1 Department of Bacteriology, Swedish Institute for Infectious Disease Control, S-17182, Solna, Sweden
2 Microbiology and Tumour Biology Centre, Karolinska Institute, S-17177, Stockholm, Sweden
3 Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-39182, Kalmar, Sweden
Correspondence
Thomas Åkerlund
Thomas.Akerlund{at}smi.ki.se
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ABSTRACT |
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INTRODUCTION |
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Latency is believed to involve a non-replicating persistence of mycobacteria or a very slow growth. One important condition believed to contribute to latency is reduced access to oxygen. M. tuberculosis is generally regarded as a strictly aerobic bacillus, although it can survive for a long time in a micro-aerophilic environment as long as the shift is not too abrupt (Wayne & Hayes, 1996). An in vitro model to study this persistent state is the so-called Wayne dormancy model, in which the bacteria downregulate their metabolism due to reduced access to oxygen (Wayne & Hayes, 1996
). Another dormancy model utilizes nutrient starvation to induce a state where M. tuberculosis arrests growth and decreases its respiration rate (Betts et al., 2002
). Recently it was demonstrated that inhibition of respiration by nitric oxide might induce a dormant state in M. tuberculosis (Voskuil et al., 2003
). One should recognize, however, that the evidence linking in vitro dormant states of M. tuberculosis to human latent infection still remains circumstantial.
The vaccine strain Mycobacterium bovis BCG has been reported to occasionally persist in a latent state in humans, and in rare instances to cause disseminated disease, mainly in patients that are immunosuppressed (Talbot et al., 1997). This is, however, an extremely rare event compared to the reactivation occurring in latent TB and could conceivably be caused by other mechanisms. The completed DNA sequences of M. tuberculosis and M. bovis BCG (Cole et al., 1998
) show that BCG lacks a number of genes present in M. tuberculosis (Brosch et al., 1998
; Mahairas et al., 1996
). Furthermore, comparative proteome analysis of M. tuberculosis and M. bovis BCG has shown differences in the expression of several protein classes (Jungblut et al., 1999
). There are also differences in protein expression between different laboratory strains, such as M. tuberculosis Erdman and H37Rv (Jungblut et al., 1999
). To study latency it is therefore important to use a model with a relevant experimental M. tuberculosis strain.
Two-dimensional (2-D) PAGE is a potent tool to analyse the proteome (total protein expression) of a certain organism. The proteome represents not only the gene product, but also translational rate and post-translational modifications. Identification of proteins that are upregulated during latency is important in order to study the mechanisms of latency and to identify vaccine targets as well as targets for drug development. There are several reports on the proteomes of mycobacterial strains where cytosolic proteins, as well as cell wall proteins and culture filtrate proteins, have been identified (Boon et al., 2001; Jungblut et al., 1999
; Mattow et al., 2001
; Monahan et al., 2001
; Rosenkrands et al., 2000
; Sonnenberg & Belisle, 1997
; Wong et al., 1999
; Yuan et al., 1996
).
Here, we studied the cytosolic proteome of the virulent clinical isolate M. tuberculosis Harlingen strain grown under aerobic and anaerobic conditions. A modified Wayne dormancy model (Wayne & Hayes, 1996) was used where the bacteria were grown aerobically for 810 days and then shifted to anaerobic growth conditions.
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METHODS |
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Culture and growth conditions.
The bacteria were thawed from a stock solution maintained in the laboratory at 70 °C and grown in LöwensteinJensen vials with pyruvate for 4 weeks. Approximately 1x106 bacteria of the M. tuberculosis isolate Harlingen were inoculated into BACTEC 13A vials (Becton & Dickinson) carrying 7H13 medium containing 14C-labelled palmitate. The vials were incubated at 37 °C, and every other day the amount of 14CO2 in the gas phase of the vial was measured in a BACTEC460 apparatus (Becton & Dickinson) and the removed gas was exchanged with an equal amount of oxygen (5 % CO2, synthetic air). After 810 days half of the cultures were shifted to anaerobic growth conditions, i.e. the measurements were performed with the air valve of the BACTEC460 apparatus connected to a non-oxygen mixture (85 % N2, 10 % H2, 5 % CO2). After 2226 days the cultures were harvested by centrifugation and the bacterial pellets were washed once in PBS and protein extracts were prepared.
Adaptation of the cells to oxygen-free conditions was monitored by daily recordings of the growth index. The metabolic activity of the adapted bacteria was monitored by measuring intracellular ATP levels. The extraction and quantification of ATP were accomplished using a Bio-Orbit 1251 luminometer, following the improved method described by Nilsson et al. (1998).
Protein extraction.
