A Proteome-Scale Identification of Novel Antigenic Proteins in Mycobacterium tuberculosis toward Diagnostic and Vaccine Development Yuqing Li, Jumei Zeng, Jianfang Shi, Mingchao Wang, Muding Rao, Chaolun Xue, Yanli Du, and Zheng-Guo He* National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Received May 21, 2010
Tuberculosis (TB) remains to be a major infectious disease throughout the world. However, the current vaccine for TB has variable protective efficacy, and there is no commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity for routine laboratory use. One of the potential strategies in developing a new diagnostic method and in improving the TB vaccine involves the identification of novel antigenic candidates. This paper aims to identify systematically the novel antigenic proteins with the greatest potential as protective or diagnostic antigens by using the differential response of Mycobacterium tuberculosis proteins to serum from TB patients and healthy individuals. Approximately 87% of the open reading frames of M. tuberculosis were successfully cloned into IPTGinducible expression vectors. The clone sets were expressed in Escherichia coli, purified under denatured conditions, and tested for antigenicity using a mixture of sera from 15 TB patients. Out of the 3480 proteins screened, 249 proteins had significant reactions with the serum samples. Among the 249 proteins, 20 proteins were identified as most reactive. Compared with the commercial test kits, 3 novel antigens from the top 20 proteins, namely, Rv1987, Rv3807c, and Rv3887c, provided better sensitivity and accuracy. These newly identified antigenic proteins may be used as candidates for serodiagnostic application and vaccine development. Overall, this study’s findings may serve as an essential reference for developing new TB diagnostic methods and more effective tuberculosis vaccines. Keywords: Mycobacterium tuberculosis • antigenic proteins • diagnostic • vaccine development
Introduction Tuberculosis (TB) is a major health threat throughout the world. The World Health Organization (WHO) estimates that approximately 9 million new TB cases occur and about 2 million individuals die of TB every year.1 Moreover, one-third of the population is latently infected with Mycobacterium tuberculosis, of which 5-10% will develop active TB when the balance between natural immunity and the pathogen changes.2,3 At the same time, the rising incidence of both multidrugresistant TB4,5 and coinfection of TB with the human immunodeficiency virus (HIV) poses serious new challenges to the response effort.6 Added to this is the inadequacy of current diagnostic tools and vaccines. Therefore, the development of a more effective vaccine and a rapid, cheap, and accurate test for the diagnosis is critical in the prevention and control of this disease, particularly in the developing world. The identification of more specific reagents to distinguish vaccination from infection and the isolation of subunit vaccine candidates for more effective vaccinations remain as research priorities. * To whom correspondence should be addressed. College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. E-mail:
[email protected] or
[email protected]. Tel: +86-27-87284300. Fax: +86-27-87280670.
4812 Journal of Proteome Research 2010, 9, 4812–4822 Published on Web 08/06/2010
One limitation to the control of tuberculosis is the lack of a sensitive and reliable diagnostic procedure. Traditional diagnostic methods based on the isolation of the bacillus are timeconsuming.7 Traditional tuberculin skin testing (TST) has low specificity because the nonspecific antigen mixture used, purified protein derivative (PPD), can cross-react with many mycobacterial species.8 Therefore, the development of a rapid, cheap, and accurate test for the diagnosis of TB is critical in the prevention and control of this disease. Several novel diagnostic methods have been investigated in recent years.9-13 However, these methods are expensive, require highly trained personnel, or yield variable and low sensitivity,14 making them of limited use in developing countries where large proportions of the population are likely to harbor latent M. tuberculosis infection.9,15 Furthermore, there is no commercially available serodiagnostic testing kit for tuberculosis with acceptable accuracy for routine laboratory use.7,16 The diagnostic test should be rapid, inexpensive, easy to perform, and user-friendly.17 It should use blood because this is easier to obtain than respiratory samples or other organic fluids and tissues.18 Enzyme-linked immunosorbent assay (ELISA) is a serological assay that is relatively simple, highthroughput, inexpensive and does not require sophisticated 10.1021/pr1005108
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Identification of Novel Antigenic Proteins in M. tuberculosis 18,19
