Immunopathology Unit1 and Protein Science Unit2, Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, Herts, SG1 2NY, UK
Author for correspondence: Joanna C. Betts. Tel: +44 1438 768138. Fax: +44 1438 764898. e-mail: jb75084{at}glaxowellcome.co.uk
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ABSTRACT |
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Keywords: mycobacteria, tuberculosis, proteome, two-dimensional gel electrophoresis, genome
Abbreviations: 2D, two-dimensional; BCG, bacille CalmetteGuérin; IPG, immobilized pH gradient; MALDI, matrix-assisted laser desorption-ionization; PGRS, polymorphic G+C rich sequence; PE, Pro-Glu; PPE, Pro-Pro-Glu; TOF, time-of-flight
a Present address: Molecular Biology, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough LE11 5RH, UK.
b Present address: Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111, USA.
c Present address: Astex Technology, 250 Cambridge Science Park, Milton Road, Cambridge CB4 0WE, UK
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INTRODUCTION |
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M. tuberculosis H37Rv is the most commonly studied laboratory strain and was first isolated in 1905. Since this time it has been continually passaged and, as its virulence in humans is unknown, there have been concerns as to whether this may have led to attenuation of virulence (Jacobs et al., 1996 ). However, it has maintained virulence in animal models. In contrast, CDC 1551 is a recent clinical isolate which was responsible for an outbreak of TB in a rural area of the USA in 1995 (Valway et al., 1998
). Due to the high rate of skin-test conversion that occurred and the large skin-test response to purified protein derivative of tuberculin (PPD) of those infected, CDC 1551 was considered to be unusually infectious in man. In addition, evaluation of the growth of CDC 1551 in lungs of mice 20 d after aerosol infection gave 100-fold higher numbers of bacilli compared to the numbers of bacilli isolated from the lungs of mice infected with the M. tuberculosis laboratory strain Erdman (Valway et al., 1998
). As a result of these findings, CDC 1551 was assumed to be highly virulent and was selected for sequencing by the National Institutes of Health (Jacobs et al., 1996
).
More recently, however, the growth of CDC 1551 has been compared to both H37Rv, the Erdman strain and two clinical isolates in a mouse aerosol infection model (Manca et al., 1999 ). These studies confirmed CDC 1551 replicates to higher number in mice than Erdman but showed it to have a comparable growth rate to H37Rv and the two additional recent clinical isolates. This study also found CDC 1551 induces granulomatous differentiation and high cytokine levels in the lungs of infected mice at an earlier time point than any of the other strains studied. When human monocytes were infected with CDC 1551, larger amounts of cytokines were produced than in monocytes infected with H37Rv, although there were similar numbers of intracellular organisms (Manca et al., 1999
). It was therefore suggested that CDC 1551 is not more virulent but induces a more rapid and vigorous immune response (Manca et al., 1999
). The two strains have also been compared in a rabbit inhalation model (Bishai et al., 1999
) where it was shown that CDC 1551 has no greater ability either to induce visible pulmonary tubercles or to multiply within these tubercles.
Comparison of the proteome of strains may therefore explain some of these phenotypic differences and may shed light on the mechanism of survival of M. tuberculosis within the host. This information is crucial for the design of new anti-tubercular vaccines and drugs. Since the virulence of M. tuberculosis of H37Rv in man is unknown, comparison of its proteome with that of a recent clinical isolate will also test its relevance for use in studies aimed at the combat of human disease. Several recent studies of the proteome of M. tuberculosis have been reported including the analysis of cell lysates, culture-filtrate proteins and the response of M. tuberculosis to different environmental conditions (Lee & Horwitz, 1995 ; Garbe et al., 1996
, 1999
; Sonnenberg & Belisle, 1997
; Urquhart et al., 1997
, 1998
; Weldingh et al., 1998
; Wong et al., 1999
). The most comprehensive study to date, in terms of protein spot identification, compared two strains of Mycobacterium bovis bacille CalmetteGuérin (BCG) and the M. tuberculosis strains H37Rv and Erdman (Jungblut et al., 1999
). These data have been deposited in a database accessible via the internet (Mollenkopf et al., 1999
; http://www.mpiib-berlin.mpg.de/2D-PAGE/). Most recently, subcellular fractions of M. tuberculosis H37Rv have been analysed by two-dimensional (2D) PAGE and several proteins identified (Rosenkrands et al., 2000
). These data are also available via the internet (http://www.ssi.dk/en/forskning/tbimmun/tbhjemme.htm).