The bacterial pellets were washed in PBS 13 times, dissolved in 20 µl 0·3 % SDS, 200 mM DTT, 28 mM Tris base either before or after bead-beating and transferred to Eppendorf tubes containing 200400 µl 0·10·5 mm zirconium beads (Techtum). The cells were disrupted in a Mini-BeadBeater (Biospec Products) for 3 min at 5000 Hz. The lysate was boiled for 2 min and cooled on ice. Four microlitres of a mix of 0·5 M Tris/HCl, 50 mM MgCl2, 1 mg DNaseI ml1, 0·25 mg RNaseA ml1 was added to the Harlingen extracts and the incubation on ice was continued for 10 min. Then, 160 µl of a mix of 9·9 M urea, 4 % NP-40, 2·2 % ampholytes, 100 mM DTT was added either before or after storage at 80 °C.
2-D PAGE.
Between 10 and 40 µg protein was loaded on IPG strips pH 47, 18 cm (Amersham Biosciences). The gel strips were focused on a Multiphor electrophoresis unit and the second dimension was run on vertical 12 % acrylamide SDS-PAGE gels (Protean II; Bio-Rad). The gels were stained by silver, dried on Novex frames and scanned in a Hewlett Packard ScanJet 3c/T scanner. For preparative gels, protein was precipitated in 10 % TCA and washed three times in acetone at 4 °C. About 0·5 mg protein was loaded on each gel and the gels were stained by Coomassie brilliant blue.
Data analysis.
The digitized gel images were imported into PDQuest (version 6.0; Bio-Rad) and were used for detection and quantification of the spot intensities, gel matching and statistical analysis. All spots were manually checked to exclude spots of low quality, e.g. spots in the periphery of the gel or in streaky areas. The analysis included spots that were either unique to one set of gels or changed in intensity by a factor of at least three. To correct for overall intensity differences between the gels, each gel was normalized to the standard according to the total quantity in valid spots.
Protein identification and MALDI-TOF.
Proteins of interest were excised in 1x1 mm pieces that were destained in 0·2 M ammonium bicarbonate in 50 % (v/v) acetonitrile for 1 h at 37 °C, followed by a second wash in 0·05 M ammonium bicarbonate in 50 % (v/v) acetonitrile (1 h, 37 °C). The gel pieces were dried by Speedvac and reswelled in 5 µl buffer containing 0·2 M ammonium bicarbonate and 0·1 µg trypsin µl1 (sequencing grade modified trypsin; Promega) for 10 min before incubation at 37 °C overnight covered by 20 µl 0·2 M ammonium bicarbonate. The cleavage was stopped by adding 0·5 µl triflouracetic acid (TFA) and peptides were extracted two times in a solution containing 60 % (v/v) acetonitrile and 0·1 % TFA followed by extraction two times in 40 % (v/v) acetonitrile and 0·1 % TFA. Peptides were concentrated by speedvac and dissolved in 13 µl 1 % acetic acid and purified using ZipTipC18 (Millipore). Purified peptides were applied to an AnchorChip (Bruker) and analysed by MALDI-TOF (Bruker Daltronic Mass Analyser Reflex III) using -4-hydroxycinnamic acid as matrix. External calibrations were made using a mix of peptides with known molecular masses and internally by using known tryptic fragments of trypsin. A search for protein candidates was made by using the apparent peptide masses, pI and molecular masses in the MS-Fit software at http://prospector.ucsf.edu/. A MOWSE score better than 1500 was judged to be significant and the whole procedure (from gel excision) was repeated to ensure that the correct protein was identified.
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RESULTS AND DISCUSSION |
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One disadvantage with our protein approach may be that protein spots may represent proteolytic degradation products rather than upregulated proteins. First, it must be stressed that not all regulation occurs at the transcriptional level and proteolysis and other post-translational modification may have a significant impact on cellular processes. Second, our estimation of general proteolytic activity was less than 10 %, but it is probably much lower. Thus, if the upregulated spots represented more stable proteins in the cell, there would not be more than a 10 % upregulation. Third, if the identified proteins were proteolytic degradation products, such as those found for HspX and GroEL2, these peptides would generally have significantly lower molecular masses. Twelve of our identified spots, excluding HspX and GroEL2, showed a mean apparent molecular mass difference of +3·9 % (range 4·9 to +16·5 %) of the predicted molecular mass, while the GroEL2 and HspX peptides showed 15 and 19/31 % reduction, respectively. The N-terminal sequences of the smallest HspX peptides identified were also determined and we recalculated the predicted molecular mass loss to 27 %, in accord with our estimated value of 31 %.