equipment. The accuracy of the ELISA test can be improved by using purified recombinant antigens specific to the TB complex or multiepitope polyproteins.18,20 In the development of serodiagnostic methods for TB, identifying specific antigens that effectively differentiate healthy individuals from patients is very important. For this purpose, several mycobacterial antigens have been evaluated.20-30 The current vaccine for TB, Bacille Calmette-Guerin (BCG), was introduced almost 90 years ago. It is efficient in preventing the most severe disseminated forms of the disease in both children and newborns, but its efficacy against pulmonary TB in adults is limited.31,32 Today, the need for a new or improved vaccine has become even more apparent because TB infection has worsened in recent years.1,3-6 Current vaccine candidates include live attenuated M. tuberculosis strains, recombinant BCG strains (e.g., rBCG30-expressing Ag85B or ∆ureCHly+rBCG, a urease deficient strain expressing listeriolysin), virus-based recombinant vaccines (e.g., MVA85A, Rv3804c expressed in replication-deficient vaccinia virus), subunit (e.g., Mtb72F, Ag85B-ESAT-6 fusion), and DNA vaccines.3,33-40 Subunit vaccines are usually comprised of dominant antigens that boost the immune response after priming with BCG.3,38,40-47 To develop an effective subunit vaccine, the identification and immunobiological characterization of the mycobacterial molecules involved in human protective immunity are of prime importance. Several attempts to screen the T cell epitopes of M. tuberculosis have been reported.48-54 However, these studies mostly used bioinformatics and are restricted to a limited number of genes or major histocompatibility complex (MHC) alleles. A more comprehensive experimental screen needs to be implemented. The availability of genome sequence information enables the systematic identification of vaccine candidates and novel antigenic proteins for serodiagnostic tests in a proteome scale.55,56 Several efforts have been made to identify novel antigenic determinants or immunodominant polypeptides of M. tuberculosis through genomics and proteomics,57-61 comparative genomics,62,63 or bioinformatics.64 Unfortunately, none of them can cover the whole genome or their intact open reading frames (ORFs). These analyses have the following limitations. (i) The ORFs in a shotgun library of the M. tuberculosis genome are usually not expressed intact. (ii) Conventional proteome analysis by 2D techniques and mass spectrometry, while highly effective, has limitations. In particular, they may miss many proteins of interest when expressed at low abundance. Low-abundance proteins are often of the greatest diagnostic interest,65-67 so these techniques may not be suitable for diagnostic applications. In addition, these methods are expensive, require highly trained personnel, and have considerable uncertainty regarding the purity and quality of the sample preparations, which are crucial in the analysis of the membrane proteome.68 The identification and characterization of M. tuberculosis membrane proteins through 2D techniques and mass spectrometry have proven to be difficult.69 (iii) Bioinformatic analysis is based on the comprehensive mining of existing data, and the identified proteins are always well-known antigens that have been previously reported. Systematic cloning and screening of antigens for reactivity with sera collected from patients represent a logical, alternative approach for the comprehensive identification of novel, potentially protective antigens.70-72 A recombination-based approach has been successfully applied (see Figure 1) in the construction of Treponema pallidum and Shewanella oneidensis
Figure 1. An outline of the protocol used for the systematic identification of M. tuberculosis antigenic proteins. Three major steps: (i) cloning, expression, and purification of M. tuberculosis proteins; (ii) high-throughput screening of M. tuberculosis antigenic proteins; and (iii) evaluation of antigenic proteins for the serodiagnosis of tuberculosis.
MR-1 clone sets in a proteome-scale.70,73 Using traditional molecular cloning techniques, 3480 (∼87%) M. tuberculosis ORFs were successfully cloned into IPTG-inducible expression vectors in the correct orientation and reading frames. Then, the clone sets were expressed in Escherichia coli, purified under denatured conditions, and tested for reactivity using a mixture of sera collected from 15 TB patients. A total of 249 proteins had significant reactions with the serum sample. Among these 249 proteins, 20 proteins were identified as most reactive. Compared with two commercial test kits, 3 of the top 20 proteins (Rv1987, Rv3807c, and Rv3887c), identified as novel antigens, offered better sensitivity and accuracy. These novel antigenic proteins may be used as candidates for serodiagnostic tests and vaccine development. Further investigation into these antigenic proteins may also provide critical information about pathogen-host interactions.
