Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M700035200 on April 11, 2007

J. Biol. Chem., Vol. 282, Issue 25, 18348-18356, June 22, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/25/18348    most recent
M700035200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lun, S.
Right arrow Articles by Bishai, W. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lun, S.
Right arrow Articles by Bishai, W. R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Characterization of a Novel Cell Wall-anchored Protein with Carboxylesterase Activity Required for Virulence in Mycobacterium tuberculosis*Formula

Shichun Lun1 and William R. Bishai2

From the Department of Medicine, Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

Received for publication, January 2, 2007 , and in revised form, March 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pooled mutant competition assays have shown that the Mycobacterium tuberculosis MT2282 gene (Rv2224c, annotated as encoding a proteinase) is required for bacterial survival in mice. To understand the mechanism of this requirement, we conducted a genetic and biochemical study of the MT2282 gene and its product. MT2282 encodes a member of the microbial esterase/lipase family with active site consensus sequences of G-X-S-X-G, and we have concluded that the MT2282 protein is, in fact, a cell wall-associated carboxylesterase rather than a proteinase, as initially annotated. The MT2282 gene product preferentially hydrolyzes ester bonds of substrates with intermediate carbon chain length. Purified MT2282 is a monomer with enzymatic catalysis properties that fit in the Michaelis-Menten kinetic model. Esterase activity was inhibited by paraoxon and dichlorvos. Replacement of Ser215, Asp450, and His477 by Ala in the consensus motifs completely abolishes esterase activity, suggesting that Ser215-Asp450-His477 forms a catalytic triad with Ser215 as an active site residue. To evaluate the role of the MT2282 in pathogenesis, the gene was deleted from the M. tuberculosis genome. BALB/c mouse aerosol infections showed reduced colony-forming unit loads in lungs and spleens and less lung pathology for the {Delta}MT2282 mutant. High dose intravenous infection of mice with the mutant resulted in a significantly delayed time to death compared with the wild type or complemented mutant. These results indicate that MT2282 encodes a cell wall-associated carboxylesterase, which is required for full virulence of M. tuberculosis. We propose that MT2282 (Rv2224c) and its adjacent paralogous gene MT2281 (Rv2223c) be named caeA and caeB respectively, for carboxylesterase A and B.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tuberculosis, one of the leading human infectious diseases claiming about 1.7 million deaths annually, is difficult to control because of human immunodeficiency virus/tuberculosis co-infection and emergence of virtually untreatable extensively drug-resistant Mycobacterium tuberculosis strains. Clearly, new drugs and effective vaccines are urgently needed, which creates a strategic demand for better understanding of the genetic basis for the virulence and pathogenesis of M. tuberculosis.

The M. tuberculosis genome sequence and annotation has provided an invaluable blueprint of the genomic organization and gene functionality for this slowly growing pathogen (1). However, annotation is unable to predict gene function with complete accuracy due to divergent evolution of protein motifs. Recent advances in genetic approaches such as transposon mutagenesis and DNA microarray techniques has facilitated initial functional screening of essential genes for in vitro and in vivo growth in a high throughput manner (2-6). Mycobacterial genes that are involved in lipid metabolism, carbohydrate transport and metabolism, cell division chromosomal partitioning, and secretion are more likely to be required for survival in the mouse (5, 6). For example, mutation of six of the mycobacterial membrane protein mmpL family genes severely compromises the ability of the respective mutants to multiply in mouse lungs (6).

M. tuberculosis escapes host defense mechanisms by inhibiting and evading the host immune system (7, 8), by arresting phagolysosome maturation (9, 10), and by establishing a state of persistence (11, 12). However, the underlying genetic expression and molecular mechanisms are incompletely understood. Virulence genes refer to those genes that are required for disease in the host, but not necessarily essential for in vitro growth. The MT2282 gene of M. tuberculosis CDC1551 (or Rv2224c of H37Rv) was identified as a putative virulence gene by high throughput techniques (5, 6). It is annotated as a putative proteinase (TIGR-CMR) or a probable exported protease (TuberculList). Interestingly, isogenic M. tuberculosis mutants with an insertionally inactivated MT2282 gene were attenuated in pooled mutant competition studies in mouse lungs and spleens (5, 6). This gene was also preferentially expressed in human macrophages (13). Under nutrient starvation, MT2282 was significantly up-regulated during 4-96 h after the onset of starvation (14).

In this study, we further characterized the virulence role of MT2282 in the mouse infection model. We demonstrated that the MT2282 protein is membrane-associated and possesses carboxylesterase activity. The functional annotation of proteinase appears incorrect because no proteinase activity was found.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Media, and Growth Conditions—M. tuberculosis CDC1551 (15) and its mutants were cultured as described previously (6). Mycobacterium smegmatis mc2155 used for mycobacterial phage propagation was cultured in 7H9 or 7H10 medium, and incubated at 30 °C when temperature-sensitive phage was recovered. Escherichia coli strains (DH5{alpha} for cloning, BL21 for expression, and HB101 for in vitro packaging) used in this study were cultured in Luria broth (LB; Difco) at 37 °C with shaking. Hygromycin B was added to the media at a final concentration of 50 µg/ml for M. tuberculosis deletion mutants or 200 µg/ml for E. coli. Kanamycin was added to the media with a final concentration of 20 µg/ml for M. tuberculosis-complemented mutants or 50 µg/ml for E. coli.

Gene Deletion, Complementation, and Characterization—A specialized phage transduction approach was used to generate the MT2282 deletion mutant (16). The complementation vector was constructed by PCR amplification of a 2-kb DNA fragment containing MT2282 and its native promoter into the EcoRI site of the mycobacterial integrating vector pMH94 (17, 18) to yield a single-copy plasmid pCOM that confers kanamycin resistance. pCOM was introduced into {Delta}MT2282 by electroporation and kanamycin selection.