Stress proteins
HspX and USP Rv2623 have been found to be upregulated in stationary phase/hypoxic conditions in strain BCG and M. tuberculosis (Boon et al., 2001; Cunningham & Spreadbury, 1998
; Desjardin et al., 2001
; Florczyk et al., 2001
; Rosenkrands et al., 2002
; Sherman et al., 2001
; Tabira et al., 1998
; Voskuil et al., 2003
; Yuan et al., 1996
, 1998
). The regulator dosR (Rv3133c) has been implicated in the control of 48 dormancy-associated genes, including hspX, Rv2623, Rv2626c, Rv2005c and Rv2629 (Boon & Dick, 2002
; Park et al., 2003
). The expression of this 48-gene regulon was also induced by low concentrations of NO (Table 2
). Eight USP homologues have been found in the genome of M. tuberculosis (Rv1636, Rv2028c, Rv2624c, Rv3134c, Rv1996, Rv2005c, Rv2026c and Rv2623) of which at least five have been found to increase during stationary phase/hypoxic conditions (O'Toole & Williams, 2003
). The USP family is conserved in many organisms and studies in, for example, Escherichia coli have shown that usp mutants are less likely to survive growth arrest and DNA damage (Kvint et al., 2003
). Also, expression of uspC, uspD and uspE in E. coli was shown to be dependent on the stringent response as well as RecA, and mutants were sensitive to UV irradiation (Gustavsson et al., 2000
, 2002
). Although the HspX, Rv2623 and Rv2626c products in M. tuberculosis are regarded as markers for dormancy and/or stationary phase, we did not detect any major difference in expression of HspX or Rv2626c, which appeared highly expressed under both conditions (Table 2
and Fig. 2
). However, we found higher abundance of the dormancy-related proteins Rv2629 and Rv2005c (Tables 1 and 2
). Thus, the expression of these commonly found dormancy markers suggests that the rapid introduction of anaerobic gas did not prevent the mounting of a dormancy programme by decreased viability, for example.
Thioredoxin reductase (Rv3913, TrxB2) has been found to be upregulated in Mycobacterium smegmatis during stationary phase (Murugasu-Oei et al., 1999). Thioredoxin and thioredoxin reductase of M. tuberculosis have been shown to reduce peroxides and dinitrobenzenes with higher efficiency under anaerobic conditions (Zhang et al., 1999
). In Streptomyces coelicolor the trxB operon has been found to be controlled by SigR (Paget et al., 1998
) and its homologue in M. tuberculosis is designated SigH. Several studies have indicated that the sigma factors SigB, SigE, SigF and SigH in M. tuberculosis and M. smegmatis are involved in adaptation to several stresses, including heat shock and oxidative stress (Chen et al., 2000
; Fernandes et al., 1999
; Hu & Coates, 1999
; Manganelli et al., 1999
). It has further been demonstrated that SigH, in response to these stresses, induces transcription of the genes encoding TrxB2, DnaK and ClpB (Raman et al., 2001
). As discussed by Zhang et al. (1999)
, it is possible that the Trx-TR system contributes to the antioxidant defence of M. tuberculosis during anaerobic or micro-aerophilic conditions.
Metabolic enzymes
The upregulated 40 kDa L-alanine dehydrogenase (Ald, Rv2780) was the first antigen reported to be produced by virulent M. tuberculosis, but not in the vaccine strain M. bovis BCG (Andersen et al., 1992; Jungblut et al., 1999
). However, its gene is present both in virulent and avirulent strains. It is a functional L-alanine dehydrogenase and thus one of the few antigens with enzymic properties that makes it attractive for diagnostic and therapeutic interventions. Proteins in the culture filtrate of M. tuberculosis are the primary targets of the immune response (Harboe, 1992
) and the 40 kDa protein is one of the earliest proteins detectable in the culture medium of M. tuberculosis, present as early as 4 days (Andersen et al., 1992
). The activity of the enzyme increases during dormancy development in M. smegmatis, in which the activity rapidly increases fivefold after depletion of oxygen. As oxygen becomes more limiting, the enzyme activity declines until it reaches a level about 3·5-fold above the baseline (Hutter & Dick, 1998
). The ald transcript of M. tuberculosis H37Rv is overproduced under hypoxic conditions (Rosenkrands et al., 2002
; Sherman et al., 2001
) and under nutrient starvation conditions (Betts et al., 2002
). The suggested main function of this enzyme is the generation of alanine for protein and peptidoglycan synthesis. As alanine synthesis is coupled to NADH oxidation, it is also possible that the induction of alanine dehydrogenase activity might support the maintenance of the NAD pool when oxygen, as terminal electron acceptor, becomes limiting (Hutter & Dick, 1998
).