Materials and Methods Bacterial Strains, Plasmids, Enzymes, and Chemicals. Restriction enzymes, T4 ligase, and modification enzymes were Journal of Proteome Research • Vol. 9, No. 9, 2010 4813
research articles from TaKaRa Biotech. DNA polymerase and deoxynucleoside triphosphates (dNTPs) were purchased from TaKaRa Biotech, whereas DNA purification kits were purchased from Watson Biotechnologies. All antibiotics were from TaKaRa Biotech. NiNTA (Ni2+-nitrilotriacetate) agarose columns were obtained from Qiagen. E. coli BL21 (DE3) cells, purchased from Novagen, were used as the host strain to express M. tuberculosis proteins. Vectors pBT and pTRG and E. coli host strains XR were purchased from Stratagene. pET28a was from Novagen. PCR Reactions. All primers were synthesized by Invitrogen. The total genome DNA of M. tuberculosis was used as the template DNA for PCR reactions. For majority of cloning and screening PCR reactions, the thermocycle program was 1 min at 96 °C, 1 min at 60 °C (58 or 62 °C for alternative), and 3 min at 72 °C for 30 cycles with a final 8 min 72 °C step. Protein Expression and Purification. For protein expression, E. coli BL21 (DE3) containing the pETEXba or pETNXba constructs was inoculated into 1 mL of LB media containing 25 µg/mL kanamycin. The cultures were grown with shaking at 37 °C for 3 h. Then, another 1 mL of LB containing 25 µg/ mL kanamycin and 1 mM IPTG was added to the culture. The cultures were grown for 6 h on a shaker at 37 °C. The cells were pelleted and stored at -80 °C before use. For protein purification, the cells were thawed for 15 min, resuspended in 1 mL of Buffer B (8 M urea, 0.1 M NaH2PO4, and 0.01 M TrisCl, pH 8.0), and incubated overnight at room temperature for cell lysis. The lysate was centrifuged at 13 000 rpm for 30 min at room temperature to collect the supernate. Then, 40 µL of per-equilibrated Ni sepharose (GE Healthcare) was mixed into the supernate. The mixture was incubated at room temperature for 2 h and centrifuged at 2000 rpm for 30 s at room temperature to collect the sepharose. The sepharose was washed twice with 1 mL of Buffer C (8 M urea, 0.1 M NaH2PO4,, and 0.01 M Tris-Cl, pH 6.3) and then with 40 µL of Buffer E (8 M urea, 0.1 M NaH2PO4,, and 0.01 M Tris-Cl, pH 4.5) to elute the His-tagged proteins. The purified proteins were stored at -80 °C until use. Serum Samples and Patients. A total of 120 serum samples from different individuals (n ) 120) was studied. The serum samples of TB patients (n ) 96) were collected from Wuhan Medical Treatment Center (Hubei, PR China), whereas those of healthy individuals (n ) 24) were obtained from the Huazhong Agricultural University Hospital. All healthy individuals did not previously suffer from TB and had negative chest X-rays. Serum Preparation and Absorption of anti-E. coli Protein Antibodies. Sera from patients and healthy individuals were collected in the hospital and stored at -80 °C until use. The serum samples were incubated with E. coli cell lysate to remove any nonspecific reactivity. E. coli BL21 (DE3) was grown overnight at 37 °C in 150 mL of LB liquid media. The cells were pelleted and freeze-thawed twice at -80 °C. The cell pellet was resuspended in 5 mL of TE buffer (pH 8.0) and subjected to cell lysis via a French press. The lysate was centrifuged at 13 000 rpm for 10 min at 4 °C. A mixture of 45 µL of serum, 405 µL of PBS (pH 7.4), and 250 µL of BL21 (DE3) cell lysate was rocked for 5 h at room temperature. Absorbed serum was stored at -20 °C until use. Indirect ELISA Protocol. Purified proteins were diluted to a final concentration of 500 ng/mL in coating buffer (0.05 M Na2CO3-NaHCO3, pH 9.6), and 0.1 mL of each protein dilution was added to each well of a 96-well plate. Plates were covered with an adhesive plastic and incubated overnight at 4 °C. Plates 4814
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Li et al. were washed thrice with 0.2 mL of PBS-T (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4,, and 0.05% Tween20) and then blocked in 0.2 mL of PBS-T and 5% dry milk for 1 h at room temperature. Plate wells were washed three times with 0.2 mL of PBS-T. Prepared absorbed serum was diluted to a final dilution of 1:400 in PBS-T, and 0.2 mL of serum dilution was added to each well for 3 h at room temperature. Then, plate wells were washed thrice with 0.2 mL of PBS-T. A 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (H + L) (Thermo) in PBS-T was added to each plate well for 1 h at room temperature. Each plate well was washed five times before 0.1 mL of TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate solution was added for 20 min at room temperature. Light emission from each plate well was read by a microplate reader (Beckman Coulter, DTX 880) at 450 nm after 0.1 mL of stop solution (2N H2SO4) was added. Each experiment was repeated thrice. The resulting measurements were used to generate a mean for each sample. Commercial Testing Kits. Two commercial testing kits, TBDOT (Upper Biotech, Shanghai, PR China) and TB-CHECK-1 (Veda.Lab, France), both of which have been widely used for clinical diagnosis and screening of TB in PR China, were used to evaluate our ELISA system for serodiagnosis of TB by using the same serum samples. The serum samples’ processing and antibody assays were performed according to the manufacturer’s instruction. Statistical Analysis. For statistical analysis, the mean OD value and standard deviation (SD) were calculated using the Statistics Package for Social Science 13.0 (SPSS, Inc., Chicago, IL). The significant levels of the fusion proteins from each plate were judged using Student’s t-test. The cutoff value was calculated from the mean OD plus two SD from the healthy control group. Sensitivity was defined as the percentage of individuals in the true-positive group that showed OD values higher than the cutoff value. Specificity was defined as the percentage of individuals in the true-negative group that had showed ELISA values lower than the cutoff value. The consistency rate of the test accuracy of two commercial testing kits and the top 20 proteins with the sputum culture results was calculated according to their consistency rate with the 29 positive results obtained by sputum culture.