To characterize the deletion and complementation, genomic DNA from wild type (WT), {Delta}MT2282, and COM was isolated, restricted with XhoI, separated by 1% agarose gel electrophoresis, and Southern blotted onto nylon membranes by standard capillary transfer. The DNA templates were hybridized with PCR-generated, digoxigenin-11-dUTP-labeled gene probes, detected with an anti-digoxigenin alkaline phosphatase conjugate, and developed with CSPD3 substrate (Roche Diagnostics). Primers used for generating probes included MT2282-specific gya-F and moi-R, hygromycin-specific hyg4-F and hyg4-R, and kanamycin-specific kanf-1 and kanr-1 (see Table 1).


View this table:
[in this window]
[in a new window]

  		TABLE 1
Primer sequences used in this study

 
MT2282 Expression, Purification, and Localization—The MT2282 gene, without its signal sequence, was PCR-cloned into the EcoRI and XhoI sites of the glutathione S-transferase-tagged expression vector pGEX-6P-1 (Amersham Biosciences) using primers EX-2 and EXP-3 (see Table 1) to yield the expression vector pSL115. To solubilize the recombinant fusion proteins that accumulate as inclusion bodies, a mildly denaturing/refolding kit was used according to the manufacturer's protocol (Novagen). The fusion proteins were affinity-purified using glutathione-Sepharose 4B gel chromatography, and the glutathione S-transferase tag was cleaved off with PreScissionTM protease following the on-column cleavage protocol (Amersham Biosciences). Protein levels were quantified using the BCA protein assay reagent kit (Pierce). Rabbit anti-MT2282 polyclonal antibodies were generated using purified MT2282 as an immunogen according to a standard protocol (Spring Valley Laboratories, Inc.).

To detect MT2282 in the culture medium, 7H9 culture supernatants were concentrated 20-fold with an Amicon ultra-centrifugal device (Millipore) before immunoblotting. M. tuberculosis cells were fractionated into cell envelope and cytosol by bead beating and centrifugation. Briefly, M. tuberculosis cell pellets were washed once and resuspended in 1 ml of phosphate-buffered saline in an O-ring tube with glass beads. After bead beating two times for 30 s each with maximum revolutions/min then centrifuging for 5 min at 13,000 revolutions/min, the upper cytosol phase was removed and passed through a 0.22-micron filter. The pellet containing the cell envelope and beads was washed once with phosphate-buffered saline and centrifuged at 2,500 revolutions/min for 2 min. The upper aqueous phase was discarded, and the middle phase containing the cell envelope fraction was collected. Standard protocols were followed for protein SDS-PAGE and semidry transfer of proteins onto nitrocellulose membranes (19). Immunohybridization and detection was carried out according to a standard ECL protocol (Amersham Biosciences) using purified polyclonal rabbit anti-MT2282 IgG as the primary antibody and goat anti-rabbit IgG horseradish peroxidase conjugate as the secondary antibody.

Carboxylesterase Activity of MT2282—Carboxylesterase activity of MT2282 was determined by observing the conversion of {alpha}-naphthyl acetate, {alpha}-naphthyl propionate, and {alpha}-naphthyl butyrate to {alpha}-naphthol according to Lanz and Williams (20). Briefly, each reaction mixture contained 0.1 ml of protein (0.5 mg/ml) and 1.5 ml of substrate (0.42 mM), 0.5 ml of Na2HPO4 buffer, pH 7.0 (0.2 M), and 0.4 ml of distilled water. The mixture was incubated for 10 min at 39 °C (20). At the end of the reaction, 0.5 ml of 10% lauryl sulfate (Sigma) solution containing 2.5 mg of fast garnet GBC (Sigma) was added. The mixture was then incubated at room temperature for 15 min for color development. Absorbance at 560 nm was measured and the yield of {alpha}-naphthol extrapolated from a standard curve (linear from 0 to 50 µM) constructed with {alpha}-naphthol (Sigma). Catalytic activity of MT2282 was also tested on the fluorogenic substrates 4-methlumbelliferyl butyrate (5 µM, 0.1 M Tris, pH 8.0), 4-methylumbelliferyl heptanoate (5 µM, 0.1 M Tris, pH 8.0), and 4-methylumbelliferyl oleate (50 µM, 0.1 M phosphate, pH 7.0). 145 µl of substrate was mixed with 4.5 µl of MT2282 (2 mg/ml) in an Eppendorf tube, and 100 µl was then pipetted into the assay wells of a 96-well plate (Costar 3694). The plate was read by the fluorometer (ICN Titertek Fluoroskan II) at room temperature with an excitation of 355 nm and an emission of 460 nm. Readings were recorded for 2 h at 3-min intervals. Initial rates were calculated and reported.

To test carboxylesterase inhibitors, paraoxon, dichlorvos, phenylmethanesulfonyl fluoride, and sodium fluoride stock solutions were prepared using the manufacturer-recommended diluents (Sigma-Aldrich). 4-Methylumbelliferyl butyrate was used as substrate at 250 µM in 0.1 M Tris pH 8.0. Each inhibitor solution was added to the substrate to make final concentrations of 1, 10, 100, and 1000 µM. 100 µl of these mixtures were used as negative controls. The percentage of inhibition was determined as the ratio of the initial rate of inhibition divided by the normal rate.

To characterize the active site serine and the catalytic triad, PCR-based site-directed mutagenesis was conducted to mutate Ser215, Asp450, and His477 to Ala215, Ala450, and Ala477, respectively, using primer pairs SER1 and SER2, Asp-F and Asp-R, and H477-1 and H477-2 (Table 1) and the plasmid pSL115. Mutagenesis was confirmed by DNA sequencing and restriction mapping.


