccharide 6 h prior to infection. Titrated mycobacterial suspensions were thawed 16 h before infection and briefly sonicated before being added to the cells at a ratio of 0.25:1. After overnight incubation, cells were washed and culture medium was replaced every 3 days. At days 4 and 7 postinfection, cells were washed with phosphate-buffered saline and lysed with cell culture lysis buffer (Promega). Serial dilutions of day 1, 4, and 7 cell lysates were plated on 7H11-oleic acid-albumin-dextrose-catalase agar to determine the number of CFU.

Survival and virulence studies. For the virulence assays, 50-ml cultures of the individual mycobacterial strains were grown in parallel in Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80; when required, antibiotics were included (20 µg/ml kanamycin or 50 µg/ml hygromycin). Bacteria were harvested, washed, and resuspended in 50 mM sodium phosphate buffer (pH 7.0). Bacterial suspensions, obtained by brief sonication, were then aliquoted and frozen at –80°C. A single defrosted aliquot was used to quantify the CFU before inoculation. Six-week-old female BALB/c mice were infected via the aerosol route, and 6-week-old male severe combined immunodeficiency (SCID) mice (Charles River) were infected intravenously via the lateral tail vein (106 CFU). Aerosol infection was performed with a suspension containing 5 x 106 bacteria/ml to obtain an estimated inhaled dose of approximately 1,000 CFU/lung. Organs from sacrificed mice were homogenized using a TissueLyser apparatus from QIAGEN and 2.5-mm-diameter glass beads. Serial fivefold dilutions in phosphate-buffered saline were plated on 7H11 agar, and the number of CFU was ascertained after 2 to 3 weeks of growth at 37°C. Time-to-death analysis was performed on 10 SCID mice. Mice were euthanized when they became moribund. The Institut Pasteur Safety Committee, in accordance with French and European guidelines, approved all animal work.

Statistical methods. Standard deviations and analysis of variance (ANOVA) of the means were calculated using StatView software.


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RESULTS
 
Essentiality of tatA, tatB, tatC, and tatD genes in M. tuberculosis. The organization of the tatA and tatC genes in a cluster suggested that these two genes were cotranscribed and formed an operon (Fig. 1). This was confirmed by RT-PCR experiments, using either primers flanking the tatA gene or primers flanking the tatA and tatC genes (data not shown).

To assess the potential essentiality of these two genes in M. tuberculosis, we first attempted to disrupt the tatAC cluster in M. tuberculosis strain H37Rv. The strategy adopted to achieve allelic replacement in M. tuberculosis used the replicative, thermosensitive shuttle vector, pPR27, carrying the sacB counterselectable marker (19). A disrupted allele of the tatA gene, tatA::Km, was constructed by inserting a kanamycin resistance gene into the coding sequence of tatA, cloned into pPR27 with xylE as a reporter gene. The resulting plasmid, pPR27-tatAC, was used in allelic exchange experiments. At the end of the procedure, putative double-crossover recombinants were expected to carry the disrupted allele tatA::Km and to have lost the sacB and xylE genes from the vector pPR27 and, therefore, to present the Sucr Kanr XylE– phenotype. However, PCR analysis of 96 clones with the desired phenotype showed that none of them exhibited the expected pattern for an allelic exchange event. As we failed to disrupt the tatAC cluster, we repeated the allelic exchange experiment with an M. tuberculosis merodiploid strain obtained by integrating functional copies of tatA and tatC into the chromosome using the clone pYUB412-IE214 (see Materials and Methods). Allelic exchange experiments were carried out as described previously; analysis by PCR (Fig. 2A) and Southern blotting of the resultant Sucr Kanr XylE– clones showed that the allelic exchange had occurred in 10% of the recombinants, as exemplified by clone 3 (Fig. 2B). These results strongly suggest that tatA and tatC are essential to M. tuberculosis.