In this study we have compared the protein profiles of cell lysates of M. tuberculosis H37Rv and CDC 1551, utilizing commercially available immobilized pH gradients (IPGs) of pH 47 and pH 310 over a three point time course. We have been able to resolve approximately 1750 distinct protein spots and show that the protein profiles of these two strains remain remarkably similar. Proteins which were identified as being different between the strains or differed with time have been characterized by MS (reviewed by Humphery-Smith et al., 1997 ). In addition, we have compared differences seen experimentally at the protein level to those predicted from comparison of the two genome sequences.
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METHODS |
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Sample preparation for two-dimensional (2D) electrophoresis.
Whole-cell extracts were prepared from M. tuberculosis by harvesting 2x10 ml culture for each strain after 5, 8 and 12 d growth. Cultures were pooled and pelleted by centrifugation (1900 g, 15 min). Cell pellets were washed twice in PBS/1 % (v/v) Tween 80. Mycobacteria were resuspended in lysis buffer containing 0·3% (w/v) SDS, 200 mM DTT, 28 mM Tris/HCl, 22 mM Tris base, 1 mM PMSF and Complete protease inhibitor cocktail (Roche Molecular Biochemicals), and heat-killed at 80 °C for 20 min prior to bead disruption in a ribolyser (Hybaid). Lysates were then clarified by centrifugation and the supernatant removed. The protein concentration of each sample was estimated using the Bradford assay (Bradford, 1976 ).
2D electrophoresis.
Isoelectric focusing sample loading buffer (8 M urea, 4%, w/v, CHAPS, 40 mM Tris base, 65 mM DTT, trace amount of bromophenol blue) was added to the samples and each separated using pH 47 and pH 310 (non-linear) 18 cm IPG strips (Amersham Pharmacia Biotech) in the first dimension. Total protein (50 µg) was loaded at the cathode and proteins focused as follows using a Multiphor II (Amersham Pharmacia Biotech): 300 V for 2 h, 1000 V for 1 h and 3500 V for 22 h. Second-dimension electrophoresis was carried out essentially as described by OFarrell (1975) , using large format (20x20 cm), 1·5 mm thick, 12% polyacrylamide gels. IPG strips were equilibrated prior to running on the second dimension as described (Bjellqvist et al., 1993
; http://www.expasy.ch/ch2d/protocols/) but using 0·375 M Tris/HCl, pH 8·8, and 15 min incubation times. Strips were then overlaid onto the second-dimension gels and sealed with 0·5% (w/v) agarose in cathode buffer, containing a trace amount of bromophenol blue. Anode and cathode buffers as described by Herbert et al. (1998)
were used. For protein spot comparisons, gels were silver stained using an ammoniacal stain as described (Bjellqvist et al., 1993
; http://www.expasy.ch/ch2d/protocols/). For subsequent MS, gels were rerun and silver stained as described by Shevchenko et al. (1996a
) with some modifications (Betts & Smith, 2000
). Gel images were digitized by scanning on a flat-bed scanner (Epson GT9000).
Sample preparation for mass spectrometry.
Protein spots of interest were excised from the gel, reduced, carboxyamidated and digested in situ with trypsin as described by Jensen et al. (1999) . After digestion overnight at 37 °C, samples were centrifuged and an aliquot of the supernatant taken for analysis by MALDI (matrix-assisted laser desorption-ionization) MS. Prior to nanoelectrospray analysis, peptides were extracted from the gel pieces as described by Jensen et al. (1999)
.
MALDI-MS.
This was performed on a VG TofSpec SE time-of-flight (TOF) mass spectrometer equipped with a delayed extraction ion source (Micromass). Samples were prepared largely as described by Jensen et al. (1996) . A saturated solution of
-cyano-4-hydroxycinnamic acid in acetone was mixed in a 4:1 ratio (v/v) with a 10 g l-1 solution of nitrocellulose (Trans-Blot transfer medium, 0·45 µm; Bio-Rad) in acetone/2-propanol (1:1, v/v). This was deposited on the stainless steel target in 0·6 µl aliquots, leaving a matrix/nitrocellulose surface by fast evaporation of the solvent. Aliquots (0·5 µl) were taken from the tryptic digest mixture and loaded into a 0·5 µl droplet of 5% (v/v) formic acid previously applied to the matrix. Samples were air dried at room temperature and washed with 1 µl 5% (v/v) formic acid prior to insertion into the instrument. Spectra were internally calibrated using the matrix ion at m/z 1060·10 and trypsin autolysis peaks at m/z 2163·06 and m/z 2289·15. Monoisotopic masses were assigned and proteins identified by peptide mass fingerprinting using PepSea software (Protana) and a mass accuracy of 0·1 Da.