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 1.
Deletion and complementation of the MT2282 (Rv2224c) gene from the M. tuberculosis CDC1551 genome. A, schematic representation of the genome region of the wild type (WT), deletion mutant (KO ={Delta}MT2282) and complemented deletion mutant (COM) strains showing the relative gene organization of MT2282 and its paralogue MT2281 (Rv2223c) and the location of relevant restriction sites and corresponding fragment lengths (hyg, hygromycin resistance marker; kan, kanamycin resistance marker; att, bacterial attachment site; int, integrase). B, Southern blotting. XhoI-digested chromosomal DNA from the WT, knock-out, and COM strains using PCR-amplified probes corresponding to the MT2282 gene fragment (upper panel), hygromycin gene fragment (middle panel), and kanamycin gene fragment (lower panel) as probes.

 
Aerosol Infection—Female, 6-week-old BALB/c mice (Charles River Laboratories, Wilmington, MA) were aerosol-infected as described previously (21). Colony-forming units (CFUs) present in lungs and spleens were counted at the indicated time points (see Fig. 8). At days 14 and 42, the lungs were fixed for histological examination with hematoxylin and eosin and acid-fast bacilli staining. All animal procedures were approved by the Institutional Animal Care and Use Committee.

Time to Death—Groups of BALB/c mice (4-6 weeks old, female; Charles River Laboratories, Wilmington, MA) were infected with 0.2 ml of M. tuberculosis by intravenous injection via the lateral tail vein. Fresh cultures grown to early log phase were used. The inoculum was 1.5 x 06 CFUs for WT (n = 13), 1.76 x 106 CFUs for {Delta}MT2282 (n = 13), and 1.66 x 106 CFUs for COM (n = 12). Five mice of each group were sacrificed at day 1 for CFU load determination in the lungs. The death of the mice was recorded, and the mice were weighed weekly. A logrank test was applied to compare the survival curves.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation and Characterization of an MT2282 Deletion Strain and Complemented Mutant—The MT2282 gene was deleted from the genome by the phage transduction and single step selection approach, yielding the deletion mutant ({Delta}MT2282). Fifteen base pairs at the 5' and 64 base pairs at the 3' of the gene remained (Fig. 1A). A single copy of MT2282 with its promoter was introduced into {Delta}MT2282 using the integrating vector pMH94 and kanamycin selection to give the complemented deletion mutant (Fig. 1A, COM). Phenotypically, {Delta}MT2282 was resistant to hygromycin but susceptible to kanamycin. COM was resistant to both hygromycin and kanamycin, whereas WT M. tuberculosis CDC1551 was susceptible to both antibiotics. Genotypically, Southern blotting demonstrated deletion of MT2282 in {Delta}MT2282 and confirmed its presence in COM and WT strains (Fig. 1B). Although {Delta}MT2282 and COM contained the hygromycin resistance gene, only COM retained the kanamycin resistance marker (Fig. 1B).

Localization of the MT2282 Protein—Cloning of MT2282 into the expression vector pGEX-6P-1 was validated by DNA sequencing. A glutathione S-transferase-MT2282 fusion protein was expressed and purified from E. coli BL21(DE3). Approximately 99% of the protein remained as an insoluble inclusion body; therefore, a mild denaturation/refolding procedure was applied to achieve a maximum yield. Affinity purification of recombinant MT2282 yielded a protein that was soluble and 96% pure based upon Coomassie-stained PAGE analysis. The affinity-purified MT2282 protein was shown to be a monomer and had an apparent molecular mass of 54 kDa as extrapolated from migration on 5, 7.5, 10, and 12.5% native PAGE (Fig. 2). A polyclonal rabbit MT2282 antiserum was raised using the purified recombinant protein. Control experiments showed that the anti-MT2282 protein A-purified polyclonal antibody reacted with a single band in M. tuberculosis whole cell lysates, indicating a high degree of specificity. Western blotting showed that a specific band was clearly present only in cell lysates from the WT and COM strains but not in the {Delta}MT2282 cell lysate (Fig. 3A), confirming the deletion. Because SignalP3.0 predicted a signal peptide sequence at the N terminus of MT2282, a WT culture supernatant was concentrated and tested for secreted MT2282. MT2282 immunoreactivity was also absent from the culture supernatant, indicating that the protein was not secreted (Fig. 3A). When whole cells were disrupted by bead beating and fractionated into cytosol and the envelope, MT2282 immunoreactivity was found only in the envelope of the WT and COM strains with <1% in the cytosol (Fig. 3B). A motif scan predicts that, at the N terminus of MT2282, an undecapeptide (amino acids 8-18, LAAVALV-LVGC) may serve as a prokaryotic membrane lipoprotein lipid attachment site. The prediction of a lipoprotein that is associated with the membrane is in agreement with our subcellular localization data (Fig. 3B).


Figure 2
View larger version (88K):
[in this window]
[in a new window]

  		FIGURE 2.
Native polyacrylamide gel electrophoresis of native MT2282 (in pH 8.8 buffer) indicated monomeric property. Shown are the percentages of acrylamide. Lanes 1-4, 20 µg of {alpha}-lactalbumin, carbonic anhydrase, and chicken and bovine albumin, respectively; lane 5, 20 µg of MT2282. On higher percentage gels, faint higher mass bands were visible. However, molecular mass extrapolation based on the migration of those bands suggested that they were not multimers of MT2282.

