The Mycobacterium tuberculosis membrane proteinRv2560 ) biochemical and functional studiesDavid F. Plaza1, Hernando Curtidor2,4, Manuel A. Patarroyo1,4, Julie A. Chapeton-Montes1,Claudia Reyes3, Jose Barreto3 and Manuel E. Patarroyo1,5

1 Molecular Biology Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia

2 Receptor–Ligand Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia

3 Chemical Synthesis Department, Fundacion Instituto de Inmunologia de Colombia, Bogota, Colombia

4 Universidad del Rosario, Bogota, Colombia

5 Universidad Nacional de Colombia, Bogota, Colombia

Tuberculosis (TB) is a major public health problem

throughout the world, affecting almost nine million

people [1] and causing more than three million deaths

per year. An increasing incidence of TB, related to the

high risk of developing the disease in immuno-

suppressed individuals and the increasing proportion

of Mycobacterium tuberculosis drug-resistant strains,

has contributed to this problem [2,3]. This makes the

Keywords

high-activity binding peptide; invasion

inhibition; Mycobacterium tuberculosis –

host cell interaction; Rv2560 membrane

protein

Correspondence

M. E. Patarroyo, Carrera 50 # 26-00,

Bogota, Colombia

Fax: +57 (1) 4815269

Tel: +57 (1) 4815219 or +57 (1) 3244672

Ext 125

E-mail: mepatarr@mail.com

(Received 31 July 2007, revised 11 October

2007, accepted 17 October 2007)

doi:10.1111/j.1742-4658.2007.06153.x

The characterization of membrane proteins having no identified function in

Mycobacterium tuberculosis is important for a better understanding of the

biology of this pathogen. In this work, the biological activity of the

Rv2560 protein was characterized and evaluated. Primers used in PCR and

RT-PCR assays revealed that the gene encoding protein Rv2560 is present

in M. tuberculosis complex strains, but transcribed in only some of them.

Sera obtained from rabbits inoculated with polymer peptides from this pro-

tein recognized a 33 kDa band in the M. tuberculosis lysate and a mem-

brane fraction corresponding to the predicted molecular mass (33.1 kDa)

of this protein. Immunoelectron microscopy analysis found this protein on

the mycobacterial membrane. Sixteen peptides covering its entire length

were chemically synthesized and tested for their ability to bind to A549

and U937 cells. Peptide 11024 (121VVALSDRATTAYTNTSGVSS140)

showed high specific binding to both cell types (dissociation constants of

380 and 800 nm, respectively, and positive receptor–ligand interaction

cooperativity), whereas peptide 11033 (284LIGIPVAALIHVYTYRKLS

GG304) displayed high binding activity to A549 cells only. Cross-linking

assays showed the specific binding of peptide 11024 to a 54 kDa membrane

protein on U937. Invasion inhibition assays, in the presence of shared

high-activity binding peptide identified for U937 and A549 cells, presented

maximum inhibition percentages of 50.53% and 58.27%, respectively. Our

work highlights the relevance of the Rv2560 protein in the M. tuberculosis

invasion process of monocytes and epithelial cells, and represents a funda-

mental step in the rational selection of new antigens to be included as

components in a multiepitope, subunit-based, chemically synthesized, anti-

tuberculosis vaccine.

Abbreviations

GRAVY, grand average of hydropathicity; HABP, high-activity binding peptide; SPf66, synthetic Plasmodium falciparum 66; SSP, simple

sequence protein; TB, tuberculosis.

6352 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS

development of new therapies and ⁄or vaccines, or the

improvement of existing ones, necessary to control this

disease.

The identification and characterization of the pro-

teins involved in the interaction of M. tuberculosis with

its target cells and the mechanisms for evading an

immune response produced against this pathogen are

thus of crucial interest. Of special interest are those

proteins found on the cell membrane and in the

secreted fraction, as they represent the ‘face’ shown by

the bacterium to its host, thereby leading to its identifi-

cation and elimination.