Another metabolic enzyme that increased during anaerobisis was succinyl-CoA : 3-oxoacid-CoA transferase (ScoB, Rv2503c), which catalyses the reversible reaction where succinyl-CoA and a 3-oxoacid are converted to succinate and 3-oxoacyl-CoA, respectively.
Cell wall
Mycobacteria have an unusual cell wall in which mycolic acids play a critical role in structure and function. This structure confers to the bacteria resistance to chemical injury, low permeability to antibiotics, resistance to dehydration and an ability to survive within the phagolysosomes of the macrophages (Barry et al., 1998). During entry into dormancy, mycobacteria enlarge due to thickening of the cell wall (Cunningham & Spreadbury, 1998
). There are at least three mycolic acid cyclopropane synthases (CmaA1, PcaA and CmaA2) that are responsible for the site-specific modifications of mycolic acids. The first one to be identified was CmaA1, based on its homology to the E. coli enzyme CFA synthase (Wang et al., 1992
). At least seven homologous genes have been identified in the genome sequence of H37Rv (Cole et al., 1998
). These are cmaA2, mmaA1-4 and umaA1-2; they share 5075 % identity with each other and are all thought to be SAM-utilizing methyl transferases. Another mycolic acid synthase, PcaA, was shown to be important for dormancy since pcaA knock-out strains lost their ability to persist, although not replicated in a mouse model (Glickman et al., 2000
). Studies in M. tuberculosis have shown that Rv0503c (CmaA2), found in this study to be fourfold upregulated during anaerobisis, is required for the synthesis of the trans-cyclopropane rings of keto- and methoxymycolates (Glickman et al., 2001
). Cyclopropanation of mycolic acids is a modification that is associated with pathogenic bacteria and is not common in the cell wall of saprophytic species such as M. smegmatis (Minnikin et al., 1982
). In wild-type M. smegmatis, less than 2 % of the mycolic acids are monocyclopropanated, whereas this increases to 25 % after induction of CmaA2 (George et al., 1995
). In addition, cyclopropane ring modifications have profound effects on the resistance of mycobacteria to oxidative stress (Yuan et al., 1995
) and the fluidity and permeability of the cell wall (George et al., 1995
). In E. coli and other species that cyclopropanate their plasma membrane, this modification occurs during the transition from active growth to stationary phase, and as a response to environmental conditions such as low pH, high incubation temperatures and low aeration rates (Grogan & Cronan, 1997
; Wang & Cronan, 1994
).
The upregulated protein -ketoacyl-ACP synthase (KasB, Rv2246) is, together with KasA, involved in the synthesis of mycolic acids (Schaeffer et al., 2001
). KasA has been shown to specifically elongate palmitoyl-CoA to monounsaturated fatty acids averaging 40 carbons in length and overproduction of KasB in the presence of KasA leads to the production of even longer chains. The production of these chains is sensitive to isoniazid, thiolactomycin and triclosan in vitro (Slayden & Barry, 2002
). Moreover, another study showed that KasAB is the target of thiolactomycin (Kremer et al., 2000
). However, since KasA is not the direct target of isoniazid, this effect may be indirect. Finally, Mycobacterium marinum KasB mutants (with transposon insertions in kasB) synthesized mycolic acids that were 24 carbons shorter than wild-type and grew poorly in macrophages (Gao et al., 2003
). Thus there appears to be an increase in full-length oxygenated mycolic acids during anaerobiosis and a requirement of long-chain mycolic acids during intracellular growth.
Protein Rv0560c shows similarity to SAM-utilizing methyl transferases and it is possible that Rv0560c is linked to synthesis of ketomycolates. Rv0560c has been shown to be induced by salicylate (Sun et al., 2001), a compound that also increases oxygen consumption when added to M. tuberculosis cultures (Bernheim, 1940
), and is clustered with other genes involved in ubiquinone biosynthesis (Sun et al., 2001
). The function of Rv3866 is not known, but it is located just upstream of the virulence-associated RD1 element on the M. tuberculosis chromosome (Sassetti & Rubin, 2003
). Finally, PHI/PSI-BLAST searches revealed that Rv2185c shows weak similarity to polyketide cyclases.
In summary, we found about 50 proteins in a clinical isolate of M. tuberculosis that were unique or more abundant under anaerobic conditions and low ATP levels. The induction pattern was different from that during a shift from exponential growth to stationary phase and the induction ratios were moderate (3- to 10-fold induction). Newly found proteins were related to, for example, mycolic acid synthesis and oxidative stress. Their roles during the survival of dormant tubercle bacilli require further research.
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ACKNOWLEDGEMENTS |
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Received 28 April 2004;
revised 16 July 2004;
accepted 17 July 2004.
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