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1 Fundación Instituto de Inmunología de Colombia, Bogotá 020304, Colombia
2 CORPOICA-CEISA, Bogotá 020304, Colombia
3 Universidad Nacional de Colombia, Bogotá 020304, Colombia
Reprint requests to: Álvaro Puentes, Fundación Instituto de Inmunología de Colombia, Carrera 50 No. 26-00, Bogotá 020304, Colombia; e-mail: puentesalvaro{at}hotmail.com; fax: 571-4815269.
(RECEIVED June 1, 2005; FINAL REVISION August 6, 2005; ACCEPTED August 15, 2005)
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Abstract |
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-helical structure. HABP–target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus–target cell interactions and implications for developing strategies for controlling this disease. Keywords: Mycobacterium tuberculosis; Rv2004c protein; high activity binding peptides; U937 cells; A549 cells
Abbreviations: HABP, high activity binding peptide • 125I-HABP, 125- iodine radiolabeled HABP • PBS, phosphate buffer saline • CD, circular dichroism • TMC, Trudeau Mycobacterial Collection • RP-HPLC, reverse-phase, high-performance liquid chromatography • BS3, bis (sulfosuccinimidyl suberate) • ATCC, American Tissue Culture Collection.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051592505.
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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Mycobacteria are intracellular microorganisms surviving and growing into host macrophages. Following phagocytosis, sustained intracellular bacterial growth depends on their ability to avoid destruction by macrophage-mediated host defenses such as lysosomal enzymes and reactive oxygen and reactive nitrogen intermediates. This suggests that host cell interaction with microbes is delicately balanced and can be tipped in favor of either organism (Mariani et al. 2000).
The immune response against Mycobacterium tuberculosis has been evaluated in human and animal models; disease pathogenesis and protective immunity development have also been studied (Andersen 1994; Andersen and Brennan 1994). It has been established that cell immunity is critical for inducing protection against tuberculosis. Defining which M. tuberculosis antigens can elicit effective immunity and, taking into account that host–pathogen molecular interactions are critical in the infection process, identifying M. tuberculosis genes expressed on the bacterial surface would greatly contribute toward developing new strategies for fighting tuberculosis (Mariani et al. 2000). Mawuenyega et al. (2005) have recently reported the identification of 1044 proteins and their corresponding subcellular localization by using a combination of high-throughput proteomics and computational approaches for elucidating those proteins being expressed in each one of the three M. tuberculosis subcellular compartments; among these proteins, the presence of Rv2004c was detected.
In the present study, the M. tuberculosis Rv2004c gene (synonyms: MT2060, Mb2027c, MTCY39.13; GenBank accession no. Q10852) has been analyzed in the M. tuberculosis complex, as has its transcription and encoded protein expression. This protein’s potential role in the mycobacterium–host cell interaction by using synthetic peptides has also been determined. In all, 25 nonoverlapping, 20-residue-long peptides spanning the complete Rv2004c protein sequence were chemically synthesized; rabbit sera raised against two of these peptides in polymerized form (Rv2004c-7, 121DAIAEVLARFHQRAQRNRCIY140; and Rv2004c-19, 361RDCGVITGEPGVLDSGLYSR380) were used for Western blot and immunoelectron microscopy studies. Their specific U937 monocyte and A549 epithelial cell line binding capacity was also determined. Five high activity binding peptides (HABPs) were identified in the Rv2004c protein as specifically interacting with U937 cells’ surface molecules. The U937-HABPs were Rv2004c-6, Rv2004c-14, Rv2004c-15, Rv2004c-16, and Rv2004c-18; while a further six HABPs (Rv2004c-5, Rv2004c-6, Rv2004c-14, Rv2004c-15, Rv2004c-18, and Rv2004c-20) were specifically identified as interacting with A549 cells’ surface molecules. Circular dichroism suggested that all HABPs display a stable -helical structure.
The present work’s most important findings concern the presence of the Rv2004c gene in all M. tuberculosis complex strains and clinical isolates, its transcription in several M. tuberculosis complex strains, and the encoded protein’s expression, its surface location on the bacilli, and its U937 and A549 cell-binding capacity. This has an important biological significance and implications for developing strategies for controlling this disease.
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Results |
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). DNA sequencing established that the Rv2004c protein was conserved in all mycobacterial strains and isolates tested (data not shown).
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). The constitutive gene (rpoB, 360 bp) used as positive control showed to be equally transcribed in all the M. tuberculosis complex’s strains (Fig. 1D).