Results and Discussion Vector Construction for Protein Expressions. Two vectors, pETEXba and pETNXba, were constructed as shown in Figure 2A for protein expression and purification. The vector pETEXba was constructed as previously described.74 For the construction of pETNXba, the multiple cloning sites of pETEXba were replaced by a cassette containing NotI, XbaI, and XhoI (Figure 2A), suitable for cloning M. tuberculosis ORFs (see below). Both pETEXba and pETNXba vectors are derived from pET28a (Novagen), resistant to kanamycin, and contain a 6× His affinity tag for the purification of recombinant proteins. Their expression is driven by the powerful T7 promoter and can be integrated with the high processivity and activity of T7 RNA polymerase in E. coli. ORF Amplification and Cloning. For the purpose of cloning in the correct orientation, different restriction enzyme cutting sites should be added during PCR amplification at the 5′ and 3′ ends for each ORF-specific forward and reverse primer, respectively (Figure 2B). The forward ORF-specific primers begin with EcoRI, NotI, or BamHI according to the sequence of each ORF, whereas the reverse ORF-specific primers begin
Identification of Novel Antigenic Proteins in M. tuberculosis
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Figure 2. Vector construction and primer design. (A) Map of expression vectors (pETEXba and pETNXba). These vectors, which were both pET28a-derived vectors, included a pBR322 origin, kanamycin resistance gene, T7 promoter, and a cassette containing the corresponding cloning sites. (B) A schematic diagram of the recombinant vectors.
with XbaI or XhoI and a stop codon. Using this design, a 6-His tag sequence in the vector was fused to each ORF encoded sequence at the N-termini of the proteins. For the generation of the M. tuberculosis clone sets, 3989 unique pairs of primers were designed, and 3896 ORFs of the right size were successfully obtained, indicating a 97.7% success rate. For constructing an M. tuberculosis protein expression library, the PCR products were then mixed into several groups (∼200 products per group) based on the size and restriction enzyme sites for each ORF (Figure 3B). The mixture was digested with the corresponding restriction enzymes and ligated into the expression vector. The ligation products were subsequently used to transform chemically competent BL21 (DE3) E. coli cells. Initially, clones on LB plates containing kanamycin were selected and sequenced. The recombinant ORF genes were confirmed by resequencing. In total, 3480 ORFs were successfully inserted into the pET expression vector series, representing approximately 87% the M. tuberculosis ORFs. Expression and Purification of M. tuberculosis Proteins. The expression of M. tuberculosis proteins in E. coli BL21 (DE3) was induced by IPTG. Many M. tuberculosis proteins have been shown to form inclusion bodies when they are expressed in E. coli,75-77 making the purification of these proteins under native conditions difficult. To avoid this problem, the M. tuberculosis proteins were purified under denaturing conditions using 8 M urea, because the interaction between Ni-NTA and His-tag of the recombinant protein does not depend on a tertiary structure. To ensure the purity of the M. tuberculosis proteins, the elution of tagged proteins was achieved by reducing the pH of the buffers. The proteins were solubilized in 8 M urea, and the His-tags were fully exposed under denaturing conditions, which led to more efficient purification and increased yields. The purified proteins were further analyzed by SDSPAGE as shown in Figure 4.
Figure 3. PCR amplification and cloning of M. tuberculosis ORFs. (A) PCR amplification of M. tuberculosis ORFs and the PCR products were separated by agarose gel electrophoresis. The M. tuberculosis ORF number is indicated at the top of the figure. (B) The PCR products were mixed into several groups based on the size and restriction enzyme sites of each ORF. The grouped PCR products were separated by agarose gel electrophoresis.
Identification of Antigenic Proteins. The reactions between purified M. tuberculosis proteins and human serum were measured by indirect ELISA as described in Materials and Methods. Of the 3480 proteins examined for reactivity with the pooled TB patient’s serum, 249 proteins showed significant reaction levels using Student’s t-test (P < 0.05) and were considered significantly antigenic. Journal of Proteome Research • Vol. 9, No. 9, 2010 4815
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Figure 4. Expression and purification of M. tuberculosis proteins. (A) Expression of M. tuberculosis proteins in E. coli BL21 (DE3). All samples were loaded onto 13.5% SDS-PAGE gel for assays. The gel was Coomassie stained. The samples are indicated at the top of the figure. Bands of the correct size are indicated by a dot on the right side of the band. (B) Purification of M. tuberculosis proteins under denaturing condition. All samples were loaded onto 13.5% SDS-PAGE gel for assays as indicated previously. The gel was Coomassie stained. The samples are indicated at the top of the figure. Bands of the correct size are indicated by a dot on the right side of the band.
