1 MRC Centre for Molecular and Cellular Biology, Department of Medical Biochemistry, University of Stellenbosch Medical School, PO Box 19063, Tygerberg, 7505, South Africa
2 GlaxoSmithKline Research and Development, Stevenage, Hertfordshire SG1 2NY, UK
Correspondence
Paul van Helden
pvh{at}sun.ac.za
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ABSTRACT |
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INTRODUCTION |
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Proteomics, the study of the protein complement of the genome, has been used extensively to identify differences with respect to virulence and pathogenesis between mycobacterial strains. Comparative proteome analysis has revealed numerous differences in the cellular protein composition of the laboratory strains M. tuberculosis H37Rv and Erdman to that of the vaccine strain Mycobacterium bovis bacille CalmetteGuérin (BCG) (Jungblut et al., 1999; Mattow et al., 2001
). In addition, a number of differences have also been observed in the expression of proteins in culture supernatants of H37Rv and BCG (Mattow et al., 2003
), and H37Rv and the attenuated strain H37Ra (He et al., 2003
). Analysis of the proteome of M. tuberculosis H37Rv and a clinical strain CDC1551, believed to elicit a more vigorous host immune response than H37Rv (Manca et al., 1999
), identified several quantitative differences in the cellular protein composition of these strains (Betts et al., 2000
).
In a previous study we compared protein expression of two M. tuberculosis clinical strains, originally isolated from patients from a community in Cape Town, South Africa, with a very high tuberculosis (TB) incidence (Warren et al., 2000), to the laboratory strain H37Rv using one-dimensional (1D) PAGE, ELISA and Western blotting (Pheiffer et al., 2002
). Based on the number of IS6110 insertions and spoligotyping, the two clinical strains were classified as belonging to the Beijing family and family 23 (F23) (Pheiffer et al., 2002
). Results from that study (Pheiffer et al., 2002
) showed that protein expression by M. tuberculosis strains was mainly growth phase dependent, although some differences between the strains were observed. However, due to the low resolving power of 1D gel electrophoresis, the identity of the differentially expressed proteins could not be ascertained. Here, we have extended our previous study through the use of two-dimensional (2D) PAGE coupled with identification of protein spot differences by MS and Western blot analysis of the expression of 14 M. tuberculosis antigens. Furthermore we have used plasma from patients infected with Beijing, F23 and family 11 (F11) strains to investigate differences in antigen recognition. F11 strains constitute another highly prevalent strain family in the study community.
This study has revealed a number of differences in cellular and culture supernatant protein composition between M. tuberculosis H37Rv, the Beijing strain and F23, particularly between the two clinical strains compared to H37Rv. Careful analysis of these differences and the differences in the antibody recognition profiles could possibly explain the different frequencies of the clinical strains in our study population, where the Beijing strain is at least tenfold more frequent than F23 strains (Warren et al., 2000). In addition to providing clues as to the differences in pathogenicity and prevalence of strains, identification of protein expression differences between strains will aid the development of vaccines, serodiagnostic tests and the choice of drug targets. This is believed to be the first study to profile expression patterns of cellular and culture supernatant proteins of a Beijing strain by 2D gel electrophoresis and the first study attempting to correlate strain prevalence with protein expression.
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METHODS |
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Isocitrate dehydrogenase (ICD) assay.
The amount of ICD in culture supernatants was measured using the Sigma Diagnostics ICD kit. In this procedure, ICD catalyses the oxidative decarboxylation of L-isocitrate to 2-oxoglutarate, and the reduced NADPH produced is measured at 340 nm.
Protein extraction.
Whole-cell lysate (WCL) proteins were extracted by bead disruption of the mycobacterial pellet in lysis buffer [0·3 % (w/v) SDS, 200 mM DTT, 50 mM Tris/HCl pH 7·0, 1 mM PMSF and complete protease inhibitor cocktail] (Roche Molecular Biochemicals) as previously described (Pheiffer et al., 2002). Culture filtrate (CF) proteins were prepared from culture supernatants sterilized by sequential filtration of the culture supernatant through 0·45 µm and 0·22 µm filter units (Corning). Filtrates were then concentrated using Centricon Plus-80 filter units (Biomax-PB membrane, 5000 MWCO), followed by Centricon units (YM membrane, 3000 MWCO) (Millipore). WCL and CF protein concentrations were estimated using the Bradford assay (Bradford, 1976
).
2D gel electrophoresis.