Nanoelectrospray MS.
Dried digest mixtures were desalted prior to nanoelectrospray analysis. Pulled glass capillaries were packed with approximately 5 µl POROS R2 sorbent (PerSeptive Biosystems). Peptides were dissolved in 0·5% (v/v) formic acid, loaded on to the sorbent and washed with 5 µl 0·5% (v/v) formic acid. Samples were eluted with approximately 2 µl 1% (v/v) formic acid, 50% (v/v) methanol and 1 µl inserted into the spraying needle. Needles for electrospraying were made with a micropipette puller (Sutter Instrument) from borosilicate glass capillaries (Clark Electromedical Instruments) as described by Wilm & Mann (1994 , 1996
) and were gold coated in a vapour desorption instrument. Electrospray mass spectra were acquired on an API III triple quadrupole machine (Perkin-Elmer Sciex) equipped with a nanoelectrospray ion source (Wilm & Mann, 1994
, 1996
). Proteins were identified by the sequence tag approach using PepSea software (Protana).
Genome comparison.
The MUMmer program (Delcher et al., 1999 ) was used to align and compare the genome sequences of M. tuberculosis H37Rv and CDC 1551 using a MUMmer cut off of 50 bp. The genome sequences of H37Rv and CDC 1551 were downloaded from the Sanger Center, Hinxton, Cambridge, release date 17 June 1998 (http://www.sanger.ac.uk/Projects/M_tuberculosis/) and The Institute for Genome Research, release date 4 June 1999 (http://www.tigr.org/tdb/CMR/gmt/htmls/SplashPage.html), respectively.
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RESULTS |
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Of the 17 spots analysed, 12 were identified unambiguously by MALDI-MS alone. Details of the number of matched peptides and the percentage sequence coverage obtained for each protein spot are given in Table 1. Criteria used to determine a positive identification were at least three matching peptides with 0·1 Da mass accuracy. The pI and Mr of the identified protein were also checked against the spot position on the gel. Further analysis of the remaining spots by nanoelectrospray failed to give an identification. Mass spectrometry analysis of these differences revealed that four spots (2, 3, 8 and 9) (Fig. 3a
) were mobility variants of the transcriptional regulator MoxR (Rv1479/MT1526). These varied in their comparative presence at each position between the strains, with H37Rv displaying two forms and CDC 1551 a different two, and may be explained by either amino acid changes or post-translational modifications. The most intense form present in H37Rv was observed in a more basic position on the gel relative to the most intense form present in CDC 1551. The other H37Rv spot corresponding to this protein was also in a more basic position than the second CDC 1551 spot. Mobility variants of this protein have also been observed between the strains H37Rv and Erdman (Jungblut et al., 1999
), with the protein being shifted to a more acidic position in the Erdman strain. One spot, identified as ribosome recycling factor (Rv2882c/MT2949), showed a marked vertical mobility shift between the two strains, being consistently in a higher Mr position on the H37Rv gels (spot 13) (Fig. 3b
). Spot 1 (Fig. 3b
), corresponding to Rv0927c, a probable alcohol dehydrogenase, was absent from the H37Rv proteome, whilst spot 10 (Fig. 3d
), corresponding to phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleotide isomerase (HisA; Rv1603/MT1639), was absent from the CDC 1551 proteome. Two spots corresponding to alkyl hydroperoxide reductase chain C (AhpC; Rv2428/MT2503) were identified and both were decreased in intensity in CDC 1551 (spots 11 and 12) (Fig. 3d
). This protein has also been found to be decreased in intensity in H37Rv relative to M. bovis BCG Chicago (Jungblut et al., 1999
).
Four proteins (spots 1417) showed increased intensity over time in both strains (Fig. 2). Spot 14 was identified as the heat-shock protein, Hsp20 (Rv0251c/MT0265) and showed increased spot intensity on days 8 and 12 compared to day 5. Spots 16 and 17 were identified as the 16 kDa antigen (alpha-crystallin) (Rv2031c/MT2031c) and showed increased intensity on day 12 compared to days 5 and 8.