Although TB is mainly a pulmonary disease, it can

also affect other organs. Few microorganisms can sur-

vive within macrophages because of the abundance of

hydrolytic enzymes contained in acid phagocytic com-

partments [4]. M. tuberculosis has developed evolution-

ary mechanisms that allow it to penetrate, survive and

grow within these compartments [5]. Adhesion and

phagocytosis are the initial steps in the entry of the

bacterium into macrophages, processes triggered by

receptor–ligand interactions. These interactions are

complex and have an important influence on the intra-

cellular fate of M. tuberculosis [6,7], being mediated

mainly by membrane proteins present on the bacillus

and its host [8]. The M. tuberculosis cell membrane,

and that of other prokaryotes, contains proteins func-

tioning as enzymes, ligands, signal transducers or viru-

lence factors, all of great importance for bacterial

survival [9].

M. tuberculosis invades alveolar epithelial cells,

thereby inducing macropinocytosis, using secreted or

membrane components [10]. The identification of the

bacterial peptides involved in this process has been

poorly studied, and represents an objective of this

work.

Membrane proteins represent a bank of antigens

available for the design of a subunit-based, multiepi-

tope vaccine, as they may be involved in the patho-

genicity of the bacterium and may also be highly

susceptible to immune recognition. The M. tuberculosis

membrane proteome has revealed the presence of mul-

tiple immunogenic components with unknown function

[11–13], which could be used for this purpose.

Bioinformatics represents a preliminary method for

the evaluation of the presence of a determined gene or

protein sequence in different organisms, and for pre-

dicting (with some degree of confidence) the existence

of membrane anchoring or signal export regions within

them, making it a fundamental tool when screening

and ⁄or selecting for a vaccine candidate [14,15]. Fur-

thermore, work carried out on the proteomics of the

bacterium has led to the identification of a large

number of membrane proteins with an as yet undeter-

mined function [11–13].

A previous study has shown the importance of the

melA gene (encoding an Rv2560 homologous protein)

in Mycobacterium marinum invasion of THP-1 cells,

indicating that its homologous gene might be relevant

in M. tuberculosis–host cell interaction [16].

This work has led to the identification of the pres-

ence of the Rv2560 proline- and glycine-rich trans-

membrane protein encoding gene and its transcripts in

the M. tuberculosis complex and clinical isolate strains,

as well as the characterization of the high-activity

binding peptide (HABP) involved in the binding to

and invasion of monocytes (U937) and type II alveolar

epithelial cells (A549), using synthetic peptides. The

protein encoded by the Rv2560 gene was synthesized

in 20-mer-long, nonoverlapping peptides, which were

analysed by a highly specific and robust methodology

(widely used in Plasmodium falciparum research) for

the identification of the HABPs interacting with U937

and A549 cells (in this case), their binding dissociation

constants, their critical binding residues and their role

in cell invasion inhibition, with the specific purpose of

employing them as probable components of a multiepi-

tope, subunit-based, synthetic vaccine. This has impor-

tant biological significance and implications for the

development of strategies to control this disease.

Results

Bioinformatic analysis

Different bioinformatics’ tools were used to predict the

cell location of Rv2560 and its transmembrane topol-

ogy. Although signalp did not predict the existence of

a signal peptide, the high grand average of hydropath-

icity (GRAVY) value (0.488) and the existence of

four possible transmembrane helices in the complete

Rv2560 amino acid sequence suggested its presence on

the bacterial surface. Membrane topology prediction

proposed four transmembrane helices (i103–125o,

o145–167i, i197–228o and o271–293I, where i denotes

inside and o denotes outside), suggesting that the iden-

tified HABPs are located in the protein’s solvent-

exposed region (Fig. 4, topology diagram, see later).

Genomic PCR assay

Two specific primers, flanking an HABP region

(encoding amino acids 80–290), were designed and syn-

thesized to determine the presence or absence of the

gene encoding the Rv2560 protein in M. tuberculosis

complex strains. A single 631 bp amplification band

D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization

FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6353

was observed in the following strains: M. tuberculosis

H37Rv, M. tuberculosis H37Ra, Mycobacterium bovis,

M. bovis BCG, Mycobacterium africanum and Myco-

bacterium microti (Fig. 1A, lanes 2–7). tblastn analy-

sis showed similar (although not identical) genes in

M. marinum and Mycobacterium ulcerans.

DNA sequencing

The amplified fragments in the different clinical iso-

lates studied were purified, and their DNA was

sequenced to determine this region’s polymorphism. It

was observed that this region was completely con-

served in all M. tuberculosis clinical isolates analysed,

independent of geographical origin. This result was

confirmed by sequencing each fragment’s complemen-

tary chain (data not shown).