 


Figure 3
View larger version (61K):
[in this window]
[in a new window]

  		FIGURE 3.
Western blotting. Immunoblotting and hybridization using rabbit anti-MT2282 polyclonal antibodies (primary) and horseradish peroxidase-labeled goat anti-rabbit-conjugated antibody (secondary) demonstrate that MT2282 is not secreted into the culture medium (A) but is retained in the cell envelope (B). In A, the cultures were centrifuged and the supernatants were concentrated 20-fold. Cell lysates were obtained by boiling mycobacterial cells in Laemmli sample buffer. In B, the cell envelope and cytosol fractions were obtained by bead beating and centrifugation. ~0.5% of the cell envelop and 2% of the cytosol fractions were applied to the Western blot and yielded roughly a total protein ratio of 1:1.5 of cell envelop over cytosol as assessed by SDS-PAGE.

 


Figure 4
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 4.
MT2282 does not possess proteinase activity. Upper panel, MT2282 was tested on the general proteinase substrate Hide Powder Azure in Tris buffer (pH 7.8). Lower panel, MT2282 was also tested on the serine proteinase substrate BZAR in HEPES buffer (pH 7.4). In both tests, MT2282 showed no proteinase activity. Three independent tests yielded the same results, and one set of data is shown.

 
MT2282 Does Not Possess Proteinase Activity as Annotated—Sequence analysis by Pfam suggests MT2282 is a member of the {alpha}/beta hydrolase fold superfamily (E = 4.2-70). This superfamily contains a typical tertiary fold, but family members lack obvious primary sequence similarity or substrate specificity (22, 23). The MT2282 protein is annotated as a putative proteinase or a probable exported protease. Using soluble, affinity-purified recombinant MT2282, we tested the protein for a variety of proteolytic activities. Purified MT2282 lacked activity when tested with either the proteinase substrate Hide Powder Azure (Calbiochem) or with the serine proteinase substrate bis-(N-CBZ-L-arginine amide)-rhodamine 110,dihydrochloride (BZAR, Biotium) (Fig. 4). Protein modeling led to the prediction that MT2282 was a proline iminopeptidase. However, assays with a proline iminopeptidase-specific substrate L-proline p-nitroanilide trifluoroacetate salt (PPNA, Sigma) were again negative (data not shown). In each substrate, we observed strongly positive proteolytic activity with the positive control enzymes (which was trypsin for Hide Powder Azure and BZAR and proline iminopeptidase for PPNA; the generous gift of Tadashi Yoshimoto, Nagasaki University, Japan).


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 5.
Carboxylesterase activity of MT2282. A, catalytic activity of MT2282 with {alpha}-naphthyl acetate (1-NA), {alpha}-naphthyl propionate (1-NP), and {alpha}-naphthyl butyrate (1-NB). Bovine serum albumin (BSA) was the negative control, and porcine liver esterase (PLE) was the positive control. Data are shown as mean ± S.E. of three independent experiments. B, enzymatic activity of MT2282 on fluorogenic substrates 4-methylumbelliferyl butyrate (4-MB), 4-methylumbelliferyl heptanoate (4-MH), and 4-methylumbelliferyl oleate (4-MO). The initial rates (relative fluorescence units/min) are indicated in parentheses for each substrate. C, pH optimum of the catalytic activity of MT2282 on 1-NP in 0.2 M Na2HPO4.

 


Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 6.
Michaelis-Menten kinetics suggesting a single active site of MT2282. Data were recorded with the fluorometer (ICN Titertek Fluoroskan II) at room temperature with an excitation of 355 nm and an emission of 460 nm. Kinetic readings were obtained for 2 h at 3-min intervals. Initial rates were calculated and reported. RFU, relative fluorescence units.

 
Carboxylesterase Activity of MT2282—In contrast to previous predictions, a sequence motif scan from the PROSITE data base predicted an esterase motif in MT2282 positioned between residues 89 and 230 with an E value = 0.00045 (24). An active site serine (Ser215) was predicted within residues 213-217 (G-Y-S-Y-G), consistent with the G-X-S-X-G consensus motif for members of the esterase/lipase superfamily (25). In view of their bioinformatics findings, we tested affinity-purified MT2282 for esterase activity in vitro and found that MT2282 cleaved ester bonds and released {alpha}-naphthol from esterase substrates, such as {alpha}-naphthyl acetate, {alpha}-naphthyl propionate, and {alpha}-naphthyl butyrate. Interestingly, the esterase activity of MT2282 increased with the increasing carbon chain length of the substrate (p < 0.001 for 1-NA versus 1-NP and p < 0.05 for 1-NP versus 1-NB). However, the positive control for this system, porcine liver esterase, did not show this pattern (Fig. 5A). Using fluorogenic substrates, we further observed that MT2282 was most active on 4-methylumbelliferyl butyrate followed by 4-methylumbelliferyl heptanoate, with weakest activity using 4-methylumbelliferyl oleate (Fig. 5B). In 0.2 M Na2HPO4 buffer, the optimum pH for MT2282 activity on {alpha}-naphthyl propionate was 7.0 within the range of pH 5.0-8.5 (Fig. 5C). Enzyme activity was routinely assayed at 39 °C because of the fact that 37 °C assays were 10% lower in activity. Enzymatic kinetics study yielded a curve that fit the classical Michaelis-Menten model, indicating a single active site of MT2282. With 4-methylumbelliferyl butyrate as the substrate, the Vmax = 128.1 relative fluorescence units/min and Km = 738 µM (Fig. 6).

Next, we conducted inhibition studies with the organophosphates paraoxon and dichlorvos that phosphorylate the active site serine of known esterases (26). These inhibitors showed concentration-dependent inhibition in the 0.1-1.0 mM range. Dichlorvos stimulated activity some 10-12% at subinhibitory concentrations. Both sodium fluoride and phenylmethylsulfonyl fluoride, which are inhibitory for serine proteases and some esterases, were non-inhibitory at low concentrations but stimulatory for enzyme activity at high concentrations. The stimulation of ester bond hydrolysis was of interest, because it would indicate that sites other than the active site serine of MT2282 were involved. Such cooperativity, in some cases, can lead to activation of enzymes (26). Plus, it has already been shown that phenylmethylsulfonyl fluoride does not inhibit all esterases (27).