Comparison of proteome data with differences observed in the genomes of M. tuberculosis H37Rv and CDC 1551
Comparison of the genome sequences of the two strains was carried out using the MUMmer program (Delcher et al., 1999 ). This enabled the mapping of single nucleotide changes and insertions and deletions present in each genome relative to the other. The majority of differences were found to be single nucleotide changes. Approximately 1000 of these were identified. One of the observed proteome differences correlates with a single nucleotide change. This occurs in the moxR gene (Rv1479/MT1526), where there is a base change from C in H37Rv to A in CDC 1551, resulting in a predicted amino acid change from histidine-26 in H37Rv to asparagine-26 in CDC 1551 and consequently differences in the predicted pI and Mr values of the two proteins. This point mutation is contained within the N-terminal tryptic peptide of the protein. However, this peptide was not mapped during the MALDI analysis to confirm the amino acid change. This can be explained by the fact that the peptide, minus the first methionine residue, has a calculated Mr of 4123·94 Da or 4101·91 Da for H37Rv or CDC 1551 respectively, meaning that it falls outside the range scanned during our MALDI analysis, as we routinely only cover a range up to m/z 4000. The predicted pI value (http://www.expasy.ch/tools/pi_tool.html) for the H37Rv protein is 5·96 and that for the CDC 1551 protein is 5·89. This is reflected in the 2D gel spot pattern, where the major MoxR spot in H37Rv (spot 9) was shifted to a more basic position relative to the major CDC 1551 MoxR spot (spot 3) (Fig. 3a
). This doesnt however explain the presence of a second form of MoxR in each strain, which could be caused by post-translational modification. The predicted Mr values for the two proteins are 40762 Da and 40739 Da for H37Rv and CDC 1551 respectively. The resolution of the gel in the Mr range is not sufficient for this difference to be observed. It is also interesting to note that, although not containing a single nucleotide change itself, the hisA gene (Rv1603/MT1639) lies directly downstream of hisH (Rv1602/MT1638), which contains a single nucleotide change resulting in an amino acid difference at the C terminus of the protein. We have shown a spot corresponding to HisA to be absent from the CDC 1551 proteome but present in the H37Rv proteome.
Insertions or deletions in the genome which result in predicted pI or Mr differences at the protein level between the two strains are summarized in Tables 2 and 3
. We have limited our comparison to regions which appeared to be unique to each genome and were at least 3 bp in size. Insertion elements differing between the two strains have not been included in Tables 2
and 3
. The majority of unique coding material found only in the H37Rv genome, with respect to CDC 1551, affected insertion elements or the acidic, glycine-rich proteins of the PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families which often contain multiple copies of the polymorphic G+C rich sequences (PGRSs) or major polymorphic tandem repeats, respectively (Cole et al., 1998
). Our analysis found eight ORFs completely unique to H37Rv, five of these belonging to the PE-PGRS family (Rv0278c, Rv0279c, Rv0746, Rv0747 and Rv1087) and one being a hypothetical protein (Rv0793). Rv0278c and Rv0279c are contained within a 5452 bp region which is missing from CDC 1551. Similarly, Rv0746 and Rv0747 are contained within a 4910 bp region which is deleted in CDC 1551 relative to H37Rv. Part of Rv0748 is also contained within this deleted region. As a result of this large deletion the ORF predictions in this region differ between the two strains. Rv0745 is present at the DNA level in CDC 1551; however, the corresponding ORF in CDC 1551 (MT0772) has a different predicted start site and is frame-shifted. MT0772 is predicted to span the deleted region and its C terminus corresponds to that of Rv0748. Deletions in CDC 1551 in the region of the remaining two ORFs (Rv0155 and Rv1928c) seem to result in frameshifts, producing numerous stop codons, which are likely to mean that these regions are non-coding in CDC 1551.
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The remaining predicted protein differences between the two strains are the result of small insertions or deletions at the DNA level, giving significant changes in predicted Mr and pI at the protein level. None of the protein spot differences observed in this study correspond to predicted changes resulting from these insertions or deletions. However, it is possible that the four remaining unidentified proteins correspond to some of these predicted protein differences.
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DISCUSSION |
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In our proteome comparison of these strains, it was hoped that any protein differences seen may give some insight into the reasons for the differing phenotypes observed, aiding the design of new drugs and vaccines against tuberculosis. Additionally, these studies provide a useful background reference for future proteome experiments and, in comparing its protein profile to that of a recent clinical isolate, test the relevance of H37Rv as a model for human infection.