RT-PCR assay

Two specific primers were designed and synthesized to

determine the transcriptional pattern of the Rv2560

gene in M. tuberculosis complex strains. A single

308 bp amplification band was observed in some

M. tuberculosis complex strains (Fig. 1B, lanes 2–7).

This gene’s transcription was exclusive to M. tuberculo-

sis H37Rv, M. tuberculosis H37Ra, M. bovis BCG and

M. africanum strains, but not M. bovis and M. microti,

suggesting that it was not transcribed, in spite of this

gene being present as assessed by PCR. Figure 1C

shows a 360 bp conserved fragment from a mycobacte-

rial genus constitutive gene (the rpoB gene encoding

the RNA polymerase b-subunit) used as transcription

control.

Western blot

Polymerized synthetic peptides corresponding to the

Rv2560 protein peptide 11027 (181ADGKPVTIATFF

RPRNLGLV200) and 11031 (242SPIDSVKASIETV

GSNIGGSVY262) amino acid sequences, located in

the extracellular solvent-exposed region (as predicted

by the TMHMM server), were used as immunogens in

rabbits, employing a 0, 20 and 40 day inoculation

scheme. Western blot (Fig. 2) was performed with

preimmune and post-third inoculation rabbit sera

against M. tuberculosis sonicate and membrane frac-

tion. Sera had previously been adsorbed on columns

with Escherichia coli and Mycobacterium smegmatis

lysates, as well as synthetic Plasmodium falcipa-

rum 66 (SPf66), prior to western blotting to eliminate

the cross-reactivity against bacterial (E. coli), myco-

bacterial (M. smegmatis) and polymerization (SPf66)

neo-antigens.

Lanes 1 and 3 in Fig. 2 show the lack of preimmune

rabbit sera protein recognition in the M. tuberculosis

sonicate and membrane fraction, respectively. Rabbits

immunized with 11031 polymerized peptide developed

polyclonal antibodies specifically recognizing a 33 kDa

molecular mass band in the M. tuberculosis sonicate,

close to the protein’s molecular mass (33.1 kDa) in

lane 2. Preimmune serum (lane 3) did not recognize

bands in the membrane fraction; however, postimmune

serum from 11027 immunized rabbits strongly recog-

nized a band close to 33 kDa in this fraction (lane 4),

suggesting that this protein becomes enriched in the

cell membrane.

Fig. 1. Genomic PCR and RT-PCR assays. (A) A 631 bp PCR prod-

uct from the gene encoding M. tuberculosis H37Rv Rv2560 protein

was only amplified on DNA from M. tuberculosis complex strains

(lanes 2–7). Lane 1, 1 kb molecular weight marker (Gibco); 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, PCR negative control. (B) M. tuberculosis

Rv2560 protein H37Rv 308 bp RT-PCR product obtained from

M. tuberculosis complex strain cDNA. Lane 1, 1 kb molecular

weight marker (Gibco); 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

[M. tuberculosis H37Rv DNA treated with DNAse Q (Promega)];

lane 9, PCR positive control (M. tuberculosis H37Rv DNA); lane 10,

PCR negative control. A single 308 bp amplification band was

observed in some M. tuberculosis complex strains. (C) PCR 360 bp

product from the same strains as (B) but with amplification of the

Mycobacterium rpoB gene as positive control for mRNA expression

and cDNA synthesis.

M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.

6354 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS

Immunoelectron microscopy

Rabbit serum raised against peptide 11027 was used

for immunolabelling. Immunoelectron microscopy

showed that colloidal gold particles (5 nm) were

mainly located on the surface of M. tuberculosis

H37Rv (Fig. 3), supporting the concept that this

expressed gene is located on the surface and is recog-

nized by serum directed against one of its forming pep-

tides. No labelling was observed in negative controls

with preimmune serum (data not shown).

High specific binding peptides

A highly specific, sensitive and robust receptor–ligand

binding assay was developed, based on previous stud-

ies with erythrocytes and hepatocytes [17–19]. This

methodology was adapted to other cell types, such as

monoblastic U937 and epithelial A549 cells, as they

are useful models for M. tuberculosis invasion studies

[20]. The binding assay was performed at four 125I-

radiolabelled peptide concentrations (10–200 nm) in

the presence or absence of at least a 400 times excess

of unlabelled peptide, as described previously [21–23].

The binding activity was defined as the slope of the

specific binding curve in the added peptide range. Pep-

tides showing binding activity greater than or equal to

1% were considered to be U937 or A549 cell HABPs.

A previously reported U937 and A549 cell HABP,

11095 (from the Rv1510c protein), was used as binding

assay control [23].

Figure 4 shows the Rv2560 synthetic peptide

sequences and the corresponding U937 or A549 cell

binding activities. The black bars represent the binding

activity. Those peptides having a binding activity ‡ 1.0

slope (dotted line) were considered to be cell line

HABPs. Peptide 11024 (121VVALSDRATTAYT

NTSGVSS140) was found to be the only U937 and

A549 cell HABP identified for the Rv2560 protein.

This peptide is predicted to be in the extracellular sol-

vent-exposed region, as determined by tmhmm and

tmpred online software. Peptide 11033 (284LIGI

PVAALIHVYTYRKLSGG304) showed high binding

activity to the epithelial A549 cell line only.

Affinity constants

Saturation assays and Hill analysis [24,25] were carried

out for shared 11024 HABPs based on initial screening

results, using a greater 125I-radiolabelled peptide

Fig. 2. Immunoblotting assay. Western blotting was performed

with rabbit preimmune and post-third inoculation sera against a

M. tuberculosis sonicate (lanes 1, 2 and 5) and a membrane frac-

tion (lanes 3 and 4). Lanes 1 and 3, absence of recognition of

M. tuberculosis proteins by rabbit preimmune sera; lane 2, serum

from rabbit inoculated with polymerized peptide 11031; lane 4,

serum from rabbit inoculated with polymerized peptide 11027;

lane 5, serum from rabbit inoculated with M. tuberculosis total son-

icate. Molecular weight markers are shown on the left-hand side.

Fig. 3. Immunoelectron microscopy. Immunogold location of

Rv2560 protein on M. tuberculosis H37Rv membrane, as assessed

by 5 nm gold-labelled anti-rabbit IgG particles. Intact cells were

incubated with a 1 : 10 dilution (1 and 2) or pure (3) rabbit sera

directed against peptide 11027, and prepared for immunoelectron

microscopy by negative contrast, as described. No labelling of the

surface was obtained in control experiments from which these anti-

bodies had been omitted, or with preimmune serum (data not

shown). The arrows indicate the location of the Rv2560 protein on

the mycobacterial surface.

D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization

FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6355

concentration range (500–2000 nm) (Fig. 5A). Scat-

chard and Hill analyses were then performed and dis-

sociation constants (Kd) were calculated for Rv2560

HABP 11024 for U937 and A549 cell lines (Kd of 380

and 800 nm, respectively). The Hill coefficient (nh) was

1.5 for U937 and 1.2 for A549 cells, suggesting positive

ligand–receptor cooperativity. The numbers of binding

sites per cell calculated for peptide 11024 were 200 000

and 3 000 000 for U937 and A549 cells, respectively.

Critical residues for cell binding in U937 cells

Glycine scanning analogue peptides were synthesized,

and competition cell binding assays were performed

between original radiolabelled peptides and their ana-

logue peptides to identify HABP critical amino acids

for U937 cell binding. Critical residues were those

which, on replacement with glycine, rendered an

invariable decrease of at least 50% in the ability to

compete with original radiolabelled peptide in a bind-

ing assay at two concentrations (16 and 400 nm). This

assumed dramatic changes in peptide binding activity.

Figure 5B shows that critical residues in peptide 11024

binding were VVALSDRATTAYTNTSGVSS (shown

in bold italic in the sequence).

Cross-linking assays

HABP 11024 bound to a protein located on the U937

cell membrane, specifically recognizing a 54 kDa hypo-

thetical receptor. Radiolabelled peptide binding to this

receptor protein was inhibited in the presence of non-

radiolabelled peptide; this was clearly shown by the

band intensities (Fig. 6). Peptide 11024 binding was

target cell specific, as it did not show any binding to

HepG2 cells or erythrocytes (data not shown).

Invasion inhibition assay using HABPs

A549 and U937 cell lines were preincubated in the

presence of HABP 11024 in two independent experi-

ments to evaluate the effect of HABP on bacterial

invasion of target lung cells. It was found that invasion

was inhibited in both cell types. The inhibitory power

of HABP 11024 was greater in assays carried out using

the A549 cell line, where it reached close to 60% inhi-

bition when such cells were preincubated in the pres-

ence of 1 lm peptide. Monocyte invasion assays

revealed around 50% maximum inhibition only when

higher peptide concentrations were used than those

employed for A549 cell assays (Fig. 7). It was also

Fig. 4. Cell binding activity of Rv2560 peptides. Amino acid sequence and specific U937 and A549 cell binding activity for 20-mer long chem-

ically synthesized Rv2560 peptides from the M. tuberculosis H37Rv strain. The peptides are given on the left-hand side, with numbers indi-

cating their position within the native protein; in peptides that did not contain tyrosine, it was added to the carboxy-terminal end. To the

right, the black bar represents each peptide binding activity, determined as the specific binding ⁄ total added peptide ratio. The dotted line

separates peptides having ‡ 1% binding activity. HABP 11095 was used as a binding activity control. Rv2560 transmembrane topology is

presented on the left-hand side. Grey shows the transmembrane helices present in the sequence according to the TMHMM prediction server.

nd, not done because of solubilization problems.

M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.

6356 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS

observed that the inhibitory activity of HABP 11024

decreased at concentrations equal to or greater than

100 lm. The secondary structure of HABP 11024 in

solution at different concentrations (including those in

which activity was seen to decrease) was verified by

CD, and it was shown that this HABP underwent

conformational changes critical for its function at the

concentrations already established in functional assays

(data not shown).

Discussion

Although the elucidation of the M. tuberculosis gen-

ome sequence has revealed the presence of many gene

families which can be classified according to their

homology to genes having a known function in other

microorganisms, no functional counterparts have been

found for many of these mycobacterial genes. Pro-

teome studies have led to the determination of some of

the proteins present on the membrane for which no

role in M. tuberculosis invasion of the host cell has yet

been determined [12,13].

The infection of epithelial cells in the alveoli requires

the interaction of the bacterium with several surface

receptors, which have been poorly studied to date [26].

Studies of nonpolarized pulmonary epithelial cells

(HEp-2) have revealed the existence of at least five

M. tuberculosis proteins that bind to biotinylated cell

fractions, including the already characterized adhesion

heparin-binding haemagglutinin [27].

A B

Fig. 5. Saturation curves and critical residues of cell binding. (A) Saturation binding curve for HABP 11024 binding to U937 and A549 cells.

The saturation curves resulted from plotting the specifically bound 125I-HABP concentration versus free 125I-HABP. The affinity constants and

maximum number of sites per cell were obtained from these curves. Inset: the abscissa is log F in the Hill plot and the ordinate is

log [B ⁄ Bm ) B], where Bm is the maximum bound peptide, B is the bound peptide and F is free peptide. (B) Competition binding assay with

analogous peptides. Specific original radiolabelled peptide binding inhibited by analogous peptide (at 16 and 400 nM) is shown. Amino acids

underlined in bold represent the critical binding residues, as their binding activity decreased by 50% or more when modified and assayed at

the two concentrations used.

Fig. 6. Cross-linking assay for peptide 11024. Autoradiographs for

U937 proteins specifically cross-linked with radiolabelled peptide

11024. Lanes 1 and 2, U937 cells; lanes 3 and 4, HepG2 cells;

lanes 1 and 3, total radiolabelled peptide binding; lanes 2 and 4,

inhibited binding. The cross-linking assays show that peptide 11024

specifically binds to a 54 kDa membrane protein on U937 cells.

D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization

FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6357

Moreover, it has been observed that M. tuberculosis

can promote monocyte differentiation into dendritic

cells that lack CD1, have low CD80 and produce inter-

leukin-10; this altered phenotype is unable to prime

effector T cells, thus becoming an escape mechanism

[28]. The identification of proteins specifically binding

to receptors on monocytes or other M. tuberculosis

infection-susceptible cells has thus become of interest

in order to determine precisely the regions in such pro-

teins that are directly involved in mycobacterial inva-

sion or immune response modulation.

Our group has characterized three mycobacterial

membrane proteins (Rv2004c [21], Rv2536 [22] and

Rv1510c [23]) in recent studies, and has determined

their target cell binding profiles. These studies repre-

sent an initial step in the rational selection of com-

ponents for a subunit-based, multiepitope, anti-TB

vaccine candidate.

Rv2560 is a proline- and glycine-rich transmembrane

protein, and may thus be classified as a simple

sequence protein (SSP). This type of sequence is fre-

quent in organisms having G + C-rich genomes, such

as M. tuberculosis, where several SSPs are organized

into large-sized families having a role in host–pathogen

interaction and are membrane-associated [29]. tblastn

analysis of the Rv2560 amino acid sequence revealed

the existence of homologous genes in M. marinum and

M. ulcerans. The homologous gene in M. marinum

(melA) was shown to be important in the adhesion,

invasion and intracellular survival of this bacterium in

THP-1 cells after insertion into the noninvasive

M. smegmatis species [16].

This work shows that the Rv2560 encoded protein,

classified as a putative membrane protein [9] in the

M. tuberculosis genome, is present in M. tuberculosis

complex strain bacilli, is transcribed, expressed and

present in greater concentration on the membrane-

enriched fraction and the mycobacterial surface, and is

involved in the mycobacterial invasion of host cells.

On PCR amplification of a 631 bp fragment, includ-

ing the sequence of nucleotides encoding a high specific

binding Rv2560 protein region (amino acids 80–290)

(Fig. 1), it was found that this fragment was amplified

in M. tuberculosis complex strains. The presence of this

gene was also determined in clinical M. tuberculosis

isolates obtained from patients having different types

of TB (pulmonary, pleural, osseous, meningeal,

abdominal, genitourinary and renal), with the 631 bp

fragment being amplified in all clinical isolates. All of

the foregoing suggests that the Rv2560 gene is present

in different M. tuberculosis complex strains, including

clinical isolates, and has no genetic variability, as

shown by DNA sequencing (data not shown).

The transcription of this gene was also confirmed

after the presence of the highly conserved gene had

been established in different M. tuberculosis complex

strains. cDNA was therefore obtained from M. tuber-

culosis complex strains and amplified by PCR, using

specific primers for the 308 bp fragment (using more

internal primers to amplify smaller fragments in order

to improve RT-PCR efficiency). The results (Fig. 1B)

led to the conclusion that the Rv2560 gene was

transcribed in M. tuberculosis H37Rv, M. tuberculosis

H37Ra, M. bovis BCG and M. africanum strains, but

not in M. bovis or M. microti.

The two polymerized peptides used in rabbit immu-

nization produced an antibody response (differing in

intensity) evident in western blot results (Fig. 2); a

Fig. 7. Invasion inhibition assays with HABP 11024. Inhibition percentages with different HABP 11024 concentrations using U937 and A549

cells. A peptide having low specific binding ability to A549 cells was used as negative control for this cell line (11021). Colchicine (a vesicular

traffic inhibitor) was used as positive invasion inhibition control. The results correspond to the average inhibition percentage calculated for

each treatment ± standard deviation. *P £ 0.05, **P £ 0.01, ***P £ 0.001.

M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.

6358 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS

33 kDa band corresponding to the theoretical molecu-

lar mass of the Rv2560 protein (33.1 kDa) was recog-

nized by these sera, suggesting that this protein is

transcribed and expressed in M. tuberculosis sonicate,

and at much higher concentration in the membrane

fraction, as inferred from the band intensity.

When using serum from rabbits immunized with poly-

merized peptide 11027, immunoelectron microscopy

confirmed the presence of this protein on the mycobac-

terial surface (which has not been reported to date).

Receptor–ligand assays led to the identification of

two high specific binding peptides, one (11024) being

shared by both U937 monoblast and A549 epithelial

cells. Peptide 11024 binding to both cell types was sat-

urable, presenting strong positive cooperativity, as

shown by the dissociation constant and Hill coefficient.

The corresponding analyses for the determination of

the receptor for 11024 HABPs and the identification of

the critical binding residues, and the invasion inhibi-

tion assays, were thus performed exclusively with this

peptide.

This peptide is located in the solvent-exposed central

region of the protein between residues 121 and 140,

displaying the VVALSDRATTAYTNTSGVSS

sequence (where the amino acids shown in bold italic

correspond to those identified as being critical in 11024

binding to U937 cells). These residues may be involved

directly in target cell binding or may be important for

the structural characteristics of the peptide, thereby

allowing specific binding. BLAST analysis of this small

region led to the identification of 78% homology with

Burkholderia cenocepacia-dependent AMP synthase,

which causes respiratory tract infection and is cyto-

toxic for alveolar epithelial cells [30].

A 54 kDa protein was found to be a receptor on

U937 cells; its binding by peptide 11024 was inhibited

by an excess of nonradiolabelled peptide. This receptor

was specific for the U937 cell line, but was not seen in

hepatocytes or erythrocytes. A receptor having a simi-

lar molecular mass (51 kDa) has been described for

the Rv1510c protein HABP 11095; however, further

studies are required to determine whether the same

membrane protein is involved.

Peptide 11024 (for which high monocyte and alveo-

lar epithelial cell binding has already been determined)

induced a 36.96–50.53% decrease in M. tuberculosis

invasion of U937 cells and a 30.98–58.27% decrease in

M. tuberculosis invasion of A549 cells (Fig. 7). This

indicates that this HABP is important in the recogni-

tion and invasion of monocytes and type II alveolar

epithelial cells by the mycobacterium.

It was observed in preliminary assays that HABP

concentrations of less than 100 lm had a greater

inhibitory effect than larger concentrations. Therefore,

additional CD secondary structural studies were per-

formed to verify possible conformational changes at

concentrations greater than 100 lm which were critical

in the inhibitory activity of this peptide. It was found

that the structure of the peptide changed at concentra-

tions equal to or greater than 100 lm (not shown),

thereby explaining the loss of inhibitory potential in

the assay carried out for alveolar epithelial cells.

The above results indicate that the Rv2560 protein

is conserved within the M. tuberculosis complex. It is

also transcribed and expressed in low abundance on

the mycobacterial surface. The protein contains a spe-

cific high binding peptide for U937 and A549 cells,

which plays an important role in mycobacterial inva-

sion. Therefore, it is concluded that the Rv2560 pro-

tein is highly relevant in pathogen–host interaction,

and that the identification of the region directly

involved in such interaction makes it an important

candidate for inclusion in the development of a

subunit-based, multiepitope, chemically synthesized

vaccine.

Experimental procedures

Bioinformatics’ analysis

M. tuberculosis Rv2560 protein sequences were obtained

from the Tuberculist Webserver (http://genolist.pasteur.fr/

TubercuList/). Proteins were aligned by BLAST (http://

www.ncbi.nlm.nih.gov/BLAST/) and clustalw (http://

npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clu

stalw.html) multiple alignments. Protein features and

GRAVY scores for the Rv2560 amino acid sequence were

calculated using the protparam tool (http://us.expasy.

org ⁄ tools ⁄protparam.html), for which a score of greater

than ) 0.4 (mean score for the cytosolic proteins) suggests

membrane association probability; the higher the score, the

greater the probability [13]. Transmembrane regions were

predicted by tmhmm (http://www.cbs.dtu.dk/services/

TMHMM) and tmpred (http://www.ch.embnet.org/

software/TMPRED_form.html). Cell localization, lipid

attachment sites and signal sequences were predicted using

psort (http://psort.nibb.ac.jp).

Mycobacterial species and strains

The following M. tuberculosis complex species and strains

were used: M. tuberculosis H37Rv (ATCC27294), M. tuber-

culosis H37Ra (ATCC25177), M. bovis (ATCC19210),

M. bovis BCG (ATCC27291, Pasteur sub strain), M. africa-

num (ATCC25420) and M. microti (kindly donated by

F. Portaels, Prince Leopold Institute of Tropical Medicine).

D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization

FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6359

Ten M. tuberculosis clinical strains were isolated from speci-

mens obtained from different patients attending the TB pro-

gramme at either the San Juan de Dios Hospital or Santa

Clara Hospital, both in Bogota, Colombia. Patients came

from several geographical areas of Colombia. All mycobacte-

rial strains were grown for 5–15 days in 7H9 Middlebrook

broth (Difco Laboratory, Detroit, MI, USA) with 0.04%

Tween 80 and in Middlebrook agar 7H10 (Difco), both sup-

plemented with oleic acid, albumin, dextrose and catalase

(BBL, Becton Dickinson, Mountain View, CA, USA).