Recognizing the Mycobacterium tuberculosis-H37Rv Rv2004c protein by polyclonal antibodies
The Rv2004c-7-synthetic peptide polymer was inoculated into three rabbits, as described in Materials and Methods. The immunoblot showed that rabbit 214’s sera specifically recognized a 54-kDa band (Fig. 2, lanes 4,5,6) in the three post-immune bleedings. No protein was recognized when pre-immune serum was being tested (Fig. 2, lane 3). The other two rabbit sera from this group showed very weak recognition of the same band (data not shown). One of the three rabbits inoculated with Rv2004c-19-synthetic peptide polymer specifically recognized a 27-kDa band (Fig. 2, lane 2). Again, no protein was recognized when pre-immune serum was being tested (Fig. 2, lane 1).
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shows the presence of colloidal gold particles, mainly on bacilli cellular surface (indicated by arrows). On the contrary, no signals were observed when pre-immune sera were used (data not shown).
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shows this serum recognizing a 54-kDa band when tested against the cell wall fraction (lane 1); no recognition against either culture supernatant (lane 2) or membrane fraction (data no shown) was observed.
Identifying high activity binding peptides (HABPs)
Binding assays were performed as described in Materials and Methods to determine which Rv2004c amino acid sequences could interact with human target cells susceptible to M. tuberculosis H37Rv invasion. The U937 monocytoblastic and A549 epithelial cell lines were used in independent experiments. 125I-peptides (100–2000 nM) were incubated with both U937 and A549 cells in the presence (nonspecific binding) or absence (total binding) of the same nonradiolabeled (cold) peptide (Fig. 4). Total binding minus nonspecific binding gave the specific binding (Yamamura et al. 1978; Rodriguez et al. 2000).
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2% was called a high activity binding peptide (HABP) (Vera-Bravo et al. 2005). Based on this methodology, five HABPs were identified in the Rv2004c protein as specifically interacting with U937 cell surface molecules (Fig. 5). Peptide Rv2004c-6 (101RDKQRLASMVTAGLPVEGAL120) was located in the N-terminal region, while peptides Rv2004c-14 (261AGYAVRSGDTAPASLRDFYI280), Rv2004c-15 (281AYRAVVRAKVECVRFSQGKP300), Rv2004c-16 (301EAAADAVRHLIIATQHLQHA320), and Rv2004c-18 (341GVAELVGAQVISTDDVRRRL360) were located in the central region, presenting high affinity binding and specificity. Peptides Rv2004c-14 and Rv2004c-15 were identified as having the highest affinity and specific binding. Six HABPs from the Rv2004c protein were also identified, specifically recognizing A549 epithelial cell surface molecules (Fig. 5). Peptide Rv2004c-5 (81AHLSDPSGGHAEPVVVMRRY100) and peptide Rv2004c-6 were located in the N-terminal region, while four peptides were localized in the protein’s central region (peptides Rv2004c-14, Rv2004c-15, Rv2004c-18, and Rv2004c-20; 381ANVVAVYQEALRKARLLLGS400). No specific binding activity was found in the Rv2004c protein C-terminal region with either of these cell lines.
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and Table 1 show the saturation assay results for each selected HABP when using a higher 125I-peptide concentration than in the screening binding assay. Scatchard and Hill analyses allowed affinity constant (Kd) calculations to be done for each Rv2004c protein HABP. All U937-HABPs presented affinity constants in the 480 and 950 nM range, and A549-HABPs presented affinity constants in the 330 and 700 nM range, indicating high affinity in peptide–cell interaction. Hill coefficients (nH) for each U937-HABP and A549-HABP were also calculated and displayed positive cooperativity (nH > 1). The number of binding sites per cell (NrSc) calculated for these U937-HABPs and A549-HABPs was 10 x 106 and 3 x 106, respectively. These values were calculated from the highest bound peptide value on the saturation curve. (Hulme 1993; Puentes et al. 2005).
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shows those critical amino acids for the interaction between the five Rv2004c protein HABPs and the U937 cells. The critical residues for each HABP are underlined, as follows: Rv2004c-6 (101RDKQRLASMVTAGLPVEGAL120), Rv2004c-14 (261AGYAVRSGDTAPASLRDFYI280), Rv2004c-15 (281AYRAVVRAKVECVRFSQGKP300), Rv2004c-16 (301EAAADAVRHLIIATQHLQHA320), and Rv2004c-18 (341GVAELVGAQVISTDDVRRRL360); these residues were located along the sequences for peptides Rv2004c-6, Rv2004c-14, Rv2004c-15, and Rv2004c-16 and for peptide Rv2004c-18 at the C terminus.