Figure 5. Classification of the antigenic proteins identified according to their annotations. (A) Classification of a total of 249 antigenic proteins. (B) Classification of the top 20 antigenic proteins.
The 249 antigenic proteins identified in the present study can be classified into eight groups based on their NCBI annotations (Figure 5A): (i) Membrane/transmembrane protein, (ii) Secreted protein, (iii) Lipid transport and metabolism, (iv) 4816
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PE/PPE family protein, (v) Regulatory protein, (vi) Hypothetical protein, (vii) Cell wall/membrane biogenesis, and (viii) Others. The list of 249 antigenic proteins included some well-known antigens, like TB8.4 (Rv1174c, a low molecular weight T-cell
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Identification of Novel Antigenic Proteins in M. tuberculosis Table 1. PE/PPE Genes Identified in the Study
Table 2. RD Genes Identified in the Study
Rv no.
gene
annotation
Rv no.
RD
gene
annotation
Rv0159c Rv1169c Rv0109 Rv0124 Rv0532 Rv1468c Rv1803c Rv2741 Rv3812 Rv1039c Rv1705c Rv3136
PE3 PE11 PE_PGRS1 PE_PGRS2 PE_PGRS6 PE_PGRS29 PE_PGRS32 PE_PGRS47 PE_PGRS62 PPE15 PPE22 PPE51
PE family protein PE family protein PE_PGRS family protein PE_PGRS family protein PE_PGRS family protein PE_PGRS family protein PE_PGRS family protein PE_PGRS family protein PE_PGRS family protein PPE family protein PPE family protein PPE family protein
Rv1512
RD6
EpiA
Rv1970
RD15
LprM
Rv1973
RD15
-
Rv1987 Rv2073c
RD2 RD12
-
Rv2074 Rv2075c
RD12 RD12
-
Rv2647 Rv2652c
RD13 RD13
-
Rv3619c
RD9
EsxV
probable nucleotide-sugar epimerase EpiA possible mce-family lipoprotein LprM (mce-family lipoprotein Mce3E) possible conserved mce associated membrane protein possible Chitinase probable shortchain dehydrogenase hypothetical protein possible hypothetical exported or envelope protein hypothetical protein probable phiRv2 prophage protein putative ESAT-6 like protein EsxV (ESAT-6 like protein 1)
antigen), EsxV (Rv3619c, a putative ESAT-6 like protein), MTC28 (Rv0040c, a proline rich 28 KDa antigen), MTB32B (Rv0983, a putative secreted serine protease antigen of M. tuberculosis78), IniB (Rv0341, a isoniazid inductible protein IniB), and FbpB and FbpC (Rv1886c and Rv0129c, also named Ag85B and Ag85C, the antigen 85 complex B and C, recently identified as plasminogen-binding proteins79). Four DosR regulon-encoded antigens (Rv0569, Rv0573c, Rv2030c and Rv3129) in the present study were shown to be antigenic.29 There are also 12 PE/PPE family proteins (Table 1), some of which are cell surface antigens associated with virulence and host immune response in several mycobacterial species and are considered as vaccine and diagnostic candidates.26,80-84 The concordance of the results with previous studies indicates the reliability of the methods used. Several studies have been carried out to define the membrane and secreted proteome of M. tuberculosis,85-88 considered as immunodominant antigens,60,69 and that can be tested as vaccine candidates.61 Among the 249 antigenic proteins identified, there are 26 secretory and 39 cell membrane associated proteins (Table S1), some of which were identified in earlier studies as antigenic.60,69 Moreover, there are several previously undefined secretory and membrane associated proteins which were shown to be antigenic (Table S1). All of these newly identified proteins can be tested as potential subunit vaccine candidates. Meanwhile, 10 RD proteins were identified in our study (Rv1512, Rv1970, Rv1973, Rv1987, Rv2073c, Rv2074, Rv2075c, Rv2647, Rv2652c, and Rv3619c) (Table 2). These RD proteins, absent in BCG, are considered attractive for the construction of a recombinant vaccine by reintroducing them to BCG or live attenuated vaccine by knock out or knock down of their coding genes in M. tuberculosis.33-40 These proteins are valuable candidates and should have the greatest potential in the development of new vaccines and diagnostic tests for tuberculosis. Further investigation into these antigenic proteins is necessary to determine their potential protective activity. Several regulatory proteins and metabolic enzymes were also identified as antigenic proteins. The presence of intracellular metabolic enzymes has been previously reported in the culture filtrate or extracellular milieu of M. tuberculosis and various pathogens.89 Possibly, they are secreted through specialized systems.90 Several extracellular metabolic enzymes of M. tuberculosis have been identified as virulence factors.79,91 A secreted transcription factor controls M. tuberculosis virulence and is itself secreted from the bacterial cell by the ESX-1 system.92 M. tuberculosis MycP1 protease, identified as an antigen, plays a dual role in the regulation of ESX-1 secretion