Figure 2
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  		FIG. 2. Allelic replacement at the tatAC and tatB loci. (A) PCR analysis using genomic DNAs from the strain H37Rv (lanes Rv), from the diploid strain H37Rv/pYUB412::IE214 (lane Di), and from the mutant Hygr Kanr Sucr strain (lane 3) as targets and primers 17 and 18 (Table 1) in all reactions. Lane (–) corresponds to the negative control without target DNA. (B) Southern blot analysis of genomic DNAs from H37Rv (lane Rv), from the diploid strain H37Rv/pYUB412::IE214 (lane Di), and from the mutant Hygr Kanr Sucr strain (lane 3) digested by SmaI and probed with the 4-kb BamHI fragment extracted from pPR27-tatA,C carrying the disrupted tatA gene. (C) PCR analysis using genomic DNAs from the diploid strain H37Rv/pYUB412::IE254 (lane Di) and from the mutant Hygr Kanr Sucr strains (lanes 1 to 3) as targets and primers 23 and 24 (Table 2) in all reactions. Lane (–) corresponds to the negative control without target DNA. (D) Southern blot analysis of genomic DNAs from the diploid strain H37Rv/pYUB412::IE254 (lane Di) and from the mutant Hygr Kanr Sucr strains (lanes 2 and 3) digested by SmaI and probed with the 4-kb BamHI fragment extracted from pPR27-tatB carrying the disrupted tatB gene. Mw, molecular weight.

Plasmids pPR27-tatB and pPR27-tatD were constructed in the same way as pPR27-tatAC. Attempts to disrupt the tatB gene in the strain H37Rv, using the same strategy as that described above, failed but were successful when performed with an H37Rv::pYUB412-IE254 merodiploid strain. Double-crossover recombinants were analyzed by PCR (Fig. 2C) and Southern blotting (Fig. 2D). Again, these results suggest that tatB is essential to M. tuberculosis. By contrast, the attempt to disrupt the tatD gene by allelic replacement in the strain H37Rv was successful at the first attempt, as shown by Southern blotting analysis (data not shown). The ability to produce a tatD knockout mutant of H37Rv indicates that tatD is nonessential, as found with E. coli. In conclusion, tatA, tatB, and most likely tatC are essential genes of M. tuberculosis while tatD is not.

Rationale for studying Rv2525c of M. tuberculosis. As outlined in Table 1, of the 11 substrates harboring the distinctive twin arginine motif, only 1 protein, Rv2525c, has been conserved in M. leprae, where its counterpart is ML1190. Furthermore, on database searching, functional orthologs of rv2525c were found in all members of the M. tuberculosis complex, all other mycobacteria whose genome sequences are available, except Mycobacterium smegmatis, and Nocardia farcinica (12) but not in Streptomyces spp. or other eubacteria. In these organisms, rv2525c is linked to fas, encoding fatty acid synthase I, and 55% of the amino acid residues in the mature protein are identical. Some mycobacteria have a second paralogous copy, in addition, that also encodes a twin arginine protein of ~275 to 300 amino acid residues which is highly related to the 240-amino-acid-residue Rv2525c protein. The paralog, but not the Rv2525c ortholog, also occurs in Corynebacterium spp. Multiple alignments performed with CLUSTAL highlight a single conserved cysteine and five conserved histidine residues that are likely to coordinate a cofactor such as zinc (Fig. 3).


Figure 3
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  		FIG. 3. CLUSTAL alignment of rv2525c orthologs. Conserved residues discussed in the text and the likely signal peptidase cleavage site are indicated by arrowheads. Abbreviations: Mm3975c, Mycobacterium marinum; MU4189c, Mycobacterium ulcerans; Rv2525c; M. tuberculosis; ML1190, M. leprae; MAP2334c, Mycobacterium avium subsp. paratuberculosis; nfa47650, Nocardia farcinica. Stars denote identical residues; periods and colons indicate functional similarity.

The conservation of the Rv2525c coding sequence in mycobacteria, and notably in M. leprae, suggests that the corresponding protein may play an important physiological role or even intervene in pathogenesis like the four Tat-dependent phospholipases C, characterized as virulence factors in M. tuberculosis (24). To explore its function, we first constructed an rv2525c knockout mutant in order to compare its phenotype with that of the parental strain H37Rv.