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HABP receptors
U937 and A549 cells, incubated earlier on with each 125I-HABP, were cross-linked, and membrane proteins were then separated by SDS-PAGE (Garcia et al. 2004; Valbuena et al. 2005). The HABPs from the Rv2004c protein bound to molecules located on U937 and A549 cell membranes were visualized by autoradiography using 125I-Rv2004c-6 and 125I-Rv2004c-14 peptides. Figure 8 shows 32- and 49-kDa bands for U937 cells (lanes 1 and 3) and five bands ranging from 40 to 97 kDa for A549 cells (lanes 5 and 7). These bands disappeared or their intensity became reduced when the binding assay was performed in the presence of excess unlabeled peptide (Fig. 8, lanes 2,4,6,8). The same assay was performed with RBCs as negative control, in which no signal appeared (data not shown).
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-helix as characterized by double minima at 208 and 220 nm and 190 nm maximum ellipticity. Figure 9 shows CD profiles for Rv2004c-6, Rv2004c-16, and Rv2004c-20 HABPs.
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Discussion |
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This work describes one of these proteins (Rv2004c), classified in group VI as being a protein having an unknown function. The Rv2004c gene consists of 1494 bp encoding a 498 amino acid long polypeptide having a 54.42 kDa calculated molecular mass (Cole et al. 1998; Mawuenyega et al. 2005). The presence of the Rv2004c gene was initially revealed in M. tuberculosis complex strains (Fig. 1A, lanes 2–7) and in the 10 clinical isolates (Fig. 1B, lanes 11–20) by PCR assays, leading to the suggestion that this gene is M. tuberculosis complex exclusive. The deciphering of the genome has determined that M. tuberculosis is highly conserved and lacks interstrain genetic diversity (Cole et al. 1998). As shown in the present study, the Rv2004c gene shares this behavior, being completely conserved in all strains tested. However, recent studies using a variety of low- and high-resolution comparative genome techniques for identifying differences in the genomes of M. bovis, the M. bovis BCG vaccine strain, and the M. tuberculosis H37Rv laboratory strain and clinical isolates have identified sequence changes among the different mycobacterial species and strains (Fleischmann et al. 2002; Gao et al. 2005). This has led to suggesting that the Rv2004c gene could be localized in a highly conserved M. tuberculosis H37Rv genome region.
The RT-PCR assays demonstrated this gene’s differential transcription in all M. tuberculosis complex strains except in M. bovis and M. microti (Fig. 1C, lanes 4 and 7, respectively). The transcription in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG (Fig. 1C, lanes 2,3,5) was more evident than transcription in Mycobacterium africanum (Fig. 1C, lane 6). Although these differences in transcription might have functional relevance, we cannot rule out the possibility that they occur as a consequence of the culture growth conditions, as previous reports have shown gene expression to be extremely sensitive to this factor (Gao et al. 2005).
Bearing in mind that not all transcribed genes are expressed, rabbit antisera raised against polymeric peptides Rv2004c-7 and Rv2004c-19 belonging to Rv2004c protein were tested against M. tuberculosis total sonicate; serum from rabbit 214 (immunized with Rv2004c-7 [121CGDAIAEVLARFHQRAQRNRCIYGC140] polymerized peptide) specifically recognized a 54-kDa band (Fig. 2, lanes 4–6) corresponding to this protein’s expected molecular weight (54.4 kDa). Antisera induced by immunization with polymerized peptide Rv2004c-19 (361CGRDCGVITGEPGVLDSGLYSRGC380) specifically recognized a 27-kDa band (Fig. 2, lane 2), possibly due to Rv2004c protein cleavage, suggesting that the antisera produced against the protein’s N-terminal portion recognized the whole molecule, while that directed against the C-terminal portion only recognized a cleavage product. Nevertheless, further experiments are needed to confirm this result.
Transmission electron microscopy studies using the anti-peptide Rv2004c-7 sera (rabbit 214) were carried out for detecting the possible presence of the Rv2004c protein on the mycobacterial cell surface. The reduced number of immunogold signals (Fig. 3A) could have been due to a low abundance of protein, perhaps, as a result of culture conditions (affecting mycobacterial protein expression) (Brennan et al. 2001; Florczyk et al. 2001; Banu et al. 2002), or the low antibody concentration present in the sera. Previous studies have demonstrated that antibodies obtained against mycobacterial proteins can (by using TEM and immunoblotting studies) determine their subcellular location (Amara et al. 1998; Banu et al. 2002). Moreover, the results obtained by Western blot confirm this protein’s presence on the mycobacterial cell surface, specifically in the cell wall; in addition, since no band was detected on culture supernatant, it seems that this protein is not being secreted. Our observations were in agreement with recently reported results determining the Rv2004c protein’s presence in the cell wall (Mawuenyega et al. 2005).