and virulence.93 Bacteria have no nuclear membrane, so the membrane association of these proteins is possible. However, extracellular localization of these proteins may also be due to autolysis during infection.61 A total of 8 ribosomal proteins was identified. A large number of ribosomal proteins in pathogens can associate with the cell membrane and demonstrate strong immunogenicity.88,94 Mycobacterial ribosomal proteins, implicated in the delayed-type skin hypersensitivity reaction (DTH) elicited by PPD,95 have also been used as an experimental vaccine against leprosy.96 This is consistent with both the specificity for mycobacteria and the high abundance of these proteins.69 These 249 proteins were selected for further analysis of reactivity with serum from patients (five samples) and healthy persons (three samples) to evaluate their potential application in the serodiagnosis of tuberculosis. This led to the identification of the 20 most reactive proteins (Table 3) which can clearly distinguish between patients and healthy individuals (based on the difference between the mean OD value of patients’ samples and the mean OD value of healthy samples). Among the top 20 antigenic proteins, the UniProt database shows that 6 proteins were localized to the cell membrane, and another 4 proteins were predicted to be membrane proteins by SOSUI.97 Diagnostic Assessment of the Top 20 Potential Antigens. The clinical diagnostic potential of the top 20 antigenic proteins was further evaluated. The sensitivity, specificity, and accuracy of these antigenic proteins were compared with those of the TB-DOT and TB-CHECK-1 tests, that are both commercially available (Figure 6). Sera from 96 patients and 24 healthy individuals were examined (Table S2). As shown in Table 3, the test sensitivity of the TB-DOT kit was 65.63%, and that of TB-CHECK-1 kit was only 42.71%; the specificity of the two kits was 66.67% and 91.67%, respectively. Similar results were obtained from another research group.18 The test results for the top 20 proteins are also shown in Table 1. The specificity of the proteins was all above 90% (91.67-100%), whereas their sensitivity ranged from 13.54 to 73.96%. Compared with the two commercial kits, three proteins (Rv1987, Rv3807c, and Rv3887c) showed better accuracy in the diagnosis of tuberculosis (Table 3). Rv1987, located in the RD2 region, is a possible Journal of Proteome Research • Vol. 9, No. 9, 2010 4817
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Table 3. Top 20 Antigenic Proteins Identified and Their Sensitivity, Specificity, and Accuracy Compared with Two Commercial Kits Rv no.
sensitivity
specificity
accuracy
gene
annotation
Rv0138 Rv1005c Rv1039c Rv1200 Rv1826 Rv1833c Rv1903 Rv1970 Rv1987 Rv1989c Rv2011c Rv2180c Rv2200c
35.42% 38.54% 33.33% 33.33% 21.88% 14.58% 27.08% 48.96% 67.71% 26.04% 36.46% 39.58% 56.25%
91.67% 91.67% 95.83% 100.00% 95.83% 100.00% 100.00% 95.83% 100.00% 95.83% 100.00% 95.83% 95.83%
46.67% 49.17% 45.83% 46.67% 36.67% 31.67% 41.67% 58.33% 74.17% 40.00% 49.17% 50.83% 64.17%
pabB PPE15 gcvH lprM ctaC
Rv2693c
39.58%
95.83%
50.83%
-
Rv2866 Rv3198A Rv3261 Rv3794
26.04% 57.29% 20.83% 13.54%
95.83% 100.00% 95.83% 91.67%
40.00% 65.83% 35.83% 29.17%
fbiA embA
Rv3807c Rv3887c Multiple-antigen TB-DOT TB-CHECK-1
73.96% 71.88% 75.00% 65.63% 42.71%
100.00% 95.83% 100.00% 66.67% 91.67%
79.17% 76.67% 80.00% 65.83% 52.50%
-
hypothetical protein para-aminobenzoate synthase component I PPE family protein probable conserved integral membrane transport protein glycine cleavage system protein H haloalkane dehalogenase probable conserved membrane protein possible mce-family lipoprotein LprM possible chitinase hypothetical protein hypothetical protein probable conserved integral membrane protein probable transmembrane cytochrome c oxidase (subunit II) CtaC probable conserved integral membrane alanine and leucine rich protein hypothetical protein possible glutaredoxin protein probable F420 biosynthesis protein FbiA integral membrane indolylacetylinositol arabinosyltransferase EmbA possible conserved transmembrane protein probable conserved transmembrane protein compose of Rv1987, Rv3807c and Rv3887c