Inactivation of Rv2525c in M. tuberculosis H37Rv. A disrupted allele, rv2525c::Km, was constructed by inserting a kanamycin resistance gene into the coding sequence and used in allelic exchange experiments with the strategy described above. At the end of this process, we selected recombinants displaying the Sucr Kanr XylE– phenotype and characterized them by PCR and Southern blotting. A typical rv2525c knockout mutant is shown in Fig. 4 and was then employed in further experiments together with H37Rv and a complemented mutant, carrying both the wild and inactivated rv2525c alleles (see Materials and Methods). The doubling time of the mutant in 7H9 broth was 24 h compared to 18 h for both H37Rv and the complemented mutant (data not shown). In addition, Tween 80 at 0.05% appeared to inhibit growth of the mutant.


Figure 4
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  		FIG. 4. Analysis of the rv2525c knockout mutant. (A) Southern blot analysis of genomic DNA from pPR27-rv2525c transformants of M. tuberculosis Kanr Sucr strains (lanes 1 to 4) and from H37Rv (lane Rv). DNA was digested by SmaI and hybridized with the radiolabeled plasmid pPR27-rv2525c. Lane 4 characterizes the knockout mutant. (B) SmaI restriction profile of the disrupted rv2525c locus.

Differential gene expression. RT-PCR was used to compare gene expressions between the mutant and the wild-type strain. No PCR product was generated from rv2525c when RNA extracted from the knockout mutant was reverse transcribed, whereas expression of the tatA gene, used for control purposes, was readily demonstrated (data not shown). Since we expected the Rv2525c protein to be secreted, via the Tat system, we compared the secretome protein profiles of the H37Rv strain and the rv2525c knockout mutant by protein chip array analysis based on SELDI. We found that a protein of 21.3 kDa was present in large quantities in the late-log-phase culture supernatant of the wild-type strain but absent from the culture supernatant of the rv2525c knockout mutant (Fig. 5A). This molecular mass is in good agreement with the size of the most probable mature form of Rv2525c, whose precursor has a predicted molecular mass of 25,369 Da; a putative site for maturation of Rv2525c by signal peptidase is before the motif Gly36-Ser37-Lys38, and cleavage there would generate a mature protein of about 21 kDa. The absence of the mature Rv2525c protein from the extracellular milieu of the rv2525c knockout mutant indicates that this Tat substrate is indeed secreted, and this result was confirmed by immunoblotting using an anti-Rv2525c polyclonal antibody since immunoreactivity was seen only with M. tuberculosis H37Rv (Fig. 5B).


Figure 5
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  		FIG. 5. Evidence for loss of Rv2525c from mutant. (A) SELDI analysis of proteins from bacterial supernatants onto CM10 ProteinChip array. Concentrated supernatants (10 µg) from late-log-phase cultures of M. tuberculosis, H37Rv (top panel) and the H37Rv rv2525c knockout mutant (bottom panel), were analyzed on CM10 arrays at pH 4. (B) Immunoblotting, using an anti-Rv2525c polyclonal antibody, of 10 µg of supernatants from late-log-phase cultures of M. tuberculosis H37Rv (H37Rv), the H37Rv rv2525c knockout mutant (KO), and the complemented strain (KO+) and of 1 µg of purified recombinant Rv2525c protein (Rv2525).

Differential transcription of rv2525c in response to isoniazid or ethionamide. Examination of transcriptional responses of M. tuberculosis to inhibitors of metabolism and specific drugs led to the identification of coordinate regulation of gene clusters (5). These correlations not only confirmed and extended the available information about the mode of action of drugs but also provided insight into the potential mechanism of action of new antituberculosis agents, thereby helping the validation of new drug targets.