Figure 7
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 7.
Site-directed mutagenesis confirmed the catalytic triad of MT2282. Esterase activity of MT2282 or its mutant forms MT2282(D450A), MT2282(H477A), and MT2282(S215A) were assayed using 1-naphthyl propionate as the substrate. MT2282-WT, wild type MT2282; PLE, porcine liver esterase (control). Lack of catalytic activity of the mutant MT2282s were also observed on fluorogenic substrate 4-methylumbelliferyl butyrate (data not shown). Data were shown as mean ± S.D. of three repeats.

 


Figure 8
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 8.
Growth curve CFUs for M. tuberculosis CDC1551 WT, {Delta}MT2282, and COM strains in BALB/c mouse lungs (A) and spleens (B) after aerosol infection. Lower CFU loads in lungs (A) and spleens (B) for {Delta}MT2282 suggest attenuation. Graph inset in A represents the total inocula of the 10-ml cultures with A600 = 0.2 for the three strains used in nebulization at day 0. Data are shown as mean ± S.D., n = 3.

 
Site-directed Mutagenesis Confirmed a Catalytic Triad—Microbial carboxylesterases share structural similarities (an open twisted beta-sheet surrounded on both sides by {alpha}-helices) and the arrangement of three catalytic residues (the catalytic triad, Ser-Asp-His) with the serine located in a sharp turn between a beta-strand and {alpha}-helix that is known as the nucleophile elbow, and the His side chain hydrogen-bonded to the carboxylic group of the Asp side chain to form a charge relay system (28). ClustalW sequence alignment indicates that the G-Y-S-Y-G motif is highly conserved not only in the genus of Mycobacterium, but also in the genera Arthrobacter, Rhodococcus, and Nocardia. The same is true for the Asp450 and His477 residues (supplemental Fig. S1). To test the Ser215-Asp450-His477 catalytic triad, site-directed mutagenesis was conducted, and replacement of Ser215, Asp450, and His477 with Ala completely abolished esterase activity as assessed on 1-naphthyl propionate (Fig. 7). The same results were seen in an assay with the fluorogenic 4-methylumbelliferyl butyrate as substrate (data not shown). These observations confirmed that the primary enzymatic function of MT2282 is esterase activity, that Ser215-Asp450-His477 is the catalytic triad, and that the 213G-Y-S-Y-G217 motif is the active site for that activity.

In Vitro Growth of the {Delta}MT2282 Mutant—Previous work had shown that a transposon insertion in MT2282 yielded an insertion mutant JHU2224c-197, but the mutant failed to survive in mouse lung competition assays (4, 6). To determine whether this phenotype could be seen in a single mutant rather than in pooled mutants, we prepared a full deletion of MT2282 (as described above) and studied its growth and virulence properties alongside that of the wild type and complemented mutant strains.

In Middlebrook 7H9 medium, the growth rates by A600 and CFU counting of {Delta}MT2282 and COM were the same as that of the WT before day 7, or A600 of ~1.2 (supplemental Fig. S2, B and C). Between days 7 and 10, the {Delta}MT2282 CFUs plateaued, whereas the WT and COM grew further. MT2282 was not essential for in vitro growth, as shown by insertional inactivation using transposon mutagenesis (3, 4). Although the optical density did not show a difference, the CFUs in this study indicated that some of the bacilli in the {Delta}MT2282 culture were not viable toward the end of growth. Introduction of the MT2282 gene with its native promoter into {Delta}MT2282, as in the COM strain, completely restored its viability phenotype. In addition to increased expression of MT2282 under nutrient starvation (14), the lower viability of {Delta}MT2282 in late log and early stationary phases implied that MT2282 was required to maintain viability under nutritional stress and that expression of MT2282 was regulated by a stress signal through an unknown pathway.

Deletion of the MT2282 Gene Resulted in Attenuation and Reduced Pathology in the Mouse Lung—To assess virulence in the mouse model, we used an aerosol infection route in BALB/c mice. An inoculum of 10 ml of A600 = 0.2 culture, which was subsequently found to have a closely matched titer of 1.46 x 107, 1.55 x 107, and 1.39 x 107 CFU/ml for WT, {Delta}MT2282, and COM, respectively, was used in the Glas-Col nebulizer for each of the three strains. We monitored CFU counts in the lungs and spleens of mice for 56 days. {Delta}MT2282 grew rapidly in the lungs for the first 14 days (Fig. 8A). From day 14 to day 28, the {Delta}MT2282 mutant replicated slowly and peaked on day 28; thereafter the CFU counts slowly decreased between days 42 and 56. Although the CFU curve of {Delta}MT2282 followed a similar pattern to that of the WT and COM, the CFUs were significantly lower for {Delta}MT2282, which indicated an attenuated mouse phenotype for {Delta}MT2282. We also noticed that on day 1, the CFU loads in the lungs of {Delta}MT2282 (2.35 log units) were lower than that of WT (3.10 log units) and COM (3.07 log units). Two possibilities could be accountable for the lower CFU loads in {Delta}MT2282 lungs. {Delta}MT2282 may be more susceptible than WT and COM to the mechanical stress of nebulization (29), or {Delta}MT2282 may be more susceptible than WT and COM to alveolar macrophages, because pulmonary alveolar macrophages are highly activated (30). To further characterize the attenuation of {Delta}MT2282 in mice, we conducted a time-to-death study using high dose intravenous mouse infection (see below). Spleen CFUs showed initial variation on day 14, but by days 28-56 a more uniform count was obtained (Fig. 8B). The 2-log difference in spleen CFUs on day 14 suggested that the MT2282 mutant may be attenuated for dissemination from the lung.