Strains were compared over a three-point time course. This is an important precursor to further proteome analyses. The protein patterns of the two strains were stable and highly similar over the time course studied. Only 13 protein spots were seen to differ between the two strains. This is consistent with the comparison of M. tuberculosis H37Rv and Erdman, which revealed 18 variant proteins (Jungblut et al., 1999 ). Four of the variants described here correspond to different mobility variants of the MoxR protein, which is a probable regulatory protein involved in the formation of active methanol dehydrogenase. Mobility variants of MoxR were also observed in the comparison of H37Rv with Erdman (Jungblut et al., 1999
). Genome sequence comparison of the moxR gene in H37Rv and CDC 1551 revealed a single nucleotide change, resulting in a predicted amino acid difference and consequent pI change, explaining the presence of mobility variants of this protein. The M. tuberculosis Erdman genome has not been sequenced. However, in the light of our findings, it is likely that the mobility variants of MoxR, described by Jungblut et al. (1999)
, are also a result of a single nucleotide change, and hence amino acid difference, in this protein between H37Rv and Erdman. A spot corresponding to Rv0927c/MT0954, a probable short-chain alcohol dehydrogenase, was found to be present on gels of CDC 1551 but not H37Rv. The gene encoding this protein is, however, present in the H37Rv genome. Similarly, a spot corresponding to HisA, an isomerase which catalyses the fourth step in the histidine biosynthetic pathway, was present on gels of H37Rv but not CDC 1551, although the corresponding gene is present in the CDC 1551 genome. The hisA gene forms part of an operon of histidine biosynthetic genes. The levels of the other proteins encoded within this operon, however, were not detected as differences in this study. These differences are most likely to be explained either by subtle expression-level differences between the two strains at the point in time studied, meaning that the absent protein spots are present but below the silver-stain detection limit, by post-translational modification or by degradation during sample preparation. Interestingly, AhpC was found to be increased in intensity in H37Rv relative to CDC 1551. This protein has also been found to be increased in intensity in M. bovis BCG compared to M. tuberculosis H37Rv (Jungblut et al., 1999
).
The protein profile of each strain also seemed similar across the three time points studied, with only a small number of protein spots changing in intensity with time. Two of these were identified as the 16 kDa antigen (-crystallin), which has been shown to be upregulated as the bacterium enters stationary phase (Yuan et al., 1996
). However, this protein is also found as an intense spot higher up the gel (Fig. 2a
) so it is likely that these spots are due to degradation or post-translational processing. This observation has been made previously (Sonnenberg & Belisle, 1997
), where lower molecular mass spots were found to correspond to N-terminal truncation or degradation by Edman sequencing. The intensity of the spot representing the main form of this protein does however seem to be increased somewhat on day 12 relative to days 5 and 8. Another heat-shock protein, Hsp20, also shows increased intensity on days 8 and 12 in both strains.
Of the differences that we have identified in this proteome comparison, only those in the MoxR protein relate to those predicted from the genome comparison. There are several explanations for this discrepancy. Firstly, some of the proteins predicted to differ between strains lie outside the region resolved on our 2D gels; that is a Mr above 100 kDa or below 10 kDa, or a pI above 10·0. Secondly, we have resolved approximately 1750 protein spots. At best this accounts for 44% of the 3924 predicted genes in M. tuberculosis H37Rv. However, some spots visualized here appear to be spot series of the same protein, perhaps caused by amino acid modifications during sample preparation. Whilst it is extremely unlikely that all predicted proteins are expressed at the same point in time, it is likely that certain low-abundance proteins are masked by the more abundant species on our gels, particularly in the acidic region where the majority of mycobacterial proteins seem to lie, or are below the detection limit of silver staining. It has been suggested that proteins present at less than 1000 copies per cell cannot currently be detected by 2D electrophoresis (Wilkins et al., 1998 ). The limitations of silver staining must also be considered. Although the ammoniacal method utilized for comparison purposes here is extremely sensitive and care was taken to ensure equal intensity of gel staining, variation between protein spots at the silver-stain detection limit was observed and these low-level differences were not included in our comparison. The dynamic range of silver staining is also limited and certain proteins have been shown to stain poorly or not at all. Fluorescent stains, compatible with MS, have recently been developed and now approach the sensitivity of silver staining whilst overcoming the issues of dynamic range and reproducibility.