On exposure of M. tuberculosis H37Rv to isoniazid (0.2 µg/ml) or ethionamide (12 µg/ml), it was found that the transcriptional rate of rv2525c increased about twofold, as previously reported (5), and this up-regulation was also observed in response to the drug PA-824 (0.4 µg/ml). Isoniazid and ethionamide inhibit enzymes of the fatty acid synthase II pathway that elongates fatty acids to mycolic acids, the major components of the cell wall, and the signature profile diagnosed by the transcriptional analysis is consistent with this mechanism. The drug PA-824 was also reported to affect mycolic acid synthesis by impeding the conversion of hydroxymycolates to ketomycolates through an indirect effect on redox potential. Expression of rv2525c is coregulated with those of a cluster of other genes encoding proteins that are secreted, found in the cell envelope or required for maintaining cell wall integrity (Table 3). Transcriptomics thus suggests a role for rv2525c in cell wall synthesis.


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  		TABLE 3. Cluster of genes coregulated with rv2525c

Lipids and fatty acids. To investigate the consequences of rv2525c gene disruption on the biosynthesis of lipids and fatty acids, the wild-type, mutant, and complemented mutant strains were radiolabeled with [1-14C]propionate or with [1,2-14C]acetic acid, which label methyl-branched fatty acids and all fatty acids, respectively. TLC and gas chromatography analyses of labeled lipids revealed no qualitative or quantitative differences in the cell envelope composition or in the secreted products of the three strains, and the same types and amounts of mycolates esterifying arabinogalactan and outer membrane lipids were recovered from all three strains (data not shown).

Envelope permeability. Although no detectable difference in the lipid profiles was observed, it remained possible that loss of the Rv2525c protein would result in an alteration of cell envelope permeability. This possibility was tested by measuring the susceptibility of the mutant and wild-type strain to a range of antituberculous drugs and to ß-lactam antibiotics. As can be seen in Table 4, compared to the wild type, the mutant displayed an increase in susceptibility to amoxicillin and ampicillin, especially in the presence of the ß-lactamase inhibitors, clavulanate and sulbactam, respectively. Susceptibility of M. tuberculosis to these combinations was reported previously (7), but loss of Rv2525c function greatly exacerbates the effect. The MICs for cephalosporins decreased slightly, while those for cycloserine and all of the other tuberculosis drugs tested (data not shown) were essentially unchanged, suggesting that there is a specific defect rather than a general increase in permeability of the cell envelope.


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  		TABLE 4. MICs for peptidoglycan inhibitors

Growth of the Rv2525c mutant in bone marrow macrophages. Since several proteins secreted by the Tat system contribute to pathogenesis (16), we investigated the effect of inactivation of rv2525c on mycobacterial virulence by comparing its growth rate with those of the complemented mutant and the wild-type strain H37Rv in resting and activated murine macrophages. In both cases, the mutant grew significantly faster than the control strains (Fig. 6A and B), suggesting that loss of Rv2525c function might lead to an increase in virulence.


Figure 6
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  		FIG. 6. Growth of strains in bone marrow macrophages. (A) Growth of M. tuberculosis H37Rv (•), the H37Rv rv2525c knockout mutant KO ({blacksquare}), and the complemented rv2525c knockout mutant strain KO+ ({diamondsuit}) in C57BL/6 murine bone marrow-derived resting macrophages. (B) Growth of M. tuberculosis H37Rv (•), the H37Rv rv2525c knockout mutant KO ({blacksquare}), and the complemented rv2525c knockout mutant strain KO+ ({diamondsuit}) in C57BL/6 murine bone marrow-derived activated macrophages. Data are the mean and standard deviation of triplicate measurements and are representative of two independent experiments. Differences in CFU means with H37Rv- and KO-infected cells were analyzed by ANOVA (NS, not significant; **, P > 0.01).

Virulence studies of the Rv2525c mutant in the mouse model of infection. First, the virulence of the wild type, the knockout mutant, and the complemented strains were compared by infection of immunocompetent BALB/c mice with 103 CFU via the aerosol route and were assessed by measuring growth rates in the lungs and spleen of infected animals (Fig. 7A). In two independent experiments, no increase in the growth kinetics of the knockout strain was detected at 36 days postinfection. Then, we compared the virulence of the three strains by intravenous infection (106 CFU) in SCID mice and again measured growth rates in the lungs and the spleen of infected animals (Fig. 7B). No significant difference in the growth kinetics was detectable.