Figure 9
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 9.
Gross pathology of mouse lungs and spleens. BALB/c mice were infected with M. tuberculosis CDC1551 WT, {Delta}MT2282, and COM strains by the aerosol route. More granulomas in lungs and larger lungs and spleens were seen in WT and COM than {Delta}MT2282, suggesting attenuation of {Delta}MT2282. A, gross pathology of lungs and spleens at day 56. B, splenic weight curves.

 
The gross pathology in the mouse lungs infected by {Delta}MT2282 was markedly less severe compared with lungs infected with WT and COM (Fig. 9A). Beginning at 4 weeks, the lungs infected with {Delta}MT2282 were smaller in size and had fewer apparent granulomas, whereas the lungs infected with WT and COM were larger and had more granulomas (Fig. 9A, upper panel). Similarly, the spleens infected with {Delta}MT2282 were also smaller in size (Fig. 9A, lower panel) and weighed less (Fig. 9B). Histopathology using hematoxylin and eosin staining indicated that, at day 14, there were more localized mononuclear cell aggregates in the lungs infected by WT and COM than in lungs infected by {Delta}MT2282 (supplemental Fig. S3A). Acid-fast-stained bacilli were identified in the infected lungs for all three groups on day 14. It was obvious that there were more bacilli in WT and COM-infected lungs than those infected by {Delta}MT2282 (supplemental Fig. S3B). By day 42, consolidation of lung tissue was apparent in the WT and COM, whereas in {Delta}MT2282-infected lungs, much more functional alveolar air space was seen (supplemental Fig. S3A). The differences in histopathology between WT and COM versus {Delta}MT2282 reflect functional changes to the lung that were confirmed by time-to-death studies in mice.

Deletion of the MT2282 Gene Conferred Prolonged Time to Death in Mice—To confirm the role of MT2282 in virulence, a high dose intravenous mouse infection experiment was carried out focusing on time-to-death analysis (18). At day 1, there were 5.62 ± 0.68 x 104, 6.03 ± 0.54 x 104, and 6.10 ± 0.29 x 104 CFUs deposited in mouse lungs infected with knock-out, WT, and COM, respectively. BALB/c mice infected with WT and COM showed significantly different median survival times as compared with {Delta}MT2282-infected mice. As seen in Fig. 10A, mice infected with WT died within 56 days (median = 47), mice infected with COM died within 64 days (median = 52.5), whereas mice infected with {Delta}MT2282 survived up to 191 days (median = 154, p < 0.0001 by Logrank test for comparison to both WT and COM). It was noteworthy that mice infected with WT and COM showed significant weight loss compared with mice infected with {Delta}MT2282 (Fig. 10B). The fact that median survival times were ~3.0-3.3x longer in {Delta}MT2282-infected mice indicates a virulence role for this carboxylesterase gene.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results revealed that deletion of the MT2282 gene in M. tuberculosis resulted in attenuation in the mouse CFU organ burden and time-to-death models of virulence. Introduction of a single copy MT2282 gene with its native promoter completely restored the wild type phenotype both in vitro and in vivo. Attenuation is unlikely due to polar effect, because expression of the downstream genes MT2281 and MT2280 (glnA2) was detected at the mRNA level, as shown by reverse transcription PCR (supplemental Fig. S4). Although insertional inactivation of MT2281 did not have a phenotype either in vitro or in vivo in a pooled mutant study (3, 5), the function of MT2281 is worthy of further study, because its predicted amino acid sequence shares homology with MT2282.


Figure 10
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 10.
Time-to-death and body weight changes of mice with intravenous infection. BALB/c mice infected with M. tuberculosis CDC1551 WT (n = 13), {Delta}MT2282 (n = 13) and COM (n = 12), respectively. Prolonged time to death (A) and sustained body weight (B) suggested attenuation for the {Delta}MT2282 mutant. Data are shown as mean ± S.D.

 
Bioinformatic analysis predicted a signal peptide sequence at the N terminus of MT2282 (SigP3.0). Further in silico analysis by LipoP1.0 and PSORT algorithms predicted a lipid attachment site at residue 18 for a membrane-anchored lipoprotein (31). By Western blotting, we showed association of MT2282 with the cell envelope without evidence of secretion into the culture medium, suggesting that MT2282 is not cleaved by signal peptidase I. Therefore, it is likely that the MT2282 immature protein is cleaved by mycobacterial signal peptidase II; a potential site exists just upstream of the Cys18 residue to which a glyceride fatty acid lipid is attached (32-34). However, further studies are necessary to demonstrate MT2282 to be a lipoprotein. Our data indicate that the MT2282 protein is anchored to the surface membrane where it may function as a catalytic protein, as suggested by this study. Furthermore, it remains possible that MT2282 may also be implicated in structural and/or nutrient transport functions.

A high degree of Mycobacterium-specific gene duplication has been observed within mycobacterial genomes (35, 36). MT2282 and its adjacent downstream paralogue MT2281 appear to be duplicated genes, because they share significant sequence similarities (51.2% identity, 64.6% similarity at the protein level) and are located adjacently in the same orientation with a gap of 62 base pairs (Fig. 1A). They also share the same structural fold and functional predictions, with the active site residues G-Y-S-Y-G being completely conserved. Although MT2282 is anchored to the membrane, MT2281 is predicted to be cleaved by signal peptidase I and secreted into the environment. Because MT2282 was characterized to be a carboxylesterase in this study, MT2281 likely has the same activity. Hence MT2282 and MT2281 appear to be proteins with similar function but possible divergent localization, one anchored to the membrane and the other secreted. A synergistic action between the two proteins may exist. We propose that the MT2282 gene be named as caeA and the MT2281 gene as caeB for carboxylesterases A and B.