Proteins were separated by 2D gel electrophoresis as previously described (Betts et al., 2000), with minor modifications. Briefly, 20 µg (60 µg for Western blotting) protein was resuspended in rehydration buffer [8 M urea, 2 % (w/v) CHAPS, 10 mM DTT, 2 % (v/v) immobilized pH gradient (IPG) buffer (pH 47), trace bromophenol blue] and applied to pH 47 IPG strips (Amersham Biosciences) for overnight rehydration. IEF was performed using a Multiphor II system (Amersham Biosciences) as follows: 100 V for 2 h, 300 V for 2 h, 1000 V for 1 h, 3500 V for 20 h (3500 V for 27·5 h for Western blotting). Second-dimension separation was carried out by placing equilibrated IPG strips (Bjellqvist et al., 1993
, http://www.expasy.ch/ch2d/protocols/) onto 12 % PAGE gels and sealing with 0·5 % (w/v) agarose in cathode buffer (defined below), containing a trace amount of bromophenol blue. Anode (375 mM Tris/HCl, pH 8·8) and cathode (192 M glycine, 0·1 %, w/v, SDS, pH 8·3) buffers as described by Herbert et al. (1998)
were used. Proteins were visualized by silver staining using an ammoniacal stain (Bjellqvist et al., 1993
, http://www.expasy.ch/ch2d/protocols/). Gels were air-dried (Bio-Rad GelAir Dryer) and compared by visualization on a light box. When protein spot differences were noted, gels were rerun, stained with a silver stain compatible with MS (Betts & Smith, 2001
) and spots of interest excised from the gel.
Sample preparation for MS.
Excised gel pieces were washed with double-distilled H2O for 10 min, followed by 100 % acetonitrile for 5 min, centrifuged and then dehydrated by vacuum centrifugation. Automated robotic digestion was carried out using a MassPREP station (Micromass), equipped with four probes for aspirating and dispensing reagent and washing solutions, and a heated incubation platform. Samples were destained with 50 mM ammonium bicarbonate/acetonitrile (1 : 1, v/v), then reduced and alkylated with 10 mM DTT and 55 mM iodoacetamide respectively. This was followed by in-gel digestion with porcine trypsin (Promega) (6 ng µl1) in 50 mM ammonium bicarbonate (25 µl) for 5 h at 37 °C. The resulting peptides were then extracted with 1 % (v/v) formic acid/acetonitrile (98 : 2, v/v).
Nanoscale liquid chromatography tandem mass spectrometry (LC/MS/MS) and database searching.
Samples were introduced using the Micromass CapLC system (Micromass), comprising a low flow capillary HPLC pump and autosampler. A 10 port valve was configured with a pre-concentration column (300 µm IDx5 mm C18 PepMap, LC Packings) and a nanoscale analytical column (75 µm IDx15 cm C18 PepMap, LC Packings). Peptides were eluted using a reverse-phase gradient of 550 % buffer B over 30 min [A=5 % (v/v) acetonitrile, 0·1 % (v/v) formic acid; B=95 % (v/v) acetonitrile, 0·1 % (v/v) formic acid] at a flow rate of approximately 200 nl min1. All data were acquired using a Q-Tof Ultima API (Micromass) hybrid quadrupole orthogonal acceleration time of flight mass spectrometer equipped with a nanospray source. Up to eight precursor ions were automatically selected from the time of flight (TOF)/MS survey scan for MS/MS per cycle, and collision energies were chosen automatically based on the m/z value and the charge state of the selected precursor ions. Peptide sequence data generated were searched against the non-redundant protein database using MASCOT (http://www.matrixscience.com). Search parameters included the fixed modification carboamidomethyl, due to the alkylation of cysteine residues by iodoacetamide and the variable modification, oxidation of methionine residues.
Monoclonal antibodies (mAbs).
Mouse mAbs against a range of M. tuberculosis proteins (Table 1) (Engers et al., 1986
, Khanolkar-Young et al., 1992
; Sonnenberg & Belisle, 1997
) were made available by the Department of Microbiology, Colorado State University, through funds from the National Institutes of Health, National Institute of Allergy and Infectious Disease, Contract NO1-AI-75320. HYB76-8 directed to ESAT6 (Sorensen et al., 1995
) was received from Karin Weldingh (Statens Serum Institut, Copenhagen, Denmark).
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2D Western blotting.
For Western blotting, 2D gels were transferred to nitrocellulose membranes (S&S Protran BA85, Schleicher & Schuell) using semi-dry blotting. Blots obtained from 2D gels were probed with mAbs IT-4 and IT-23 using the same conditions as for 1D Western blotting. For blots using plasma from TB patients, plasma was diluted 1 : 150 and screened for antibody binding using alkaline-phosphatase-conjugated goat anti-human IgG (Kirkegaard & Perry Laboratories) as described above. Immunoreactive antigens were detected using the BCIP/NBT substrate (Kirkegaard & Perry Laboratories) as described above.
Patients.