One of our laboratory’s research aims is to identify pathogen surface protein regions that are involved in the complex processes of host cell recognition and invasion. We have recently reported several studies aimed at characterizing receptor–ligand interactions between synthetic peptides derived from pathogen proteins specifically recognizing the host cell. This method was used for determining specific binding regions in proteins from simple pathogens such as hepatitis C virus (Garcia et al. 2002), Human papillomavirus (Vera-Bravo et al. 2003), and Epstein-Barr virus (Urquiza et al. 2004). It has also been used in more complex protozoa such as Leishmania (Puentes et al. 1999), Plasmodium falciparum and Plasmodium vivax (Ocampo et al. 2002; Rodriguez et al. 2002; Urquiza et al. 2002), malarial blood (Curtidor et al. 2005; Ocampo et al. 2005), and hepatic stages (Garcia et al. 2004; Puentes et al. 2004).
In the case of tuberculosis, it has been assumed that the bacterium is ingested by alveolar macrophages and subsequently gains access to the bloodstream by being transported by the alveolar macrophages and blood monocytes through the alveolar wall. However, several groups have recently demonstrated that M. tuberculosis invades and survives within human type II alveolar epithelial cells in vitro (Bermudez and Goodman 1996). This background has led us to determine those Rv2004c protein regions that could be involved in the mycobacteria–host cell interaction. Binding assays were done using monocyte-like U937 and A549 type II alveolar epithelial cells. This protein’s U937 and A459 cell-binding capacity was determined by using synthetic peptides. Three types of target-cell-binding behavior were found for these peptides (Fig. 4). Figure 4, A and B, shows HABP-U937 interaction for a high binding peptide, that is, Rv2004c-14 (261AGYAVRSGDTAPASLRDFYI280). Figure 4, C and D, shows high non-specific binding for the Rv2004c-19 peptide (361RDCGVITGEPGVLDSGLISR380), because this bound to U937 cells but there was no inhibition with the same nonradio-labeled peptide (Fig. 4D). Figure 4, E and F, shows a peptide that did not show binding to U937 cells, that is, Rv2004c-12 (221LLDCLEFEDELRYLDRIDDA240). The same behavior was observed in A549 epithelial cell-binding assays (data not shown); the affinity was generally characteristic of high-affinity interactions for both types of cell used (binding curve slope 2%).
The assay using U937 monocytoblastic cells revealed five HABPs for this protein; four of them were located in the central region: Rv2004c-14 (261AGYAVRSGDTAPASLRDFTI280), Rv2004c-15 (281AYRAVVRAKVECVRFSQGKP300), Rv2004c-16 (301EAAADAVRHLIIATQHLQHA320), and Rv2004c-18 (341GVAELVGAQVISTDDVRRRL360); another one, Rv2004c-6 (101RDKQRLASMVTAGLPVEGAL120), was located in the N-terminal region. Six A549 epithelial cell HABPs were also found; Rv2004c-14 (261AGYAVRSGDTAPASLRDFTI280), Rv2004c-15 (281AYRAVVRAKVECVRFSQGKP300), Rv2004c-18 (341GVAELVGAQVISTDDVRRRL360), and Rv2004c-20 (381ANVVAVYQEALRKARLLLGS400 were located in the protein’s central region; and Rv2004c-5 (81AHLSDPSGGHAEPVVVMRRY100) and Rv2004c-6 in the N-terminal region (Fig. 5). These results suggest that target cell interaction with the Rv4004c protein is preferentially presented by the protein’s central region.
Determining physico-chemical binding constants represents one way of characterizing HABP interaction with target cells (Kd, affinity constant and the number of binding sites per cell). Table 1 shows that HABP affinity for cellular surface molecules was greater for A549 epithelial cells than U937 monocyte cells; however, this effect was compensated for by the greater number of binding sites per cell found for U937 cells than for A549 cells. Saturation assays (Fig. 6) also established that HABP binding to U937 and A549 target cells was specific and saturable.
The exact role of amino acids directly involved in interaction with target cell surface molecules has not been completely determined; however, it has been recently reported that identifying them could represent a novel tool for designing immunogenic and protection-inducing molecules (Espejo et al. 2001; Purmova et al. 2002). Critical residues for the binding process were found for each HABP-U937 in glycine peptide analog competition binding assays (Fig. 7). It is likely that these critical residues may not only correspond to those amino acids directly involved in binding to receptors on U937 cells but may also be responsible for keeping the peptide conformation that is necessary for binding. Such observations suggest that each peptide requires different residues for its interaction with a particular target cell and that most HABPs are localized along a particular peptide’s sequence (showing such sequence’s specificity). A clear example would be that shown by the arginine residues in the Rv2004c-6 peptide, where peptide analog binding remained unchanged when substituting R101 for Gly, while binding became drastically reduced when substituting R105. The same thing happened with alanine residues in the Rv2004c-16 peptide when binding remained unchanged on substituting A320, while changing residues A303, A306, and A313 led to noticeably reduced binding. This confirmed that each HABP’s binding activity was influenced more by a particular HABP’s spatial conformation than by the nature of the amino acid involved in the binding.
Although the nature of U937 cell and A549 cell receptors for HABPs remains unknown, it was found that HABPs Rv2004c-6 and Rv2004c-14 bound to 32- and 49-kDa bands on the U937 cell surface (Fig. 8, lanes 1,3) and to 40-, 66-, 74-, 90-, and 97-kDa bands on the A549 cell surface (Fig. 8, lanes 5,7). This confirmed that interaction with these bands was specific since they disappeared on each 125-I-HABP’s binding being inhibited by the respective nonradiolabeled HABP (Fig. 8, lanes 2,4,6,8). The fact that this protein contained regions specifically interacting with epithelial and monocyte cells suggests that it could be implicated in mycobacterial binding to host cells. Previous works have posed the possibility that receptors on epithelial cell and macrophage surfaces could be adhesion molecules such as CD11a, CD11b, VLA4, or ICAM-1. Even though our results were very close to some of these adhesion molecules’ molecular weights, more studies must be done to enable their biochemical characterization and their role in bacillus and target cell recognition and interaction (Bermudez et al. 2002).
Experimental results coincided with the Rv2004c protein’s secondary structure prediction according to HABP circular dichroism structural analysis (Fig. 9), where 64% of the amino acid sequence will tend to form -helix structures (Combet et al. 2000). Their high target cell binding activity also suggests some structure–affinity relationship since all HABPs could conserve the same type of arrangement independently of the target cell with which they are interacting.
The present work’s most important findings concern Rv2004c gene presence in all M. tuberculosis complex strains and clinical isolates, its transcription and expression in several M. tuberculosis complex strains, and the surface location of the protein on the bacillus and its ability to bind to monocyte (U937) and type II alveolar epithelial (A549) cells. This has important biological significance and implications for developing strategies for controlling this disease. More detailed studies are still needed for determining HABPs’ role in the complex process of bacillus interaction with its target cell.
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Materials and methods |
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Chromosomal DNA extraction
Chromosomal DNA was isolated by using a previously described method (Mahairas et al. 1996). DNA was precipitated with 0.6 volumes of 2-propanol. The pellet was washed with 70% (v/v) ethanol and suspended in 1x TE (Del Portillo et al. 1991; Parra et al. 1991).
PCR conditions and primers
PCR amplification was done in a thermal cycler Gene Amp PCR system 9600 (Perkin Elmer) using 100 ng of M. tuberculosis genomic DNA. The mixture contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.1 mM each deoxynucleoside triphosphate, 0.4 µM both forward IN-1 (5'-GATGGCGAACCGGCGCTG-3') and reverse IN-2 (5'-TAGAGCCCGGAGTCCAAA-3') internal primers, and 1.5 U of Taq DNA polymerase (Promega). Then 30 cycles of the following thermal profile were carried out after 5 min of DNA denaturing at 95°C: 59°C for 30 sec, 72°C for 30 sec, and 94°C for 30 sec. A final 5-min extension cycle performed at 72°C was used for PCR amplification.
DNA sequencing
The dideoxy chain-termination method was followed by a sequencing reaction with the Taq FS DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) using a GeneAmp PCR system 9600 (Perkin-Elmer) and run on a DNA Analysis System model 373 (Applied Biosystems). The sequencing strategy involved using M. tuberculosis Rv2004c putative protein forward and reverse primers.
Reverse transcriptase-PCR
Total RNA was extracted from different mycobacterial strains by Trizol (GIBCO), then treated with RNase-free DNase RQ1, and purified by standard phenol-chloroform procedure (Alland et al. 1998). RNA preparation, concentration, and purity were spectrophotometrically measured. Target RNA (20 µg/mL) was reverse-transcribed in a single tube containing distilled water and 10 µg/mL random primers (GIBCO). This mixture was incubated at 70°C for 10 min and chilled on ice. Then 1x RT buffer (0.14 M KCl, 8 mM MgCl2, and 50 mM Tris-HCl at pH 8.1), 10 mM dithiothreitol (DTT), 0.5 mM dNTPs, and 40 U of Human Placenta Ribonuclease Inhibitor (Platinum Taq) were added. Following this, 200 U of M-MLV reverse transcriptase (GIBCO) was added to a final 40-µL volume. This mixture was kept at 37°C for 1 h. The enzyme was denatured at 95°C for 5 min. Then 5% of the total RT mixture was used as template for PCR, which was carried out as described above. Appropriate positive and negative controls were included in each experiment. The rpoB gene was used as the transcription positive control (direct, 5'-TCAAGGAGAAGCGCTACGA-3'; and reverse, 5'-GGATGTTGATCAGGGTCTGC-3' primers). This gene, encoding RNA polymerase subunit B, is present in all myco-bacterial strains (Lee et al. 2000); it is also one of the genes implicated in its metabolism. DNAse-Q treated M. tuberculosis H37Rv was used as the cDNA synthesis negative control. Sterile distilled water and M. tuberculosis H37Rv DNA were used as the PCR negative and positive controls, respectively.
Cell-binding assays
Cell lines
Two cell lines, susceptible to invasion by M. tuberculosis, were used in this study: nonadherent monocyte-like U937 cells and A549 cells (adherent epithelial cell line derived from a pulmonary carcinoma). The lines were obtained from ATCC and kept in culture using RPMI 1640 (GIBCO) and 10% fetal bovine serum (Hyclone) at 37°C and 5% CO2 (at 105 cell/mL density) in 275-mL culture dishes. Adherent A549 cells were dislodged with 0.1% EDTA-PBS. Both cell lines were collected in 50-mL tubes and centrifuged at 2000g for 5 min, washed with PBS, and counted in a Neubauer chamber (Sato et al. 2000; Passmore et al. 2001; Liu et al. 2004).
Peptide synthesis
Twenty-five sequential, 20-mer-long, amino acid peptides, spanning the complete M. tuberculosis Rv2004c putative protein sequence, were chemically synthesized using the Multiple Peptide Synthesis method in Solid Phase. MBHA resin (0.7 meq/g), t-Boc amino acids, and low-high cleavage techniques were used (Merrifield 1963; Tam et al. 1983; Houghten 1985). Peptide identity and purity were analyzed by MALDI-TOF mass spectrometry and analytical reverse-phase, high-performance liquid chromatography (RP-HPLC) and freeze-dried. The synthesized peptide sequences are shown in Figure 3 in one-letter code. Tyr was added at the C-terminal end of those peptides that did not contain it to enable radiolabeling.
Radiolabeling
125I-radiolabeling was done according to previously described techniques (Yamamura et al. 1978; Hulme 1993), in which chloramine T (2.25 mg/mL) and 3.2 µL of Na125I (100 mCi/mL) were added to 5 µL of peptide solution (1 µg/µL). Then 15 µL of sodium bisulfite (2.75 mg/mL) and 50 µL of NaI (0.16 M) were added after 5 min of reaction at 18°C. The 125I-peptide was then separated from reaction subproducts on a Sephadex G-10 column (Pharmacia; 80 x 5.0 mm).
Binding assay
U937 or A549 cell lines kept in RPMI 1640 culture medium (1 x 106 cells), previously washed in isotonic PBS and incubated with increasing quantities of each 125I-radiolabeled M. tuberculosis putative Rv2004c protein peptide (between 100 and 2000 nM), were used for binding assays in a 100-µL total volume for 90 min at 4°C, in the presence or absence of 40 µM unlabeled peptide to determine binding specificity (binding assays were done for each of the Rv2004c protein peptides). Unbound peptide was removed from cells following incubation by sedimentation through a dibutylphthalate–dioctylphthalate cushion mixture (d = 1.015 g/mL) and spun at 9000g for 2 min (Garcia et al. 2004; Vera-Bravo et al. 2005). The cell-bound peptide was measured in an automatic gamma counter (4/200 plus ICN Biomedicals, Inc.). The assay was carried out in triplicate under identical conditions; the mean results of the triplicate assays are reported and graphically represented in Figures 4 and 5.
Determining physico-chemical constants for the HABP–target cell interaction
Saturation assay
Cells were first washed twice with RPMI 1640 medium and then washed twice with isotonic PBS before carrying out the binding assays. Saturation binding assays were carried out for each peptide presenting high specific target cell binding activity. In accordance with a previously described method (Ocampo et al. 2000; Rodriguez et al. 2000; Garcia et al. 2002), 1 x 106 U937 or A549 cells were incubated with increasing 125I-peptide concentrations between 100 and 3000 nM in a 120-µL total volume for 90 min at 4°C, in the presence or absence of 40 µM unlabeled peptide to determine binding specificity. After incubation, unbound peptide was removed as described above. As before, each assay was performed in triplicate; bound and free 125I-peptides were determined by gamma counter measurement. The curves so obtained were analyzed by Scatchard analysis, and affinity constants were determined by the Hill equation (Garcia et al. 2004; Vera-Bravo et al. 2005).
Determining critical amino acids in the HABP–U937 cell interaction
Competition binding assay
HABP Rv2004c-6, Rv2004c-14, Rv2004c-15, Rv2004c-16, and Rv2004c-18 glycine scanning analogs were synthesized to recognize critical target cell binding residues. Then 1 x 106 U937 cells were incubated with increasing quantities (0.2, 0.4, 4.0, and 40.0 µM) of each unlabeled analog peptide or original unlabeled peptide in the presence of native 125I-peptide for the competition binding assays at a final 255-µL volume. After 90 min of incubation at 4°C, the mixture was centrifuged at 9000g for 2 min through a dibutylphalate cushion. The radioactivity bound to U937 or A549 cells was measured in a gamma counter. Data from triplicate assays were averaged (Ocampo et al. 2000; Rodriguez et al. 2000; Garcia et al. 2002).
Cross-linking assays between U937-HABP cells
Some HABPs were cross-linked to cells to identify U937 cell-binding sites, based on the methodology previously reported (Garcia et al. 2002). Briefly, 2 x 106 U937 cells were subjected to a conventional binding assay (incubation for 90 min at 4°C) with 125I- Rv2004c-6 and 125I-Rv2004c-14 HABPs. Cells were washed with PBS following incubation and subjected to cross-linking with 25 µM, Bis(sulfosuccinimidyl suberate) (BS3, Pierce) for 20 min at 4°C. The reaction was stopped with 40 nM Tris-HCl (pH 7.4), and cells were washed again with PBS. They were then treated with lysis buffer (5% SDS, 10 nM iodoacetamide, 1% Triton X-100, 100 mM EDTA, 10 mM PMSF). The obtained membrane proteins were solubilized in Laemmli buffer and separated in SDS-PAGE. Those proteins cross-linked with radiolabeled peptides were exposed on a Bio-Rad Imaging Screen K (Bio-Rad Molecular Imager FX; Bio-Rad Quantity One Quantitation Software) for 7 d, and the apparent molecular weight was determined by using 6.4–198-kDa or 14–97-kDa molecular weight markers (Bio-Rad).
CD spectroscopy
Circular dichroism (CD) was carried out for each HABP to determine whether there was any conformational–functional correlation. CD spectra were recorded for each HABP at 20°C on a Jasco J-810 spectropolarimeter at wavelengths ranging from 260 to 190 nm in 1.00-cm cuvettes (Provencher and Glockner 1981). The peptides were dissolved at 0.1 mM concentration in pure water or in aqueous TFE solutions containing 30% TFE by volume. Each spectrum was obtained from averaging three scans taken at a 20-nm/min scan rate with 1 nm spectra bandwidth, corrected for baseline. The results were expressed as the mean residue ellipticity [
], the units being degrees x centimeters squared per decimole according to the function [] = 
/(100lcn), where is the measured ellipticity, l is the optical pathlength, c is the peptide concentration, and n is the number of amino acid residues contained in the sequence (Sreerama et al. 1999).
Rabbit immunization
The numbers-in-the-bag method was used for randomly selecting one peptide out of the eight from the Rv2004c protein’s N-terminal region and one out of eight from its carboxy region (none of the nine from its central region were chosen). These were synthesized as described above but CG residues were added to each extreme to facilitate their polymerization. Three New Zealand strain rabbits per peptide, having previously been determined to be nonreactive to M. tuberculosis sonicate by Western blotting, were subcutaneously immunized with 500 µg of Rv2004c-7 polymer peptide, to amino acid sequence 121CGDAIAEVLARFHQRAQRNRCIYGC140 and Rv2004c-19 polymer peptide, corresponding to amino acid sequence 361CGRDCGVITGEP GVLDSGLYSRGC380. Polymerized peptides were emulsified with Freund’s Incomplete Adjuvant (1:1 v/v) and used for immunizations on days 0, 20, and 40. Final bleeding was carried out on day 60, and the sera were collected. The immunizations and bleedings were all carried out according to the handling procedures required by the Colombian Ministry of Public Health.
Mycobacterial sonicate and subcellular fractions
Ten grams (wet weight) of mycobacteria was suspended in 20 mL of phosphate-buffered saline (PBS) containing DNase, RNase, and a proteinase inhibitor cocktail (phenylmethylsulphonyl fluoride [PMSF] and EDTA at a final 1 mM concentration of each one and 1 µg/mL leupeptin and 1 µg/mL pepstatin A). Sonication was done in a Branson Sonifier 450 for 20 min with amplitude set at 4 and 90% duty cycle. The sonicate was centrifuged at 650g for 20 min. The supernatant was then centrifuged at 36,000g for 45 min at 4°C. The protein concentration was determined by bicinchoninic acid assay (BCA kit; Pierce) and stored in aliquots at –70°C until needed. The mycobacterial subcellular fractions (cell wall, membrane, and culture filtrate proteins) were purchased from Colorado State University.
Rabbit sera adsorption with Escherichia coli and Mycobacterium smegmatis sonicate
M. smegmatis proteins were obtained from 5-d-old Middlebrook 7H9 broth culture, washed, suspended, sonicated for 10 min (as described above), and centrifuged for 10 min at 4500g at 4°C. E. coli (DH5 strain) proteins were obtained from overnight culture in Luria Bertani medium, washed, suspended, sonicated for 2 min at 4°C, and centrifuged for 10 min at 4500g. Both pellets were suspended in coupling buffer (0.1 M NaHCO3 at pH 8.3). The suspended lysates were collected and used individually for coupling to CNBr-activated Sepharose 4B (Pharmacia Biotech), according to the manufacturer’s recommendations. Each rabbit serum (pre-immune and immune) was pre-adsorbed on E. coli-Sepharose and M. smegmatis-Sepharose affinity columns to eliminate cross-reactivity. Briefly, 5 mL of each serum was added to 4 mL of lysate-Sepharose affinity columns and left in a gentle rotating/shaking mode for 20 min at room temperature. This procedure was done twice using a new lysate-Sepharose affinity column each time.
SDS-PAGE and immunoblotting
Proteins from the M. tuberculosis sonicate (36,000g supernatant fraction) and subcellular fractions were separated in a discontinuous SDS-PAGE system, using a 10% to 20% (w/v) acrylamide gradient. A total of 1 mg of sample was loaded per gel and transferred to nitrocellulose membranes (Hybond 203C, Pharmacia) using the semidry blotting technique (Kyhse-Andersen 1984). Commercial molecular mass markers (New England Biolabs) were used for calibration. The filters were incubated with a 1:100 dilution of the sera obtained from rabbits immunized with polymerized peptides Rv2004c-7 and Rv2004c-19. Pre-adsorbed sera were diluted in TBST (0.02 M Tris-HCl at pH 7.5, 0.05 M NaCl, 1% Tween 20) and 5% skimmed milk. Then they were incubated for 1 h with 1:3000 alkaline phosphatase conjugated anti-rabbit IgG antibody (ICN) following five TBST washes. The reaction was developed with NBT/BCIP (Promega).
Electron microscopy
Transmission electron microscopy (TEM) studies were carried out on a Philips CM 10 TEM. Fifty microliters of M. tuberculosis H37Rv wet pellet was fixed with a 4% paraformaldehyde–0.5% glutaraldehyde solution for 2 h at 4°C for sample preparation. The pellet was dehydrated in graded ethanol (50%, 70%, 80%, 90%, and twice 100%) after being fixed and was then embedded in LR-white hard-grade acrylic resin (Sigma) for 4 d at 4°C. Then 400-nm-thin sections were cut and mounted on 300 mesh nickel grids. These sections were incubated in a saturated sodium metaperiodate solution for antigen retrieval (Stirling and Graff 1995). Grids were then floated sections down in a beaker containing 0.01 M sodium citrate buffer for 15 min at 80°C. After 1 h of blocking in Tris-buffered saline (TBS) (0.05 M Tris in isotonic saline at pH 7.6) containing 0.05% BSA, grids were incubated in pure rabbit polyclonal serum for 1 h at 37°C. Following a TBS–0.025% Tween 20 wash, grids were immersed in a 1:50 dilution of 5 nm gold-labeled anti-rabbit IgG (Sigma) for 1 h at room temperature, according to the manufacturer’s instructions. Grids were then washed with TBST and fixed in 2.5% glutaraldehyde. Then a 15-min incubation was carried out in 1% uranile acetate after fixing. Grids were washed with distilled water and dried at room temperature before observation.
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—) U937 cells incubated with 125I-peptide in the presence of 400 excess of unlabeled peptide. (B,D,F) Specific binding resulting from subtracting total binding from nonspecific binding. Rv2004c-14, Rv2004c-19, and Rv2004c-12 peptides showed high U937 cell binding, nonspecific binding, and non-binding activity, respectively.