membrane-anchored chitinase with 1 transmembrane helix as predicted by SOSUI.97 Rv3807c and Rv3887c are both transmembrane proteins as annotated in the UniProt database. These three proteins may exert unique functions compared with the homologue species, making them ideal serodiagnostic candidates. Further studies are essential for determining the specific role of these proteins in the pathogenesis of TB. The difference in accuracy and consistency rate of the top 20 proteins from those of the commercial kits may be due to the different antigenic proteins used. A multiple-antigen ELISA system can be superior to a single antigen-based assay.18,20Therefore, the accuracy of a multipleantigen test, a mixture of Rv1987, Rv3807c, and Rv3887c, was compared to that of the commercial kits (TB-DOT and TBCHECK-1). As shown in Table 3, the multiple-antigen test provided the highest accuracy (80.00%). From 96 patients in Wuhan Medical Treatment Center (Hubei, PR China), 68 sputum cultures were obtained (Table S3), 29 of which were positive (a low sensitivity of 42.65%, Table
S3). The sputum culture results are a direct evidence of TB, so we further compared the test accuracy of the two commercial testing kits and top 20 proteins with that of the sputum culture. As shown in Figure S1, the TB-DOT testing kit had the highest consistency rate (93.10%), whereas those of the TB-CHECK-1 testing kit, three single antigen tests (Rv1987, Rv3807c and Rv3887c), and the multiple-antigen test ranged from 65.52 to 72.41%. Given the high false negative rate of the sputum culture, the consistency rate only reflects the accuracy of detection in a limited number of TB patients. When comparing the sensitivity and specificity in the diagnosis of 96 TB patients, the multiple-antigen test performed better (Table 3). Therefore, multiple-antigen testing has potential in the serodiagnosis of TB. There is an acknowledged need for other highly sensitive, specific, and accessible high-throughput experimental techniques for protein detection analysis. In the current study, the following improvements were made: (i) Approximately 87% of the full length ORFs of M. tuberculosis were cloned in the
Figure 6. Test accuracy of the top 20 antigenic proteins and two commercial testing kits. 4818
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Identification of Novel Antigenic Proteins in M. tuberculosis correct orientation and correct reading frame, ensuring the integrity of expression of M. tuberculosis genes. (ii) The expression of M. tuberculosis genes was driven by the T7 promoter, enabling the detection of the “low-abundance” proteins. (iii) To obtain more efficient purification and increased yields, the proteins were all purified under denatured conditions; this also enabled the isolation of transmembrane proteins. (iv) The antigenicity of M. tuberculosis proteins was tested by ELISA, which has been used to identify antigens in T. pallidum systematically and is a proven simple, sensitive, and high-throughput assay. This is the first study on the systematic cloning and screening of antigens of an intractable pathogen which have a large size, high GC content of genome, and large number of ORFs. Some well-established antigens were not identified in this study. However, note that Rv3619c-esxV, an ESAT-6 homologue, was identified as a novel antigen. This may due to three reasons: (i) 13% of the open reading frames of M. tuberculosis were not successfully cloned. This may be related to the size of the PCR products. (ii) Some M. tuberculosis proteins were not successfully expressed. One possibility is that these M. tuberculosis proteins are toxic when expressed in E. coli. In addition, some M. tuberculosis proteins require their bindingpartner proteins for folding and stability. Expression of these proteins alone in E. coli has been shown to be unsuccessful.69 (iii) ELISA was performed with denatured proteins. As the noncovalent bonds in the proteins were disrupted by urea, the three-dimensional surface epitopes may be affected, making the detection of these epitopes difficult. However, linear epitopes which are determined by the amino acid sequence (the primary structure) rather than the 3D shape (tertiary structure) of a protein were not affected by urea. Within a pathogen-host system, protein-protein interactions (PPIs) between surface or secretory proteins form the foundation for communication between the host immune system and the pathogen, therefore, playing a vital role in initiating infection.98 To better understand how pathogens infect their hosts, there is a need to identify the proteins important for attachment and invasion of human cells, as well as the potential targets for therapeutics. In this study, a possible serine/threonine phosphatase (PPP, Rv0018c) was identified. Interestingly, many prokaryotic serine/threonine kinases and the phosphatases appear to be involved in host-pathogen interaction.99,100 Experiments are underway to explore the potential interactions between M. tuberculosis antigenic proteins and human proteins.