Patients were recruited from a high TB incidence community and were homogeneous with respect to social class and ethnicity. All patients were culture-positive for M. tuberculosis drug-sensitive organisms. The smear status, disease episode, anti-tuberculosis chemotherapy status and IS6110 genotype of the infecting M. tuberculosis strain were documented for all patient samples (Table 2). Blood was collected by clinical staff; plasma was collected by centrifugation at 2500 g for 5 min, and stored at 20 °C. Ethical approval for this study was obtained from the University of Stellenbosch Faculty of Health Sciences ethics committee and samples were only taken after informed consent was given.
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RESULTS |
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1D and 2D Western blotting identified PstS1 as the protein responsible for the decreased expression of spot 12 (Fig. 1, Table 3
) in the Beijing strain compared to the F23 strain and H37Rv (Fig. 2d
, Fig. 3b
). 2D Western blotting showed the existence of more than one species of PstS1, which has been previously reported by Sonnenberg & Belisle (1997)
and is probably due to post-translational modifications such as glycosylation, phosphorylation or acetylation. The absence of PstS1 on 2D blots of CFs of the F23 strain (Fig. 3b
) could possibly be due to decreased expression of PstS1 in CFs compared to WCLs, as observed on 1D Western blots (Fig. 2d
). Additionally, proteins may be more concentrated in bands on 1D gels than in spots on 2D gels and proteins may transfer more efficiently from 1D gels than from 2D gels. Several species corresponding to
-crystallin were observed and were expressed more highly in the Beijing strain compared to F23 and H37Rv (Fig. 2a and 3a
). Consistent with the 2D results, expression of
-crystallin was increased in the WCL compared to the CF in all the strains. Expression of the 47 kDa protein was decreased in the Beijing strain compared to H37Rv and F23 (Fig. 2h
). Two proteins, Hsp65 and the Ag85 complex, were downregulated in both clinical strains relative to H37Rv (Fig. 2c, f
), while Hsp65 was also downregulated in the Beijing strain compared to F23. Multiple bands of Hsp65, possibly different isoforms or degradation products, were observed in both WCLs and CFs. Hsp65 observed in CFs is possibly due to bacterial lysis. L-Alanine dehydrogenase and ESAT6 (Fig. 2b, 2i
) showed decreased expression in H37Rv compared to both clinical strains.
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Reactivity of plasma samples to proteins extracted from the three M. tuberculosis strains
To establish whether there was differential antigen expression by the M. tuberculosis strains, plasma-derived antibodies of TB patients were tested against WCL proteins extracted from M. tuberculosis H37Rv, the Beijing strain and F23. After 1D PAGE separation and transfer to PVDF membranes, proteins were probed with plasma from TB patients infected with Beijing, F23 or F11 strains (Table 2, Fig. 4
). As expected, plasma from TB patients reacted with a wide variety of M. tuberculosis proteins, ranging from 10 kDa to over 105 kDa (Fig. 4
). Plasma samples were tested on antigens fractionated on different gels; therefore figures were aligned according to molecular mass markers, which were included on each gel, and mobility of immunodominant antigens. As noted previously by others, patient-to-patient variation, where each patient had a characteristic banding pattern (Fig. 4
), was evident. However, some prominent differences between the antigens expressed by the strains were observed; these are circled in Fig. 4
and listed in Table 4
. A band of approximately 60 kDa (circle 1) was recognized by patient 880 in H37Rv but not in F23 or the Beijing strain (Fig. 4f
). Patient 880 also reacted with a protein of about 40 kDa (circle 2) in protein extracts of F23 and the Beijing strain only (Fig. 4f
), with more prominent expression in the Beijing strain. Similarly, a band of approximately 15 kDa (circle 5) was recognized more strongly in the Beijing strain compared to the other strains by patient 880. Proteins of approximately 28 and 25 kDa which were recognized strongly by patient 97 in H37Rv are indicated by circles 3 and 4 (Fig. 4a
).
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DISCUSSION |
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The high prevalence of the Beijing genotype worldwide is indicative of the success of this M. tuberculosis strain type as a human pathogen (Glynn et al., 2002). It has been suggested that the global dissemination of this strain genotype may be linked to an altered phenotype, thereby conferring an advantage over M. tuberculosis strains belonging to other genotypes (Lopez et al., 2003
). This is supported by the observation that Beijing strains are more virulent and elicit a non-protective immune response compared to other genotypes, during experimental disease in mice (Lopez et al., 2003
). Infection with Beijing genotypes has also been linked to the development of fever during the early phase of treatment (van Crevel et al., 2001
). These findings highlight the need to elucidate the mechanisms underlying the success of Beijing strains compared to other M. tuberculosis strains. It has been suggested that the wide dispersion of the Beijing strain family compared to other less prevalent clinical isolates may be related to differential protein expression (Bifani et al., 2002
).