At least two possibilities exist regarding the molecular mechanism of attenuation of the M. tuberculosis caeA mutant. Surface lipids are important for the virulence of M. tuberculosis (37, 38). For example, the peptidoglycan-linked mycolic acids that are esterified to arabinogalactan are key virulence determinants (39), and lipoproteins of M. tuberculosis have been shown to be capable of triggering activation of humoral and cellular immune responses to mycobacteria (40, 41). As a membrane-anchored protein, CaeA may play a structural role in modifying envelope composition through its esterase activity (42). Alternatively, it is also possible that CaeA is primarily the hydrolase of free esters in the local environment. Certain fatty acyl esters, for example, may possess mycobactericidal activity (43), and hence CaeA may play a protective role in degrading potential toxic lipids. Additionally, esterase action on free esters in the microenvironment could be a source of fatty acids to be used nutritionally. Further assessment of the in vivo substrates of CaeA and CaeB will be useful in distinguishing among these hypotheses.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI36973, -37856, and -43846 (to W. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4. Back

1 A recipient of a postdoctoral fellowship from the Heiser Program of the New York Community Trust. Back

2 To whom correspondence should be addressed: Dept. of Medicine, Center for Tuberculosis Research, Johns Hopkins University School of Medicine, 1550 Orleans St., Baltimore, MD 21231. Tel.: 410-955-3507; Fax: 410-614-8173; E-mail: wbishai{at}jhmi.edu.

3 The abbreviations used are: CSPD, disodium 3-[4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3,3,13,7]decan}-4-yl]phenyl phosphate; (Fast Garnet) GBC, (4'-amino-2,3-dimethylazobenzene O-aminoazotoluene); CFU, colony-forming unit; WT, wild type. Back