Conclusion The potential antigens of M. tuberculosis were systematically cloned and screened in a proteome scale. A total of 249 novel antigenic proteins was identified, which may be used as candidates for serodiagnostic application and vaccine development. In particular, three membrane proteins (Rv1987, Rv3807c, and Rv3887c) and a multiple-antigen (mixture of Rv1987, Rv3807c, and Rv3887c) were clearly effective in the serodiagnosis of tuberculosis. These results are expected to serve as an essential reference for further developing new TB diagnosis methods and more effective vaccines for combating tuberculosis. Abbreviations: BCG, Bacille Calmette-Guerin; DTH, delayedtype hypersensitivity; ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; IPTG, isopropyl β-D-1-thiogalactopyranoside; LC, liquid chromatography; LPE, liquid phase electrophoresis; MHC, major histocompatibility
complex; MS, mass spectrometry; PCR, polymerase chain reaction; PPD, purified protein derivative; PPIs, protein-protein interactions; SPR, surface plasmon resonance;TB, tuberculosis; TST, tuberculin skin testing.
Acknowledgment. We thank Dr. Jie Xiang (Wuhan Medical Treatment center, Wuhan, China) for providing serum samples. This work was supported by 973 Program (2006CB504402), the National Natural Science Foundation of China (30930003) and the National Special Key Project of China on Major Infectious Diseases (2008ZX10003-005). Supporting Information Available: Consistency rates of the test accuracy of two commercial testing kits and the top 20 proteins with the sputum culture results; tables of 249 antigenic proteins identified in this study; the diagnostic test results of the top 20 antigenic proteins and two commercial testing kits; and analysis of the diagnostic test results of the top 20 antigenic proteins and two commercial testing kits. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) World Health Organization. WHO Report 2007: Global Tuberculosis Control, Surveillance, Planning, Financing; Geneva: WHO, 2007; p 277. (2) Dolin, P. J.; Raviglione, M. C.; Kochi, A. Global tuberculosis incidence and mortality during 1990-2000. Bull. W. H. O. 1994, 72, 213–220. (3) Andersen, P. Vaccine strategies against latent tuberculosis infection. Trends Microbiol. 2007, 15, 7–13. (4) Johnson, R.; Streicher, E. M.; Louw, G. E.; Warren, R. M.; van Helden, P. D.; Victor, T. C. Drug resistance in Mycobacterium tuberculosis. Curr. Issues Mol. Biol. 2006, 8, 97–112. (5) Aziz, M. A.; Wright, A.; Laszlo, A.; De Muynck, A.; Portaels, F.; Van Deun, A.; Wells, C.; Nunn, P.; Blanc, L.; Raviglione, M. Epidemiology of antituberculosis drug resistance (the Global Project on Anti-tuberculosis Drug Resistance Surveillance): an updated analysis. Lancet 2006, 368, 2142–2154. (6) Kaufmann, S. H.; McMichael, A. J. Annulling a dangerous liaison: vaccination strategies against AIDS and tuberculosis. Nat. Med. 2005, 11, S33–44. (7) Abebe, F.; Holm-Hansen, C.; Wiker, H. G.; Bjune, G. Progress in serodiagnosis of Mycobacterium tuberculosis infection. Scand. J. Immunol. 2007, 66, 176–191. (8) Harboe, M. Antigens of PPD, old tuberculin, and autoclaved Mycobacterium bovis BCG studied by crossed immunoelectrophoresis. Am. Rev. Respir. Dis. 1981, 124, 80–87. (9) Ling, D. I.; Flores, L. L.; Riley, L. W.; Pai, M. Commercial nucleicacid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression. PLoS One 2008, 3, e1536. (10) Menzies, D.; Pai, M.; Comstock, G. Meta-analysis: new tests for the diagnosis of latent tuberculosis infection: areas of uncertainty and recommendations for research. Ann. Intern. Med. 2007, 146, 340–354. (11) Pai, M.; Zwerling, A.; Menzies, D. Systematic review: T-cell-based assays for the diagnosis of latent tuberculosis infection: an update. Ann. Intern. Med. 2008, 149, 177–184. (12) Nienhaus, A.; Schablon, A.; Diel, R. Interferon-gamma release assay for the diagnosis of latent TB infection - analysis of discordant results, when compared to the tuberculin skin test. PLoS One 2008, 3, e2665. (13) Leyten, E. M. S.; Mulder, B.; Prins, C.; Weldingh, K.; Andersen, P.; Ottenhoff, T. H. M.; van Dissel, J. T.; Arend, S. M. Use of enzyme-linked immunospot assay with Mycobacterium tuberculosissspecific peptides for diagnosis of recent infection with M. tuberculosis after accidental laboratory exposure. J. Clin. Microbiol. 2006, 44, 1197–1201. (14) Smith, K. C.; Starke, J. R.; Eisenach, K.; Ong, L. T.; Denby, M. Detection of Mycobacterium tuberculosis in clinical specimens from children using a polymerase chain reaction. Pediatrics 1996, 97, 155–160. (15) Barth, R. E.; Mudrikova, T.; Hoepelman, A. I. Interferon-gamma release assays (IGRAs) in high-endemic settings: could they play
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