In agreement with previous studies of protein expression across different M. tuberculosis strains (Betts et al., 2000; Jungblut et al., 1999
; Mattow et al., 2001
), this study revealed that a large portion of the proteome is similarly expressed. Despite this, we have been able to detect several differences in protein expression and shown distinct differences in antigen expression levels across the three strains, with several changes being particular to the Beijing strain. Several species of
-crystallin, a M. tuberculosis virulence factor (Monahan et al., 2001
; Sherman et al., 2001
; Yuan et al., 1998
), were more highly expressed in the Beijing strain compared to H37Rv and F23. Altered expression of this protein may be a factor contributing to the virulence of Beijing strains. The success of the Beijing family as a human pathogen may in part be due to decreased expression of Hsp65, PstS1 and the 47kDa protein as it has previously been suggested that reduced expression of certain major antigens may allow strains to evade the host immune response (Stewart et al., 2001
). Moreover, differences in antigen expression levels between the strains, as observed from 1D and 2D Western blots with patient plasma, may be significant and could assist the Beijing strain to minimize recognition by the host immune response, thereby facilitating the increased prevalence of Beijing strains. In order to further assess the importance of the antigens identified here in general strain prevalence or whether these differences are unique to the Beijing family, expression levels could be determined in other prevalent strains. Importantly, certain antigen expression differences between strains may only become evident when the bacteria are growing within the host environment. Therefore, it will be necessary to analyse proteins extracted from mycobacteria isolated from conditions that mimic the host environment, for example macrophages or animal models. In addition, to further validate the antigens found to be differentially expressed here and determine whether they also show expression differences in vivo, sera from patients infected with the Beijing strain could be used to probe against purified recombinant proteins of the antigens of interest, with the caveat that patient variation and extent of disease may influence antigen recognition.
Differential protein expression may explain the heterogeneous host humoral immune response and why no serodiagnostic test for TB has yet been developed (Lyashchenko et al., 1998). The most marked difference in protein expression between the Beijing and F23 was observed for PstS1. PstS1 is a 38 kDa M. tuberculosis complex-specific phosphate-binding lipoprotein, and a known B- and T-cell stimulant (Harboe & Wiker, 1992
). To date, PstS1 has shown promise for serological diagnosis of TB, with sensitivities of 70 % and 73 % for smear-negative pulmonary TB and extrapulmonary TB patients, respectively (Wilkins & Ivanyi, 1990
). However, in a recent evaluation of commercially available tests the highest sensitivity achieved with PstS1 was 55 % (Pottumarthy et al., 2000
). The absence of PstS1 antibodies in some TB patients may be due to decreased expression of PstS1 in some clinical strains, as observed for the Beijing strain in this study. These findings suggest that the development of a serodiagnostic test for TB may be hindered by variable protein expression by M. tuberculosis strains, and support the development of a test measuring the levels of antibodies to a panel of antigens that are common to different M. tuberculosis genotypes (Al Zahrani et al., 2000
; Amicosante et al., 1999
). Furthermore, differential expression of PstS1 by different M. tuberculosis genotypes suggests that this antigen may not be a good choice for a vaccine, and may partially explain the discrepant results obtained when using PstS1 as a subunit or DNA vaccine candidate (Falero-Diaz et al., 2000
).
This study has demonstrated extensive humoral heterogeneity between patients, even though they were homogeneous with respect to social class and ethnicity. Humoral heterogeneity has previously been shown (Lyashchenko et al., 1998) and attributed, in part, to differential protein expression by M. tuberculosis genotypes. Variation in antigen recognition was observed even between patients infected with the same genotype, highlighting the importance of other factors, in addition to differential protein expression, in dictating the host humoral immune response. These factors could be host factors (Bothamley et al., 1989
), extent of disease (Jackett et al., 1988
), or even multiple infections with different M. tuberculosis genotypes (Warren et al., 2004
). Taken together, these results suggest that detection of mycobacterial antigens (Wallis et al., 1998
) may be more valuable than antibody detection, since antigen detection will not be influenced by the significant humoral heterogeneity between patients.
In conclusion, this study has shown that proteome analysis of M. tuberculosis genotypes may contribute to our understanding of the pathogenesis of tuberculosis by identifying differentially expressed proteins, and potentially helping us to understand why certain strain families, such as the Beijing family, are more successful than others. In addition to aiding an understanding of pathogenicity and strain prevalence, identification of antigens differentially expressed between strains provides important information for consideration in the design of serodiagnostic tests, vaccines and even drug target selection.
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ACKNOWLEDGEMENTS |
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Received 27 July 2004;
revised 10 December 2004;
accepted 23 December 2004.
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