   ACKNOWLEDGMENTS
 
The assistance of Naomi Gauchet is gratefully acknowledged. We thank Norman Morrison for critical reading of this manuscript. We also thank Sandeep Tyagi for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M. A., Rajandream, M. A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J. E., Taylor, K., Whitehead, S., and Barrell, B. G. (1998) Nature 393, 537-544[CrossRef][Medline] [Order article via Infotrieve]
  2. Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12712-12717[Abstract/Free Full Text]
  3. Sassetti, C. M., Boyd, D. H., and Rubin, E. J. (2003) Mol. Microbiol. 48, 77-84[CrossRef][Medline] [Order article via Infotrieve]
  4. Lamichhane, G., Zignol, M., Blades, N. J., Geiman, D. E., Dougherty, A., Grosset, J., Broman, K. W., and Bishai, W. R. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 7213-7218[Abstract/Free Full Text]
  5. Sassetti, C. M., and Rubin, E. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 12989-12994[Abstract/Free Full Text]
  6. Lamichhane, G., Tyagi, S., and Bishai, W. R. (2005) Infect. Immun. 73, 2533-2540[Abstract/Free Full Text]
  7. Pai, R. K., Convery, M., Hamilton, T. A., Boom, W. H., and Harding, C. V. (2003) J. Immunol. 171, 175-184[Abstract/Free Full Text]
  8. Post, F. A., Manca, C., Neyrolles, O., Ryffel, B., Young, D. B., and Kaplan, G. (2001) Infect. Immun. 69, 1433-1439[Abstract/Free Full Text]
  9. Pethe, K., Swenson, D. L., Alonso, S., Anderson, J., Wang, C., and Russell, D. G. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13642-13647[Abstract/Free Full Text]
  10. Vergne, I., Chua, J., Lee, H. H., Lucas, M., Belisle, J., and Deretic, V. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4033-4038[Abstract/Free Full Text]
  11. Wayne, L. G. (1994) Eur. J. Clin. Microbiol. Infect. Dis. 13, 908-914[CrossRef][Medline] [Order article via Infotrieve]
  12. Parrish, N. M., Dick, J. D., and Bishai, W. R. (1998) Trends Microbiol. 6, 107-112[CrossRef][Medline] [Order article via Infotrieve]
  13. Dubnau, E., Fontan, P., Manganelli, R., Soares-Appel, S., and Smith, I. (2002) Infect. Immun. 70, 2787-2795[Abstract/Free Full Text]
  14. Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A., and Duncan, K. (2002) Mol. Microbiol. 43, 717-731[CrossRef][Medline] [Order article via Infotrieve]
  15. Valway, S. E., Sanchez, M. P., Shinnick, T. F., Orme, I., Agerton, T., Hoy, D., Jones, J. S., Westmoreland, H., and Onorato, I. M. (1998) N. Engl. J. Med. 338, 633-639[Abstract/Free Full Text]
  16. Bardarov, S., Bardarov, S., Jr., Pavelka, M. S., Jr., Sambandamurthy, V., Larsen, M., Tufariello, J., Chan, J., Hatfull, G., and Jacobs, W. R., Jr. (2002) Microbiology (Read.) 148, 3007-3017[Abstract/Free Full Text]
  17. Lee, M. H., Pascopella, L., Jacobs, W. R., Jr., and Hatfull, G. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3111-3115[Abstract/Free Full Text]
  18. Chen, P., Ruiz, R. E., Li, Q., Silver, R. F., and Bishai, W. R. (2000) Infect. Immun. 68, 5575-5580[Abstract/Free Full Text]
  19. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (2006) Current Protocols in Molecular Biology, Vol. 2, pp. 10.8.4-10.8.7, John Wiley & Sons, Inc., New York
  20. Lanz, W. W., and Williams, P. P. (1973) J. Bacteriol. 113, 1170-1176[Abstract/Free Full Text]
  21. Nuermberger, E. L., Yoshimatsu, T., Tyagi, S., Williams, K., Rosenthal, I., O'Brien, R. J., Vernon, A. A., Chaisson, R. E., Bishai, W. R., and Grosset, J. H. (2004) Am. J. Respir. Crit. Care Med. 170, 1131-1134[Abstract/Free Full Text]
  22. Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I., Schrag, J., Sussman, J. L., Verschueren, K. H. G., and Goldman, A. (1992) Protein Eng. 5, 197-211[Abstract/Free Full Text]
  23. Nardini, M., and Dijkstra, B. W. (1999) Curr. Opin. Struct. Biol. 9, 732-737[CrossRef][Medline] [Order article via Infotrieve]
  24. Falquet, L., Pagni, M., Bucher, P., Hulo, N., Sigrist, C. J., Hofmann, K., and Bairoch, A. (2002) Nucleic Acids Res. 30, 235-238[Abstract/Free Full Text]
  25. Fojan, P., Jonson, P. H., Petersen, M. T., and Petersen, S. B. (2000) Biochimie (Paris) 82, 1033-1041
  26. Rosenfeld, C. A., and Sultatos, L. G. (2006) Toxicol. Sci. 90, 460-469[Abstract/Free Full Text]
  27. Patel, D., and Scott, C. S. (1991) Biochem. Pharmacol. 42, 1577-1585[CrossRef][Medline] [Order article via Infotrieve]
  28. Liu, P., Wang, Y. F., Ewis, H. E., Abdelal, A. T., Lu, C. D., Harrison, R. W., and Weber, I. T. (2004) J. Mol. Biol. 342, 551-561[CrossRef][Medline] [Order article via Infotrieve]
  29. Sun, R., Converse, P. J., Ko, C., Tyagi, S., Morrison, N. E., and Bishai, W. R. (2004) Mol. Microbiol. 52, 25-38[CrossRef][Medline] [Order article via Infotrieve]
  30. Dannenberg, A. M. (2006) Pathogenesis of Human Pulmonary Tuberculosis. pp. 120-152, ASM Press, Washington D.C.
  31. Juncker, A. S., Willenbrock, H., Von Heijne, G., Brunak, S., Nielsen, H., and Krogh, A. (2003) Protein Sci. 12, 1652-1662[CrossRef][Medline] [Order article via Infotrieve]
  32. Hayashi, S., and Wu, H. C. (1990) J. Bioenerg. Biomembr. 22, 451-471[CrossRef][Medline] [Order article via Infotrieve]
  33. Mattar, S., Scharf, B., Kent, S. B., Rodewald, K., Oesterhelt, D., and Engelhard, M. (1994) J. Biol. Chem. 269, 14939-14945[Abstract/Free Full Text]
  34. Klein, P., Somorjai, R. L., and Lau, P. C. (1988) Protein Eng. 2, 15-20[Abstract/Free Full Text]
  35. Kinsella, R. J., Fitzpatrick, D. A., Creevey, C. J., and McInerney, J. O. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10320-10325[Abstract/Free Full Text]
  36. Qamra, R., Prakash, P., Aruna, B., Hasnain, S. E., and Mande, S. C. (2006) Biochemistry 45, 6997-7005[CrossRef][Medline] [Order article via Infotrieve]
  37. Khuller, G. K., Taneja, R., Kaur, S., and Verma, J. N. (1982) Aust. J. Exp. Biol. Med. Sci. 60, 541-547[CrossRef][Medline] [Order article via Infotrieve]
  38. Reed, M. B., Domenech, P., Manca, C., Su, H., Barczak, A. K., Kreiswirth, B. N., Kaplan, G., and Barry, C. E., III (2004) Nature 431, 84-87[CrossRef][Medline] [Order article via Infotrieve]
  39. Draper, P., and Daffe, M. (2005) in Tuberculosis and the Tubercle Bacillus (Cole, S. T., Eisenach, K. D., McMurray, D. N., and Jacobs, W. R., Jr., eds) pp. 261-274, ASM Press, Washington, D.C.
  40. Young, D. B., and Garbe, T. R. (1991) Res. Microbiol. 142, 55-65[Medline] [Order article via Infotrieve]
  41. Sinha, S., Kosalai, K., Arora, S., Namane, A., Sharma, P., Gaikwad, A. N., Brodin, P., and Cole, S. T. (2005) Microbiology (Read.) 151, 2411-2419[Abstract/Free Full Text]
  42. Trivedi, O. A., Arora, P., Vats, A., Ansari, M. Z., Tickoo, R., Sridharan, V., Mohanty, D., and Gokhale, R. S. (2005) Mol. Cell 17, 631-643[CrossRef][Medline] [Order article via Infotrieve]
  43. Kondo, E., and Kanai, K. (1977) Jpn. J. Med. Sci. Biol. 30, 171-178[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 FASEB J.Home page
N. P. West, F. M. E. Chow, E. J. Randall, J. Wu, J. Chen, J. M. C. Ribeiro, and W. J. Britton
Cutinase-like proteins of Mycobacterium tuberculosis: characterization of their variable enzymatic functions and active site identification
FASEB J, June 1, 2009; 23(6): 1694 - 1704.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
O. H. Vandal, J. A. Roberts, T. Odaira, D. Schnappinger, C. F. Nathan, and S. Ehrt
Acid-Susceptible Mutants of Mycobacterium tuberculosis Share Hypersusceptibility to Cell Wall and Oxidative Stress and to the Host Environment
J. Bacteriol., January 15, 2009; 191(2): 625 - 631.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Rengarajan, E. Murphy, A. Park, C. L. Krone, E. C. Hett, B. R. Bloom, L. H. Glimcher, and E. J. Rubin
Mycobacterium tuberculosis Rv2224c modulates innate immune responses
PNAS, January 8, 2008; 105(1): 264 - 269.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/25/18348    most recent
M700035200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lun, S.
Right arrow Articles by Bishai, W. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lun, S.
Right arrow Articles by Bishai, W. R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement