Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to...

Identifying putative Mycobacterium tuberculosis

Rv2004c protein sequences that bind specificallyto U937 macrophages and A549 epithelial cells

MARTHA FORERO,1,4 ALVARO PUENTES,1,4 JIMENA CORTES,1

FABIO CASTILLO,3 RICARDO VERA,1,3 LUIS E. RODRIGUEZ,1

JOHN VALBUENA,1 MARISOL OCAMPO,1 HERNANDO CURTIDOR,1,3

JAIVER ROSAS,1 JAVIER GARCIA,1,3 GLORIA BARRERA,2 ROSALBAALFONSO,3 MANUEL A. PATARROYO,1,3 AND MANUEL E. PATARROYO1,3

1Fundacion Instituto de Inmunologıa de Colombia, Bogota 020304, Colombia2CORPOICA-CEISA, Bogota 020304, Colombia3Universidad Nacional de Colombia, Bogota 020304, Colombia

(RECEIVED June 1, 2005; FINAL REVISION August 6, 2005; ACCEPTED August 15, 2005)

Abstract

Virulence and immunity are still poorly understood inMycobacterium tuberculosis. TheH37RvM. tubercu-losis laboratory strain genome has been completely sequenced, and this along with proteomic technologyrepresent powerful tools contributing toward studying the biologyof target cell interactionwith a facultativebacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosisprotein that could have specific mycobacterial functions. This study has revealed that the encoding gene ispresent in all mycobacterium species belonging to theM. tuberculosis complex. Rv2004c gene transcriptionwas observed in all of this complex’s strains except Mycobacterium bovis and Mycobacterium microti.Rv2004c protein expression was confirmed by using antibodies able to recognize a 54-kDa molecule byimmunoblotting, and its location was detected on the M. tuberculosis surface by transmission electronmicroscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing highactivity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically boundto A549 cells. HABP circular dichroism suggested that they had an a-helical structure. HABP–target cellinteractionwasdetermined tobe specific and saturable; someof themalsodisplayedgreater affinity forA549cells thanU937 cells. The critical amino acids directly involved in their interaction withU937 cells were alsodetermined. Two probable receptor molecules were found on U937 cells and five on A549 for the twoHABPsanalyzed.These observations have important biological significance for studyingbacillus–target cellinteractions and implications for developing strategies for controlling this disease.

Keywords: Mycobacterium tuberculosis; Rv2004c protein; high activity binding peptides; U937 cells;A549 cells

Millions of people have died and continue to die fromtuberculosis, a chronic infectious disease caused by thetubercle bacillus. Tuberculosis causes nearly four-mil-lion deaths worldwide, and nine-million new casesappear per year, the highest mortality and morbidityrate of any disease caused by a single infectious organ-ism (WHO 2005). Although progress is being madetoward controlling the disease by using directly observedshort-course treatment, this strategy may not be suitablein many regions having high rates of HIV infection

4These authors contributed equally to this work.Reprint requests to: Alvaro Puentes, Fundacion Instituto de Inmu-

nologıa de Colombia, Carrera 50 No. 26-00, Bogota 020304, Colom-bia; e-mail: [email protected]; fax: 571-4815269.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 CultureCollection.Article published online ahead of print. Article and publication date

are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051592505.

Protein Science (2005), 14:2767–2780. Published by Cold Spring Harbor Laboratory Press. Copyright � 2005 The Protein Society 2767

ps0515925 Forero et al. Article RA

accompanied by drug-resistant tuberculosis (Raviglioneet al. 1995; Small and Fujiwara 2001). Worldwide effortsare thus being made in terms of different strategiesaimed at controlling tuberculosis. One of these hasbeen the detailed study of the microorganism, leadingto the complete genome sequence being recently deter-mined to improve the understanding of this pathogen’sbiology (Cole et al. 1998).

Mycobacteria are intracellular microorganisms sur-viving and growing into host macrophages. Followingphagocytosis, sustained intracellular bacterial growthdepends on their ability to avoid destruction by macro-phage-mediated host defenses such as lysosomal en-zymes and reactive oxygen and reactive nitrogen inter-mediates. This suggests that host cell interaction withmicrobes is delicately balanced and can be tipped infavor of either organism (Mariani et al. 2000).

The immune response against Mycobacterium tubercu-losis has been evaluated in human and animal models;disease pathogenesis and protective immunity develop-ment have also been studied (Andersen 1994; Andersenand Brennan 1994). It has been established that cellimmunity is critical for inducing protection against tuber-culosis. Defining which M. tuberculosis antigens can eliciteffective immunity and, taking into account that host–pathogen molecular interactions are critical in the infec-tion process, identifying M. tuberculosis genes expressedon the bacterial surface would greatly contribute towarddeveloping new strategies for fighting tuberculosis (Mar-iani et al. 2000). Mawuenyega et al. (2005) have recentlyreported the identification of 1044 proteins and theircorresponding subcellular localization by using a combi-nation of high-throughput proteomics and computationalapproaches for elucidating those proteins being express-ed in each one of the three M. tuberculosis subcellularcompartments; among these proteins, the presence ofRv2004c was detected.

In the present study, the M. tuberculosis Rv2004c gene(synonyms: MT2060, Mb2027c, MTCY39.13; GenBankaccession no. Q10852) has been analyzed in the M. tuber-culosis complex, as has its transcription and encodedprotein expression. This protein’s potential role in themycobacterium–host cell interaction by using syntheticpeptides has also been determined. In all, 25 nonoverlap-ping, 20-residue-long peptides spanning the completeRv2004c protein sequence were chemically synthesized;rabbit sera raised against two of these peptides in poly-merized form (Rv2004c-7, 121DAIAEVLARFHQRAQRNRCIY140; and Rv2004c-19, 361RDCGVITGEPGVLDSGLYSR380) were used for Western blot and immunoelec-tron microscopy studies. Their specific U937 monocyteand A549 epithelial cell line binding capacity was alsodetermined. Five high activity binding peptides (HABPs)were identified in the Rv2004c protein as specifically

interacting with U937 cells’ surface molecules. TheU937-HABPs were Rv2004c-6, Rv2004c-14, Rv2004c-15, Rv2004c-16, and Rv2004c-18; while a further sixHABPs (Rv2004c-5, Rv2004c-6, Rv2004c-14, Rv2004c-15, Rv2004c-18, and Rv2004c-20) were specifically iden-tified as interacting with A549 cells’ surface molecules.Circular dichroism suggested that all HABPs display astable a-helical structure.

The present work’s most important findings concernthe presence of the Rv2004c gene in all M. tuberculosiscomplex strains and clinical isolates, its transcription inseveral M. tuberculosis complex strains, and the encodedprotein’s expression, its surface location on the bacilli,and its U937 and A549 cell-binding capacity. This has animportant biological significance and implications fordeveloping strategies for controlling this disease.

Results

Molecular assays

Genomic DNA from 19 different mycobacterial strains,including those belonging to the M. tuberculosis com-plex and 10 M. tuberculosis clinical isolates, was tested.PCR amplification using IN-1 (5¢-GATGGCGAACCGGCGCTG-3¢) and IN-2 (5¢-TAGAGCCCGGAGTCCAAA-3¢) internal primers revealed the presence of a single490-bp amplification band in all M. tuberculosis com-plex strains and clinical isolates used (Fig. 1A,B). DNAsequencing established that the Rv2004c protein was con-served in all mycobacterial strains and isolates tested(data not shown).

RNA was isolated from all tuberculosis complexstrains to determine whether Rv2004c was being tran-scribed by these mycobacteria. RT-PCR (using IN-1 andIN-2 internal primers) revealed the presence of the 490-bp band in all M. tuberculosis complex strains, exceptMycobacterium bovis and Mycobacterium microti (Fig.1C). The constitutive gene (rpoB, 360 bp) used as posi-tive control showed to be equally transcribed in all theM. tuberculosis complex’s strains (Fig. 1D).

Recognizing the Mycobacterium tuberculosis-H37RvRv2004c protein by polyclonal antibodies

The Rv2004c-7-synthetic peptide polymer was inocu-lated into three rabbits, as described in Materials andMethods. The immunoblot showed that rabbit 214’s seraspecifically recognized a 54-kDa band (Fig. 2, lanes4,5,6) in the three post-immune bleedings. No proteinwas recognized when pre-immune serum was beingtested (Fig. 2, lane 3). The other two rabbit sera fromthis group showed very weak recognition of the sameband (data not shown). One of the three rabbits

2768 Protein Science, vol. 14

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inoculated with Rv2004c-19-synthetic peptide polymerspecifically recognized a 27-kDa band (Fig. 2, lane 2).Again, no protein was recognized when pre-immuneserum was being tested (Fig. 2, lane 1).

Localizing Rv2004c on M. tuberculosis H37Rv surface

Electron microscopy studies determined the location ofthe Rv2004c protein on the M. tuberculosis bacilli sur-face, using anti-sera 214 from the rabbit immunized withpolymerized peptide Rv2004c-7. Figure 3A shows thepresence of colloidal gold particles, mainly on bacillicellular surface (indicated by arrows). On the contrary,no signals were observed when pre-immune sera wereused (data not shown).

A Western blot assay using anti-sera 214 against theM. tuberculosis H37Rv cell wall, membrane fraction,and culture supernatant was carried out in order toverify the results obtained by electron microscopy. Fig-ure 3B shows this serum recognizing a 54-kDa bandwhen tested against the cell wall fraction (lane 1); norecognition against either culture supernatant (lane 2) ormembrane fraction (data no shown) was observed.

Identifying high activity binding peptides (HABPs)

Binding assays were performed as described in Materialsand Methods to determine which Rv2004c amino acidsequences could interact with human target cells suscep-tible to M. tuberculosis H37Rv invasion. The U937

monocytoblastic and A549 epithelial cell lines were usedin independent experiments. 125I-peptides (100–2000 nM)

Figure 1. (A,B) PCR amplification of genomic DNA from different mycobacterial strains for evaluating Rv2004c gene presence, using internal primers

IN-1 (5¢-GATGGCGAACCGGCGCTG-3¢) and IN-2 (5¢-TAGAGCCCGGAGTCCAAA-3¢). PCR products were analyzed by electrophoresis on a

1% agarose gel. (A, lane 1) Negative control; (lane 2)M. africanum; (lane 3)M. bovis; (lane 4)M. bovisBCG; (lane 5)M.microti; (lane 6)M. tuberculosis

H37Ra; (lane 7) M. tuberculosis H37Rv; (lane 8) molecular weight markers; (lane 9) M. avium; (lane 10) M. chelonae abcesus; (lane 11) M. chelonae

chelonae; (lane 12)M. diernhoferi; (lane 13)M. flavescens; (lane 14)M. fortuitum; (lane 15)M. gastri; (lane 16)M. gordonae; (lane 17)M. intracellulare;

(lane 18)M. kansassi; (lane 19)M. marinum; (lane 20)M. non-chromogenicum. (B) (Lane 1)M. peregrinum; (lane 2)M. phlei; (lane 3)M. scrofulaceum;

(lane 4) M. simiae; (lane 5) M. smegmatis; (lane 6) M. szulgae; (lane 7) M. terrae; (lane 8) M. vaccae; (lane 9) M. xenopi; (lane 10) molecular weight

markers. (Lanes 11–20) M. tuberculosis clinical samples: (lane 11) SC-1; (lane 12) SC-8; (lane 13) SC-18; (lane 14) SC-26; (lane 15) SJ-1; (lane 16) SJ-7;

(lane 17) SJ-8; (lane 18) SJ-21; (lane 19) SJ-26; (lane 20) E-4. (C,D) Analysis of M. tuberculosis complex strains for evidence that the Rv2004c gene is

being transcribed in these mycobacteria. (C) RT-PCR results, showing a 490-bp band when the Rv2004c gene is being transcribed. (Lane 1) Molecular

weight markers; (lane 2)M. tuberculosisH37Rv; (lane 3)M. tuberculosisH37Ra; (lane 4)M. bovis; (lane 5)M. bovisBCG; (lane 6)M. africanum; (lane 7)

M. microti; (lane 8) negative control; (lane 9) PCR positive control (H37Rv DNA); (lane 10) PCR negative control. (D) rpoB gene RT-PCR results,

showing a 360-bp band (transcription positive control). (Lane 1) Molecular weight markers; (lane 2) M. tuberculosis H37Rv; (lane 3) M. tuberculosis

H37Ra; (lane 4) M. bovis; (lane 5) M. bovis BCG; (lane 6) M. africanum; (lane 7) M. microti; (lane 8) negative control; (lane 9) PCR positive control

(H37Rv DNA); (lane 10) PCR negative control. M. tuberculosis DNA processed with DNAse Q (Promega) was used as the negative control.

Figure 2. Immunoreactivity of sera from rabbits 214 and 80 immunized

with Rv2004c-7 and Rv2004c-19 synthetic polymerized peptides, respec-

tively. (Lane 1) Rabbit 80 pre-immune sera; (lane 2) rabbit 80 post III/20

sera; (lane 3) rabbit 214pre-immune sera; (lane 4) rabbit 214 post II/20 sera;

(lane 5) rabbit 214 post III/15 sera; (lane 6) rabbit 214 post III/20 sera; (lane

7) hyperimmune sera from a rabbit immunized with M. tuberculosis total

sonicate. The antisera were analyzed againstM. tuberculosis total sonicate.

www.proteinscience.org 2769

Mycobacterial Rv2004c peptides bind to target cell

were incubated with both U937 and A549 cells in thepresence (nonspecific binding) or absence (total binding)of the same nonradiolabeled (cold) peptide (Fig. 4). Totalbinding minus nonspecific binding gave the specific bind-ing (Yamamura et al. 1978; Rodriguez et al. 2000).

The specific binding for each cell line was calculatedfrom the specific binding curve slope as described inMaterials and Methods. Any peptide showing specificbinding activity $2% was called a high activity bindingpeptide (HABP) (Vera-Bravo et al. 2005). Based on thismethodology, five HABPs were identified in the Rv2004cprotein as specifically interacting with U937 cell surfacemolecules (Fig. 5). Peptide Rv2004c-6 (101RDKQRLASMVTAGLPVEGAL120) was located in the N-terminalregion, 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. PeptidesRv2004c-14 and Rv2004c-15 were identified as having thehighest affinity and specific binding. Six HABPs from theRv2004c protein were also identified, specifically recog-nizing 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 theprotein’s central region (peptides Rv2004c-14, Rv2004c-15, Rv2004c-18, and Rv2004c-20; 381ANVVAVYQEAL

RKARLLLGS400). No specific binding activity wasfound in the Rv2004c protein C-terminal region witheither of these cell lines.

Physico-chemical constants for theHABP–target cell interaction

Figure 6 and Table 1 show the saturation assay results foreach selected HABP when using a higher 125I-peptide con-centration than in the screening binding assay. ScatchardandHill analyses allowed affinity constant (Kd) calculationsto be done for each Rv2004c protein HABP. All U937-HABPs presented affinity constants in the 480 and 950 nMrange, andA549-HABPs presented affinity constants in the330 and 700 nM range, indicating high affinity in pep-tide–cell interaction. Hill coefficients (nH) for each U937-HABPand A549-HABP were also calculated and displayed posi-tive cooperativity (nH>1). The number of binding sites percell (NrSc) calculated for these U937-HABPs and A549-HABPs was 10· 106 and 3· 106, respectively. These valueswere calculated from the highest bound peptide value onthe saturation curve. (Hulme 1993; Puentes et al. 2005).

Critical amino acids in the U937-HABP cell interaction

Glycine scanning analog peptides were synthesized andtested in competition binding assays between originallabeled peptides and their analog peptides to identifyHABP critical amino acids for U937 cell binding. Critical

Figure 3. (A) Subcellular localization ofRv2004c protein inM. tuberculosisH37Rv. Cross-sections ofM. tuberculosiswere incubated

with anti-sera from rabbit 214 as primary antibody followed by a 1:50 dilution of 10 nm of gold-labeled anti-rabbit IgG as a

secondary antibody. No surface labeling was obtained in the control experiment in which the pre-immune serum was used. 240,000·magnification. (B) Western blot analysis of Post III/20 serum from rabbit 214 immunized with Rv-2004c-7 synthetic polymerized

peptide. (Lane 1) Analyzed against M. tuberculosis cell wall fraction; (lane 2) analyzed against M. tuberculosis culture supernatant.


Forero et al.

amino acids were defined as being those amino acids that,when replaced by glycine in HABPs, were not able toinhibit original peptide binding by at least 50% in theconditions used in this assay (Ocampo et al. 2002; Vera-Bravo et al. 2003). Figure 7 shows those critical aminoacids for the interaction between the five Rv2004c proteinHABPs and the U937 cells. The critical residues for eachHABP are underlined, as follows: Rv2004c-6 (101RDKQRLASMVTAGLPVEGAL120), Rv2004c-14 (261AGYAVRSGDTAPASLRDFYI280), Rv2004c-15 (281AYRAVVRAKVECVRFSQGKP300), Rv2004c-16 (301EAAADAVRHLIIATQHLQHA320), and Rv2004c-18 (341GV

AELVGAQVISTDDVRRRL360); these residues werelocated along the sequences for peptides Rv2004c-6,Rv2004c-14, Rv2004c-15, and Rv2004c-16 and for pep-tide Rv2004c-18 at the C terminus.

A549 cells could not be used in competition bindingassays because of the lack of glycine analog peptides(poor efficiency when being synthesized).

HABP receptors

U937 and A549 cells, incubated earlier on with each 125I-HABP, were cross-linked, and membrane proteins were

Figure 4. Peptide-U937 cell-binding behavior for Rv2004c-14, Rv2004c-19, and Rv2004c-12. (A,C,E) Total binding (—•—)

U937 cells incubated with 125I-peptide, and nonspecific binding (—&—) 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.



then separated by SDS-PAGE (Garcia et al. 2004; Val-buena et al. 2005). The HABPs from the Rv2004c proteinbound to molecules located on U937 and A549 cell mem-branes were visualized by autoradiography using 125I-Rv2004c-6 and 125I-Rv2004c-14 peptides. Figure 8 shows32- and 49-kDa bands for U937 cells (lanes 1 and 3) andfive bands ranging from 40 to 97 kDa for A549 cells (lanes5 and 7). These bands disappeared or their intensity becamereduced when the binding assay was performed in thepresence of excess unlabeled peptide (Fig. 8, lanes 2,4,6,8).The same assay was performed with RBCs as negativecontrol, in which no signal appeared (data not shown).

CD spectroscopy

Circular dichroism spectra were obtained in the presenceor absence of TFE when determining HABP secondarystructure. All HABPs displayed typical random struc-ture spectra in water (data not shown). All HABPpeptides’ CD profiles indicated a clear shift toward anordered structure in more hydrophobic medium, possi-bly a typical a-helix as characterized by double minimaat 208 and 220 nm and 190 nm maximum ellipticity.Figure 9 shows CD profiles for Rv2004c-6, Rv2004c-16, and Rv2004c-20 HABPs.

Discussion

Elucidating the M. tuberculosis genome sequence (Coleet al. 1998) and innovative proteomic technology,together with advances made in the field of bioinfor-matics, have allowed a great number of proteins to beidentified, making them the focus of studies aimed atdetermining their biological function. This systematicanalysis may lead to identifying new targets (individualproteins or functional links) or virulence factors respon-sible for the persistence of tuberculosis and its patho-genicity, which can be used both in diagnosing tuber-culosis and for proteomic-based vaccine development.The identification of 1044 proteins and their corre-sponding subcellular localization have been recently re-ported; a combination of high-throughput proteomicsand computational approaches were used for elucidatingthe globally expressed proteins (Mawuenyega et al. 2005).

This work describes one of these proteins (Rv2004c),classified in group VI as being a protein having anunknown function. The Rv2004c gene consists of 1494bp encoding a 498 amino acid long polypeptide having a54.42 kDa calculated molecular mass (Cole et al. 1998;Mawuenyega et al. 2005). The presence of the Rv2004cgene was initially revealed in M. tuberculosis complexstrains (Fig. 1A, lanes 2–7) and in the 10 clinical isolates

Figure 5. Rv2004c protein peptides’ amino acid sequences and binding profiles to target cells. The specific binding ·100 slope

value curve is indicated as black bars for U937 cells and gray bars for A549 cells. Peptides with slope values ·100 $2% were

selected as being high specific binding peptides (HABPs).


Forero et al.

(Fig. 1B, lanes 11–20) by PCR assays, leading to thesuggestion that this gene isM. tuberculosis complex exclu-sive. The deciphering of the genome has determined thatM. tuberculosis is highly conserved and lacks interstraingenetic diversity (Cole et al. 1998). As shown in the pre-sent study, the Rv2004c gene shares this behavior, beingcompletely conserved in all strains tested. However,recent studies using a variety of low- and high-resolutioncomparative genome techniques for identifying differ-ences in the genomes of M. bovis, the M. bovis BCG

vaccine strain, and the M. tuberculosis H37Rv laboratorystrain and clinical isolates have identified sequencechanges among the different mycobacterial species andstrains (Fleischmann et al. 2002; Gao et al. 2005). Thishas led to suggesting that the Rv2004c gene could belocalized in a highly conserved M. tuberculosis H37Rvgenome region.

The RT-PCR assays demonstrated this gene’s differen-tial transcription in all M. tuberculosis complex strainsexcept inM. bovis and M. microti (Fig. 1C, lanes 4 and 7,respectively). The transcription inM. tuberculosisH37Rv,M. tuberculosisH37Ra, andM. bovis BCG (Fig. 1C, lanes2,3,5) was more evident than transcription in Mycobac-terium africanum (Fig. 1C, lane 6). Although these differ-ences in transcription might have functional relevance, wecannot rule out the possibility that they occur as a con-sequence of the culture growth conditions, as previousreports have shown gene expression to be extremely sen-sitive to this factor (Gao et al. 2005).

Bearing in mind that not all transcribed genes areexpressed, rabbit antisera raised against polymeric pep-tides Rv2004c-7 and Rv2004c-19 belonging to Rv2004cprotein were tested against M. tuberculosis total soni-cate; serum from rabbit 214 (immunized with Rv2004c-7

Figure 6. Saturation assay using peptide concentrations in the presence or absence of unlabeled peptide. The results for the

specific binding to U937 cells are shown on the top and to A549 cells are shown at the bottom. The larger graph is the saturation

curve and the smaller one a Hill plot. In the Hill plot the abscissa is Log F and the ordinate is Log [B/(Bmax-B)], where B is the

bound peptide concentration, Bmax is the maximum bound peptide concentration, and F corresponds to the free peptide

concentration.

Table 1. Binding constants of Rv2004c-HABPs to U937 and

A549 cells

Macrophage (U937 cells) Epithelial (A549) cells

Peptide Kd (nM) NrSc (1 · 106) Kd (nM) NrSc (1· 106)

Rv2004c-5 — — 700 2.1

Rv2004c-6 760 19.1 550 3.3

Rv2004c-14 950 7.6 400 1.7

Rv2004c-15 580 3.0 330 1.7

Rv2004c-16 750 14.0 — —

Rv2004c-18 480 12.3 400 4.6

Rv2004c-20 — — 550 4.0



[121CGDAIAEVLARFHQRAQRNRCIYGC140] poly-merized peptide) specifically recognized a 54-kDa band(Fig. 2, lanes 4–6) corresponding to this protein’sexpected molecular weight (54.4 kDa). Antisera inducedby immunization with polymerized peptide Rv2004c-19(361CGRDCGVITGEPGVLDSGLYSRGC380) specifi-cally recognized a 27-kDa band (Fig. 2, lane 2), possiblydue to Rv2004c protein cleavage, suggesting that theantisera produced against the protein’s N-terminal por-tion recognized the whole molecule, while that directed

against the C-terminal portion only recognized a cleav-age product. Nevertheless, further experiments are need-ed to confirm this result.

Transmission electron microscopy studies using theanti-peptide Rv2004c-7 sera (rabbit 214) were carriedout for detecting the possible presence of the Rv2004cprotein on the mycobacterial cell surface. The reducednumber of immunogold signals (Fig. 3A) could havebeen due to a low abundance of protein, perhaps, asa result of culture conditions (affecting mycobacterial

Figure 7. Competition assay with U937-HABP glycine analog peptides. The height of the black bars is proportional to the

binding of 125I-HABP (100 nM) being inhibited by the unlabeled HABP (*) (0.4 mM and 4.0 mM) or unlabeled Gly-analog

peptides. The amino acid at the bottom represents the residue replaced by glycine in the analog peptide. The critical residues for

each HABP are shown underlined.


Forero et al.

protein expression) (Brennan et al. 2001; Florczyk et al.2001; Banu et al. 2002), or the low antibody concentra-tion present in the sera. Previous studies have demon-strated that antibodies obtained against mycobacterialproteins can (by using TEM and immunoblotting stu-dies) determine their subcellular location (Amara et al.1998; Banu et al. 2002). Moreover, the results obtainedby Western blot confirm this protein’s presence on themycobacterial cell surface, specifically in the cell wall; inaddition, since no band was detected on culture super-natant, it seems that this protein is not being secreted.Our observations were in agreement with recentlyreported results determining the Rv2004c protein’s pre-sence in the cell wall (Mawuenyega et al. 2005).

One of our laboratory’s research aims is to identifypathogen surface protein regions that are involved in thecomplex processes of host cell recognition and invasion.We have recently reported several studies aimed at char-acterizing receptor–ligand interactions between syntheticpeptides derived from pathogen proteins specificallyrecognizing the host cell. This method was used fordetermining specific binding regions in proteins fromsimple pathogens such as hepatitis C virus (Garcia etal. 2002), Human papillomavirus (Vera-Bravo et al.2003), and Epstein-Barr virus (Urquiza et al. 2004). Ithas also been used in more complex protozoa such asLeishmania (Puentes et al. 1999), Plasmodium falciparumand Plasmodium vivax (Ocampo et al. 2002; Rodriguez

et al. 2002; Urquiza et al. 2002), malarial blood (Curti-dor 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 thebacterium is ingested by alveolar macrophages and subse-quently gains access to the bloodstream by being trans-ported by the alveolar macrophages and blood monocytesthrough the alveolar wall. However, several groups haverecently demonstrated that M. tuberculosis invades andsurvives within human type II alveolar epithelial cells invitro (Bermudez and Goodman 1996). This backgroundhas led us to determine those Rv2004c protein regions thatcould be involved in the mycobacteria–host cell interac-tion. Binding assays were done using monocyte-like U937and A549 type II alveolar epithelial cells. This protein’sU937 and A459 cell-binding capacity was determined byusing synthetic peptides. Three types of target-cell-bindingbehavior were found for these peptides (Fig. 4). Figure 4,A and B, shows HABP-U937 interaction for a high bind-ing 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 U937cells but there was no inhibition with the same nonradio-labeled peptide (Fig. 4D). Figure 4, E and F, shows apeptide that did not show binding to U937 cells, thatis, Rv2004c-12 (221LLDCLEFEDELRYLDRIDDA240).The same behavior was observed in A549 epithelial cell-binding assays (data not shown); the affinity was generallycharacteristic of high-affinity interactions for both types ofcell used (binding curve slope $2%).

The assay using U937 monocytoblastic cells revealedfive HABPs for this protein; four of them were located in

Figure 8. Cross-linking assay between two HABPs and target cells.

U937 and A549 cells were incubated with 125I-labeled peptides in the

absence (total binding, lanes 1,3,5,7) or presence (inhibited binding,

lanes 2,4,6,8) of unlabeled peptide. Rv2004c-6 and Rv2004c-14 pep-

tides bind to 32–49 kDa bands onto U937 cells and bind to 40, 66, 74,

90, and 97 kDa bands onto A549 cells.

Figure 9. Circular dichroism spectra. All U937- and A 549-HABPs

displayed a characteristic curve suggesting an a-helix conformation.

The figure shows the three most representative curves graphed for

Rv2004c-6, Rv2004c-16, and Rv2004c-20.



the central region: Rv2004c-14 (261AGYAVRSGDTAPASLRDFTI280), Rv2004c-15 (281AYRAVVRAKVECVRFSQGKP300), Rv2004c-16 (301EAAADAVRHLIIATQHLQHA320), and Rv2004c-18 (341GVAELVGAQVISTDDVRRRL360); another one, Rv2004c-6 (101RDKQRLASMVTAGLPVEGAL120), was located in theN-terminal region. Six A549 epithelial cell HABPs werealso found; Rv2004c-14 (261AGYAVRSGDTAPASLRDFTI280), Rv2004c-15 (281AYRAVVRAKVECVRFSQGKP300), Rv2004c-18 (341GVAELVGAQVISTDDVRRRL360), and Rv2004c-20 (381ANVVAVYQEALRKARLLLGS400 were located in the protein’s centralregion; and Rv2004c-5 (81AHLSDPSGGHAEPVVVMRRY100) and Rv2004c-6 in the N-terminal region (Fig.5). These results suggest that target cell interaction withthe Rv4004c protein is preferentially presented by theprotein’s central region.

Determining physico-chemical binding constantsrepresents one way of characterizing HABP interactionwith target cells (Kd, affinity constant and the number ofbinding sites per cell). Table 1 shows that HABP affinityfor cellular surface molecules was greater for A549epithelial cells than U937 monocyte cells; however, thiseffect was compensated for by the greater number ofbinding sites per cell found for U937 cells than forA549 cells. Saturation assays (Fig. 6) also establishedthat HABP binding to U937 and A549 target cells wasspecific and saturable.

The exact role of amino acids directly involved in inter-action with target cell surface molecules has not beencompletely determined; however, it has been recentlyreported that identifying them could represent a noveltool for designing immunogenic and protection-inducingmolecules (Espejo et al. 2001; Purmova et al. 2002).Critical residues for the binding process were found foreach HABP-U937 in glycine peptide analog competitionbinding assays (Fig. 7). It is likely that these critical resi-dues may not only correspond to those amino acids directlyinvolved in binding to receptors on U937 cells but mayalso be responsible for keeping the peptide conformationthat is necessary for binding. Such observations suggestthat each peptide requires different residues for its interac-tion with a particular target cell and that most HABPs arelocalized along a particular peptide’s sequence (showingsuch sequence’s specificity). A clear example would be thatshown by the arginine residues in the Rv2004c-6 peptide,where peptide analog binding remained unchanged whensubstituting R101 for Gly, while binding became drasticallyreduced when substituting R105. The same thing happenedwith alanine residues in the Rv2004c-16 peptide whenbinding remained unchanged on substituting A320, whilechanging residues A303, A306, and A313 led to noticeablyreduced binding. This confirmed that each HABP’s bind-ing activity was influenced more by a particular HABP’s

spatial conformation than by the nature of the amino acidinvolved in the binding.

Although the nature of U937 cell and A549 cell recep-tors for HABPs remains unknown, it was found thatHABPs Rv2004c-6 and Rv2004c-14 bound to 32- and49-kDa bands on the U937 cell surface (Fig. 8, lanes 1,3)and to 40-, 66-, 74-, 90-, and 97-kDa bands on the A549cell surface (Fig. 8, lanes 5,7). This confirmed that inter-action with these bands was specific since they disap-peared on each 125-I-HABP’s binding being inhibited bythe respective nonradiolabeled HABP (Fig. 8, lanes2,4,6,8). The fact that this protein contained regionsspecifically interacting with epithelial and monocytecells suggests that it could be implicated in mycobacter-ial binding to host cells. Previous works have posed thepossibility that receptors on epithelial cell and macro-phage surfaces could be adhesion molecules such asCD11a, CD11b, VLA4, or ICAM-1. Even though ourresults were very close to some of these adhesionmolecules’ molecular weights, more studies must bedone to enable their biochemical characterization andtheir role in bacillus and target cell recognition andinteraction (Bermudez et al. 2002).

Experimental results coincided with the Rv2004cprotein’s secondary structure prediction according toHABP circular dichroism structural analysis (Fig. 9),where 64% of the amino acid sequence will tend toform a-helix structures (Combet et al. 2000). Theirhigh target cell binding activity also suggests some struc-ture–affinity relationship since all HABPs could con-serve the same type of arrangement independently ofthe target cell with which they are interacting.

The present work’s most important findings concernRv2004c gene presence in all M. tuberculosis complexstrains and clinical isolates, its transcription and expres-sion in several M. tuberculosis complex strains, and thesurface location of the protein on the bacillus and itsability to bind to monocyte (U937) and type II alveolarepithelial (A549) cells. This has important biologicalsignificance and implications for developing strategiesfor controlling this disease. More detailed studies arestill needed for determining HABPs’ role in the complexprocess of bacillus interaction with its target cell.

Materials and methods

Molecular assays

Mycobacterial strainsThe ATCC and Trudeau Mycobacterial 88 Collections

(TMC) were the sources of most of those mycobacterial strainsused, except M. microti, which was kindly provided by Dr. F.Portaels from the Institute of Tropical Medicine. In addition,10 different M. tuberculosis isolates were obtained from


Forero et al.

patients attending the TB program at either the San Juan deDios Hospital or the Santa Clara Hospital, both in Bogota. Allmycobacterial strains were grown for 5–15 d (rapid growth) inMiddlebrook 7H9 and Middlebrook 7H10 agar (Difco), bothsupplemented with OADC (oleic acid, albumin, dextrose, andcatalase) (BBL; BD).

Chromosomal DNA extractionChromosomal DNA was isolated by using a previously

described method (Mahairas et al. 1996). DNA was precipi-tated with 0.6 volumes of 2-propanol. The pellet was washedwith 70% (v/v) ethanol and suspended in 1· TE (Del Portillo etal. 1991; Parra et al. 1991).

PCR conditions and primersPCR amplification was done in a thermal cycler Gene Amp

PCR system 9600 (Perkin Elmer) using 100 ng of M. tubercu-losis genomic DNA. The mixture contained 50 mM KCl, 10mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.1 mM each deox-ynucleoside triphosphate, 0.4 mM both forward IN-1 (5¢-GATGGCGAACCGGCGCTG-3¢) and reverse IN-2 (5¢-TAGAGCCCGGAGTCCAAA-3¢) internal primers, and 1.5U of Taq DNA polymerase (Promega). Then 30 cycles of thefollowing thermal profile were carried out after 5 min of DNAdenaturing at 95˚C: 59˚C for 30 sec, 72˚C for 30 sec, and 94˚Cfor 30 sec. A final 5-min extension cycle performed at 72˚C wasused for PCR amplification.

DNA sequencingThe dideoxy chain-termination method was followed by a

sequencing reaction with the Taq FS DyeDeoxy TerminatorCycle Sequencing kit (Applied Biosystems) using a GeneAmpPCR system 9600 (Perkin-Elmer) and run on a DNA AnalysisSystem model 373 (Applied Biosystems). The sequencing strat-egy involved using M. tuberculosis Rv2004c putative proteinforward and reverse primers.

Reverse transcriptase-PCRTotal RNA was extracted from different mycobacterial

strains by Trizol (GIBCO), then treated with RNase-freeDNase RQ1, and purified by standard phenol-chloroformprocedure (Alland et al. 1998). RNA preparation, concentra-tion, and purity were spectrophotometrically measured. TargetRNA (20 mg/mL) was reverse-transcribed in a single tubecontaining distilled water and 10 mg/mL random primers(GIBCO). This mixture was incubated at 70˚C for 10 min andchilled on ice. Then 1· 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 RibonucleaseInhibitor (Platinum Taq) were added. Following this, 200 U ofM-MLV reverse transcriptase (GIBCO) was added to a final40-mL volume. This mixture was kept at 37˚C for 1 h. Theenzyme was denatured at 95˚C for 5 min. Then 5% of thetotal RT mixture was used as template for PCR, which wascarried out as described above. Appropriate positive and nega-tive controls were included in each experiment. The rpoB genewas 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 genesimplicated in its metabolism. DNAse-Q treatedM. tuberculosis

H37Rv was used as the cDNA synthesis negative control.Sterile distilled water and M. tuberculosis H37Rv DNA wereused as the PCR negative and positive controls, respectively.

Cell-binding assays

Cell linesTwo cell lines, susceptible to invasion by M. tuberculosis,

were used in this study: nonadherent monocyte-like U937 cellsand A549 cells (adherent epithelial cell line derived from apulmonary carcinoma). The lines were obtained from ATCCand kept in culture using RPMI 1640 (GIBCO) and 10% fetalbovine serum (Hyclone) at 37˚C and 5% CO2 (at 105 cell/mLdensity) in 275-mL culture dishes. Adherent A549 cells weredislodged with 0.1% EDTA-PBS. Both cell lines were collectedin 50-mL tubes and centrifuged at 2000g for 5 min, washedwith PBS, and counted in a Neubauer chamber (Sato et al.2000; Passmore et al. 2001; Liu et al. 2004).

Peptide synthesisTwenty-five sequential, 20-mer-long, amino acid peptides,

spanning the complete M. tuberculosis Rv2004c putative pro-tein sequence, were chemically synthesized using the MultiplePeptide Synthesis method in Solid Phase. MBHA resin (0.7meq/g), t-Boc amino acids, and low-high cleavage techniqueswere used (Merrifield 1963; Tam et al. 1983; Houghten 1985).Peptide identity and purity were analyzed by MALDI-TOFmass spectrometry and analytical reverse-phase, high-perfor-mance liquid chromatography (RP-HPLC) and freeze-dried.The synthesized peptide sequences are shown in Figure 3 inone-letter code. Tyr was added at the C-terminal end of thosepeptides that did not contain it to enable radiolabeling.

Radiolabeling125I-radiolabeling was done according to previously

described techniques (Yamamura et al. 1978; Hulme 1993), inwhich chloramine T (2.25 mg/mL) and 3.2 mL of Na125I (100mCi/mL) were added to 5 mL of peptide solution (1 mg/mL).Then 15 mL of sodium bisulfite (2.75 mg/mL) and 50 mL ofNaI (0.16 M) were added after 5 min of reaction at 18˚C. The125I-peptide was then separated from reaction subproducts ona Sephadex G-10 column (Pharmacia; 80· 5.0 mm).

Binding assayU937 or A549 cell lines kept in RPMI 1640 culture medium

(1 · 106 cells), previously washed in isotonic PBS and incubatedwith increasing quantities of each 125I-radiolabeled M. tuber-culosis putative Rv2004c protein peptide (between 100 and2000 nM), were used for binding assays in a 100-mL totalvolume for 90 min at 4˚C, in the presence or absence of 40mMunlabeled peptide to determine binding specificity (bindingassays were done for each of the Rv2004c protein peptides).Unbound peptide was removed from cells following incubationby sedimentation through a dibutylphthalate–dioctylphthalatecushion mixture (d=1.015 g/mL) and spun at 9000g for 2 min(Garcia et al. 2004; Vera-Bravo et al. 2005). The cell-boundpeptide was measured in an automatic gamma counter (4/200plus ICN Biomedicals, Inc.). The assay was carried out intriplicate under identical conditions; the mean results of thetriplicate assays are reported and graphically represented inFigures 4 and 5.



Determining physico-chemical constantsfor the HABP–target cell interaction

Saturation assayCells were first washed twice with RPMI 1640 medium and

then washed twice with isotonic PBS before carrying out thebinding assays. Saturation binding assays were carried out foreach peptide presenting high specific target cell binding activ-ity. In accordance with a previously described method(Ocampo et al. 2000; Rodriguez et al. 2000; Garcia et al.2002), 1 · 106 U937 or A549 cells were incubated with increas-ing 125I-peptide concentrations between 100 and 3000 nM in a120-mL total volume for 90 min at 4˚C, in the presence orabsence of 40 mM unlabeled peptide to determine bindingspecificity. After incubation, unbound peptide was removedas described above. As before, each assay was performed intriplicate; bound and free 125I-peptides were determined bygamma counter measurement. The curves so obtained wereanalyzed by Scatchard analysis, and affinity constants weredetermined by the Hill equation (Garcia et al. 2004; Vera-Bravo et al. 2005).

Determining critical amino acids inthe HABP–U937 cell interaction

Competition binding assayHABP Rv2004c-6, Rv2004c-14, Rv2004c-15, Rv2004c-16,

and Rv2004c-18 glycine scanning analogs were synthesized torecognize critical target cell binding residues. Then 1 · 106U937 cells were incubated with increasing quantities (0.2,0.4, 4.0, and 40.0 mM) of each unlabeled analog peptide ororiginal unlabeled peptide in the presence of native 125I-pep-tide for the competition binding assays at a final 255-mLvolume. After 90 min of incubation at 4˚C, the mixture wascentrifuged at 9000g for 2 min through a dibutylphalate cush-ion. The radioactivity bound to U937 or A549 cells wasmeasured in a gamma counter. Data from triplicate assayswere 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 U937cell-binding sites, based on the methodology previouslyreported (Garcia et al. 2002). Briefly, 2 · 106 U937 cellswere subjected to a conventional binding assay (incubationfor 90 min at 4˚C) with 125I- Rv2004c-6 and 125I-Rv2004c-14HABPs. Cells were washed with PBS following incubationand subjected to cross-linking with 25 mM, Bis(sulfosucci-nimidyl suberate) (BS3, Pierce) for 20 min at 4˚C. Thereaction was stopped with 40 nM Tris-HCl (pH 7.4), andcells were washed again with PBS. They were then treatedwith lysis buffer (5% SDS, 10 nM iodoacetamide, 1% Tri-ton X-100, 100 mM EDTA, 10 mM PMSF). The obtainedmembrane proteins were solubilized in Laemmli buffer andseparated in SDS-PAGE. Those proteins cross-linked withradiolabeled peptides were exposed on a Bio-Rad ImagingScreen K (Bio-Rad Molecular Imager FX; Bio-Rad Quan-tity One Quantitation Software) for 7 d, and the apparentmolecular weight was determined by using 6.4–198-kDa or14–97-kDa molecular weight markers (Bio-Rad).