Figure 7
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  		FIG. 7. Results of virulence studies of mice. (A) Growth of M. tuberculosis H37Rv (black bars), the H37Rv rv2525c knockout mutant (gray bars), and the complemented rv2525c knockout mutant strain (white bars) in BALB/c mouse organs following infection via the aerosol route. (B) Growth of M. tuberculosis H37Rv (black bars), the H37Rv rv2525c knockout mutant (gray bars), and the complemented rv2525c knockout mutant strain (white bars) in SCID mouse organs following intravenous infection with 106 CFU. For each time point of experiments A and B, data are the mean ± standard deviation from three to four mice per group. (C) Percent survival of cohorts of 10 SCID mice infected with 106 CFU of either M. tuberculosis H37Rv (solid line) or the H37Rv rv2525c knockout mutant (broken line). Data are the mean and standard deviation of CFU measured from four animals per group. Differences in CFU means with the H37Rv- and KO-infected groups were analyzed by ANOVA (*, P > 0.05; **, P > 0.01).

However, mice infected by the knockout mutant were clearly in much poorer condition than animals infected with the wild-type strain of M. tuberculosis, and many were moribund at 21 days postinfection. On account of this observation, we also determined the mortality within cohorts of 10 SCID mice intravenously infected (106 CFU) with either the wild-type or mutant strain (Fig. 7C). In this time-to-death experiment, the knockout mutant proved to be significantly more virulent than the wild-type strain, as all mice in this group died 5 days earlier than those infected with M. tuberculosis H37Rv.


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DISCUSSION
 
Here, we provide evidence for the essentiality of the tatAC and tatB translocon genes in M. tuberculosis and thus demonstrate that the Tat secretion system of M. tuberculosis is important for the growth and survival of the bacterium. While this work was in progress, McDonough et al. reached similar conclusions about the Tat system in the close relative Mycobacterium smegmatis and demonstrated that while tatAC could be deleted, the mutants displayed multiple growth defects, forming small colonies only after lengthy incubation (14). Very recently, this observation was replicated by other investigators (21), but to our knowledge, there are no studies to date of inactivation of tat genes in M. tuberculosis other than those of saturation mutagenesis with the Himar1 transposon, performed by Sassetti et al. (28), that largely agree with the findings presented here. The one discrepancy concerns tatB, which we were unable to inactivate by allelic exchange, whereas a transposon insertion in this gene has been reported but not precisely located. If the Himar1 element had inserted at a distal site, it is conceivable that the truncated tatB may have retained biological activity. Our construction of a tatD knockout mutant with no distinct phenotypic difference compared to the wild-type strain indicates that TatD is not an essential component of the Tat system of M. tuberculosis. This is in good agreement with the finding that the TatD protein of E. coli is endowed with DNase activity (30) and not required for the secretion of Tat substrates. It has not yet been demonstrated whether tatD is dispensable in M. smegmatis.

The tat genes are present in most bacteria whose genomes have been completely sequenced (11). The tatC gene seems to play a critical role in the Tat secretion pathway of several bacteria, including E. coli (4), Pseudomonas aeruginosa (16), Streptomyces lividans (29), and Bacillus subtilis (13). Moreover, TatC displays a high degree of sequence conservation among sequenced organisms. Although the minimal Tat system requires one copy of tatA and one copy of tatC, the different Tat apparatuses characterized so far exhibit a large diversity of subunit combinations. On the basis of our genetic data, the Tat machinery of M. tuberculosis comprises a minimal set of proteins, TatA, TatB, and TatC, but each of them appears to be essential.