Some of the proteins predicted to differ are membrane proteins and it is very likely that these hydrophobic proteins are underrepresented on our gels as standard 2D techniques, such as those used in our study, often result in substantial losses of hydrophobic proteins due to solubility problems during sample preparation, precipitation in the first-dimension IPG or inefficient transfer to the second-dimension gel (Adessi et al., 1997 ; Wilkins et al., 1998
). There have recently been a number of methods reported which improve protein solubilization in 2D electrophoresis (Rabilloud et al., 1997
; Herbert et al., 1998
; Molloy et al., 1998
; Santoni et al., 1999
). Such methods are likely to be required in order to visualize more of the mycobacterial proteome. To this end, it may also be necessary to study subcellular fractions of M. tuberculosis as described recently by Rosenkrands et al. (2000)
. Another approach may be to utilize several overlapping narrow-pH-range IPGs to build up a composite proteome map and increase spot resolution (Cordwell et al., 2000
).
Lastly, many of the predicted differences are within PE or PPE genes, for which there is little functional information available and it is still unclear as to whether they are all expressed and at what level. The recently published mycobacterial 2D PAGE internet database, which contains information on approximately 260 proteins identified from 2D gels of M. tuberculosis and M. bovis BCG by MS, does not include any PE or PPE proteins (Mollenkopf et al., 1999 ; http://www.mpiib-berlin.mpg.de/2D-PAGE/) However, inspection of the amino acid sequence of many of these proteins reveals them to have few trypsin cleavage sites. This would make identification by MS following tryptic digestion, as routinely used, almost impossible. Since the PE and PPE families of proteins have been implicated in antigenic variation in M. tuberculosis (Cole et al., 1998
), it is interesting to speculate that the variation in these proteins between the two strains may be significant with regard to their differing immunogenic properties (Manca et al., 1999
).
The genome comparison also reveals that CDC 1551 contains an extra gene (MT2420) that bears homology to the ESAT-6 family. The ESAT-6 protein is a major T-cell antigen which has been purified from M. tuberculosis culture filtrates (Sorensen et al., 1995 ) and is recognized strongly by a high percentage of tuberculosis patients (Mustafa et al., 1998
; Ulrichs et al., 1998
; Ravn et al., 1999
). More recently, two further members of this family have been identified within culture filtrates and shown to induce interferon gamma release from lymphocytes isolated from tuberculosis patients (Skjøt et al., 2000
). There are 14 members of the ESAT-6 family within H37Rv and all are situated in genomic loci showing strongly conserved structure and organization (Tekaia et al., 1999
). Each of these genes are conserved in CDC 1551. It is possible that the extra copy of this gene in CDC 1551 may contribute to the increased host immune response to this strain (Manca et al., 1999
). Our study was focused on the analysis of cellular proteins. It is likely that an analysis of the culture filtrate would be required to reveal differences in the secreted antigens such as the ESAT-6 family of proteins. Our current studies are now directed toward the analysis of membrane and secreted proteins from M. tuberculosis.
Of the 17 silver-stained spots analysed here, 12 were identified by MALDI-MS alone giving a success rate of about 70%. The availability of the complete genome sequence of M. tuberculosis means that, in theory, it should be possible to identify the majority of proteins by peptide mass fingerprinting using the MALDI data alone. We have used a protein loading of approximately 50 µg for our analyses and pooled spots from duplicate gels. The protein spots which remain unidentified are low in abundance as judged by the silver-stain intensity and it is therefore likely to be advantageous to increase protein loading on gels for subsequent mass spectrometry analysis. Another alternative is to use HPLC combined with tandem mass spectrometry (LC/MS/MS) for the analysis of these protein spots. We have recently found this method to have increased sensitivity over the nanoelectrospray technique employed in this study (unpublished results).
In conclusion, we have performed a rigorous proteome comparison of two virulent strains of M. tuberculosis for which there is genome sequence available. The proteome patterns of the two strains grown in broth are highly similar which is in accordance with their high sequence similarity. This suggests that M. tuberculosis H37Rv is indeed a valid laboratory model for the study of the mechanisms of human disease. To gain more insight into the differing phenotypes displayed by these two strains it may be necessary to perform proteome analysis on mycobacteria isolated from conditions where these phenotypes are observed; for example macrophages or animal models of infection. In contrast to the genome level, differences seen at the proteome level are likely to reflect variability in translational control and post-translational modification between the strains. Despite the limitations discussed here, 2D gel-based proteomics remains a powerful approach toward the functional understanding of the proteins encoded within the mycobacterial genome. Technologies are constantly being improved and the problems associated with 2D gels are being eliminated. This study provides a useful and necessary background reference for future studies where the genes responsible for virulence, intracellular survival or drug resistance may be identified through the analysis of mutant strains or mycobacteria subjected to differing environmental conditions. In turn, this may provide us with targets for the development of novel therapies against tuberculosis.
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Received 14 March 2000;
revised 5 June 2000;
accepted 19 August 2000.