CD spectroscopy

Circular dichroism (CD) was carried out for each HABP todetermine whether there was any conformational–functional cor-relation. CD spectra were recorded for each HABP at 20˚C on aJasco J-810 spectropolarimeter at wavelengths ranging from 260to 190 nm in 1.00-cm cuvettes (Provencher and Glockner 1981).The peptides were dissolved at 0.1 mM concentration in purewater or in aqueous TFE solutions containing 30% TFE byvolume. Each spectrum was obtained from averaging three scanstaken at a 20-nm/min scan rate with 1 nm spectra bandwidth,corrected for baseline. The results were expressed as the meanresidue ellipticity [Q], the units being degrees· centimeters squaredper decimole according to the function [Q]=Ql/(100lcn), whereQl is the measured ellipticity, l is the optical pathlength, c is thepeptide concentration, and n is the number of amino acid residuescontained in the sequence (Sreerama et al. 1999).

Rabbit immunization

The numbers-in-the-bag method was used for randomly select-ing 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). Thesewere synthesized as described above but CG residues wereadded to each extreme to facilitate their polymerization.Three New Zealand strain rabbits per peptide, having pre-viously been determined to be nonreactive to M. tuberculosissonicate by Western blotting, were subcutaneously immunizedwith 500 mg of Rv2004c-7 polymer peptide, to amino acidsequence 121CGDAIAEVLARFHQRAQRNRCIYGC140 andRv2004c-19 polymer peptide, corresponding to amino acidsequence 361CGRDCGVITGEP GVLDSGLYSRGC380. Poly-merized peptides were emulsified with Freund’s IncompleteAdjuvant (1:1 v/v) and used for immunizations on days 0, 20,and 40. Final bleeding was carried out on day 60, and the serawere collected. The immunizations and bleedings were all car-ried out according to the handling procedures required by theColombian Ministry of Public Health.

Mycobacterial sonicate and subcellular fractions

Ten grams (wet weight) of mycobacteria was suspended in 20mL of phosphate-buffered saline (PBS) containing DNase,RNase, and a proteinase inhibitor cocktail (phenylmethylsul-phonyl fluoride [PMSF] and EDTA at a final 1 mM concen-tration of each one and 1 mg/mL leupeptin and 1 mg/mLpepstatin A). Sonication was done in a Branson Sonifier 450for 20 min with amplitude set at 4 and 90% duty cycle. Thesonicate was centrifuged at 650g for 20 min. The supernatantwas then centrifuged at 36,000g for 45 min at 4˚C. The proteinconcentration was determined by bicinchoninic acid assay(BCA kit; Pierce) and stored in aliquots at -70˚C until needed.The mycobacterial subcellular fractions (cell wall, mem-

brane, and culture filtrate proteins) were purchased from Col-orado State University.

Rabbit sera adsorption with Escherichia coli andMycobacterium smegmatis sonicate

M. smegmatis proteins were obtained from 5-d-old Middleb-rook 7H9 broth culture, washed, suspended, sonicated for 10


Forero et al.

min (as described above), and centrifuged for 10 min at 4500gat 4˚C. E. coli (DH5a strain) proteins were obtained fromovernight culture in Luria Bertani medium, washed, sus-pended, sonicated for 2 min at 4˚C, and centrifuged for 10min at 4500g. Both pellets were suspended in coupling buffer(0.1 M NaHCO3 at pH 8.3). The suspended lysates were col-lected and used individually for coupling to CNBr-activatedSepharose 4B (Pharmacia Biotech), according to the man-ufacturer’s recommendations. Each rabbit serum (pre-immuneand immune) was pre-adsorbed on E. coli-Sepharose and M.smegmatis-Sepharose affinity columns to eliminate cross-reac-tivity. Briefly, 5 mL of each serum was added to 4 mL of lysate-Sepharose affinity columns and left in a gentle rotating/shak-ing mode for 20 min at room temperature. This procedure wasdone twice using a new lysate-Sepharose affinity column eachtime.

SDS-PAGE and immunoblotting

Proteins from the M. tuberculosis sonicate (36,000g superna-tant fraction) and subcellular fractions were separated in adiscontinuous SDS-PAGE system, using a 10% to 20% (w/v)acrylamide gradient. A total of 1 mg of sample was loaded pergel and transferred to nitrocellulose membranes (Hybond203C, Pharmacia) using the semidry blotting technique(Kyhse-Andersen 1984). Commercial molecular mass markers(New England Biolabs) were used for calibration. The filterswere incubated with a 1:100 dilution of the sera obtained fromrabbits immunized with polymerized peptides Rv2004c-7 andRv2004c-19. Pre-adsorbed sera were diluted in TBST (0.02 MTris-HCl at pH 7.5, 0.05 M NaCl, 1% Tween 20) and 5%skimmed milk. Then they were incubated for 1 h with 1:3000alkaline phosphatase conjugated anti-rabbit IgG antibody(ICN) following five TBST washes. The reaction was devel-oped with NBT/BCIP (Promega).

Electron microscopy

Transmission electron microscopy (TEM) studies were car-ried out on a Philips CM 10 TEM. Fifty microliters of M.tuberculosis H37Rv wet pellet was fixed with a 4% parafor-maldehyde–0.5% glutaraldehyde solution for 2 h at 4˚C forsample preparation. The pellet was dehydrated in gradedethanol (50%, 70%, 80%, 90%, and twice 100%) afterbeing fixed and was then embedded in LR-white hard-gradeacrylic resin (Sigma) for 4 d at 4˚C. Then 400-nm-thin sectionswere cut and mounted on 300 mesh nickel grids. These sec-tions were incubated in a saturated sodium metaperiodatesolution for antigen retrieval (Stirling and Graff 1995).Grids were then floated sections down in a beaker containing0.01 M sodium citrate buffer for 15 min at 80˚C. After 1 h ofblocking in Tris-buffered saline (TBS) (0.05 M Tris in isotonicsaline at pH 7.6) containing 0.05% BSA, grids were incubatedin pure rabbit polyclonal serum for 1 h at 37˚C. Following aTBS–0.025% Tween 20 wash, grids were immersed in a 1:50dilution of 5 nm gold-labeled anti-rabbit IgG (Sigma) for 1 hat room temperature, according to the manufacturer’sinstructions. Grids were then washed with TBST and fixedin 2.5% glutaraldehyde. Then a 15-min incubation was car-ried out in 1% uranile acetate after fixing. Grids were washedwith distilled water and dried at room temperature beforeobservation.

Acknowledgments

This work was supported by the Colombian President’s Officeand the Colombian Ministry of Public Health. We are gratefulto Jason Gary for reading and revising the work.

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