In E. coli, the Tat system is required primarily for the export of a series of redox enzymes that adopt their tertiary structure in the cytoplasm following the incorporation of their cofactors (17), whereas in P. aeruginosa, its substrates also include virulence factors, such as phospholipases (16). On inspection of the likely Tat substrates of M. tuberculosis (Table 1), four phospholipases C were found and these zinc-containing enzymes have been shown to intervene in mycobacterial virulence (24) and probably contribute to the lipolytic lifestyle of the tubercle bacillus by liberating phospholipids from host membranes for use as sources of carbon and energy. While a few of the other likely Tat substrates of M. tuberculosis may be involved in scavenging metal ions (Table 1), no functional information could be deduced for the others by bioinformatics. Among this group is Rv2525c, which is well conserved in all mycobacteria except M. smegmatis, where the gene appears to have several null mutations, and the orthologous gene also occurs in Nocardia (Fig. 3). Genes paralogous to rv2525c were detected in Corynebacterium spp., but no orthologs were found. These paralogs encode likely substrates for the Tat system and are also present in mycobacteria with larger genomes, such as M. smegmatis, M. paratuberculosis, M. marinum, and M. ulcerans.

Of particular importance is the fact that the Rv2525c ortholog has been conserved in M. leprae in the face of massive gene loss and decay, suggesting that the protein must play an important biological role. From its primary structure, with clusters of conserved histidine residues, one could infer a role as a possible metalloprotein or heme-containing enzyme, as histidine residues are frequently involved in the coordination of these cofactors. This would explain the necessity for secretion via the Tat system.

Genetic, molecular, and biochemical analyses showed the Rv2525c protein to be secreted, as expected, and also shed some light on its possible activity. Transcriptomics revealed that transcription of rv2525c is up-regulated by drugs that perturb cell envelope biogenesis and that the gene belongs to an expression cluster which includes several genes encoding secreted proteins or enzymes involved in various aspects of cell wall metabolism, such as mycolic acid and peptidoglycan synthesis. These observations suggest that Rv2525c might also be involved in biogenesis of the cell envelope even though it is not an essential protein, as predicted by the results of saturation transposon mutagenesis (28). The cell envelope of the mutant appears to be more permeable to ß-lactam antibiotics, and possibly to cephalosporins, but not to other drugs, suggesting that Rv2525c might intervene in a specific step in peptidoglycan synthesis (Table 4). Biochemical characterization of the protein and structure determination are now required.

Contradictory rates of growth were seen in vitro, where the rv2525c mutant grew more slowly than the wild-type strain, and in vivo, where it multiplied more vigorously, being significantly more virulent than the parent in both resting and activated macrophages, suggesting that Rv2525c inactivation somehow confers an enhanced capacity to proliferate in vivo. In accordance with this finding, the pathogenicity of the knockout mutant was more pronounced than that of the wild-type strain in SCID mice. Although differential growth between the two strains in lungs and spleen was minimal, the increased virulence of the mutant was particularly apparent in this immunodeficient mouse model as the onset of mortality occurred 5 days earlier than with wild-type M. tuberculosis (Fig. 7C). In careful studies, North and colleagues have previously described that growth kinetics in lungs and spleen do not always correlate with mycobacterial pathogenesis (15).

It is conceivable that loss of Rv2525c function results in an alteration of the cell envelope or even the release of components such as muramyl peptides, lipoglycans, and glycolipids that have an immunomodulatory effect, as has been described for the phenolic glycolipid produced by certain tubercle bacilli (25). This would result in dampening of the innate immune response, which is consistent with the results presented here. However, no evidence of enhanced pathogenicity was observed in immunocompetent mice with the mutant strain, compared to wild-type M. tuberculosis, weakening this possibility. Further research into the physiological role of Rv2525c is under way.


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ACKNOWLEDGMENTS
 
We thank Jacques D'Alayer for help with SELDI-TOF and Roland Brosch for providing cosmid clones.

This work received the financial support of the Institut Pasteur (GPH-5), the European Community (QLRT-2001-02018 and LHSP-CT-2005-018923), the Institut National de la Santé et de la Recherche Médicale, and the Association Française Raoul Follereau.


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FOOTNOTES
 
* Corresponding author. Mailing address: Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex, France. Phone: 33-1-45688446. Fax: 33-1-40613583. E-mail: stcole{at}pasteur. Back


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Journal of Bacteriology, September 2006, p. 6669-6679, Vol. 188, No. 18
0021-9193/06/$08.00+0     doi:10.1128/JB.00631-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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