Genomic DNA extraction

Cells were harvested in Tris–EDTA buffer (TE, pH 8.0)

and suspended in 25% sucrose in 10 · TE. Lysozyme

(100 lgÆmL)1) was added and the preparation was incubated

at 37 �C for 2 h with subsequent addition of 100 lgÆmL)1

proteinase K and N-lauroylsarcosine (Sigma, St Louis, MO,

USA) at 1% final concentration. Following 18 h incubation

at 65 �C, the mixture was extracted with chloroform–iso-

amyl alcohol (24 : 1, v ⁄ v), once with phenol–chloroform

(1 : 1, v ⁄ v) and twice more with chloroform–isoamyl alco-

hol. DNA was precipitated with 2-propanol, washed with

70% (v ⁄ v) ethanol and suspended in 1 · TE [31,32].

PCR assay

PCR amplifications were performed in a thermal cycler

GeneAmp PCR System 9600 (Perkin-Elmer Life Sciences

Inc., Boston, MA, USA), using 100 ng mycobacterial geno-

mic DNA for amplifications. The mixture contained 50 mm

KCl, 10 mm Tris ⁄HCl (pH 8.3), 1.5 mm MgCl2, 0.1 mm of

each dNTP, 0.4 mm of both direct 5¢-CAATCGTCG

GGTTTTAGCG-3¢ and reverse 5¢-GTGTAGACGTG

GATGAGCG-3¢ oligonucleotide primers and 1.5 U Taq

DNA polymerase (Invitrogen, Carlsbad, CA, USA).

Twenty-five cycles of the following thermal profile were car-

ried out after DNA had been denatured for 5 min at 94 �C:56 �C for 30 s, 72 �C for 40 s and 95 �C for 40 s. A final

5 min extension cycle was then performed at 72 �C. The

amplification products were loaded onto 1% agarose gel,

electrophoresed at 70 V in 1 · Tris/acetate/EDTA, stained

with ethidium bromide and photographed.

DNA sequencing

The dideoxy chain termination method was used for

sequencing reactions with a Taq FS DyeDeoxy Terminator

cycle sequencing kit (Applied Biosystems, Foster City, CA,

USA) on a GeneAmp PCR System 9600 (Perkin-Elmer Life

Sciences Inc.), and run on a 373 DNA sequencer model

(Applied Biosystems). The sequencing strategy involved for-

ward and reverse specific primers for the 631 bp fragment

of the Rv2560 gene.

RNA isolation

The bacilli were harvested by spinning at 12 000 g for

15 min at 4 �C. Sodium azide (10 mm) was added to the

culture just before harvesting. The cell pellet was suspended

in 2 mL of cold lysis buffer for each 200 mg wet weight of

cells [33], and sonicated twice for 15 min. Two volumes of

Trizol (Gibco BRL, Gaithersburg, MD, USA) were then

added and extracted according to the manufacturer’s

instructions. The pellet was suspended in 100 lL of distilled

water and stored in aliquots at ) 80 �C.

RT-PCR

Total RNA was quantified by a GeneQuant spectropho-

tometer (Pharmacia Biotech, Piscataway, NJ, USA), treated

with RNAse-free DNAse RQ1 at 37 �C for 3 h, precipi-

tated with isopropanol, washed with 70% ethanol and sus-

pended in distilled water. M. tuberculosis H37Rv DNA was

included as DNAse Q activity control (1 UÆlg)1 DNA).

Target RNA (500 lg) was reverse transcribed in a tube

containing distilled water and 10 lgÆmL)1 random primers

(Gibco BRL). This mixture was incubated for 10 min at

70 �C; 1 · RT buffer, 10 mm dithiothreitol, 0.5 mm dNTPs

and 40 U human placenta ribonuclease inhibitor (Promega,

Madison, WI, USA) were then added on ice; 200 U

M-MLV reverse transcriptase (Gibco-Life Technologies,

Grand Island, NY, USA) was then added at a final volume

of 30 lL. This mixture was kept at 37 �C for 1 h. The

enzyme was finally denatured for 5 min at 95 �C. PCR was

carried out as described above.

The rpoB gene was used as positive transcription control.

This gene, encoding the RNA polymerase b-subunit, is

present in all mycobacterial species [33]. DNAse-Q-treated

M. tuberculosis H37Rv was used as cDNA synthesis nega-

tive control. Distilled water and M. tuberculosis H37Rv

DNA were used as negative and positive PCR controls,

respectively.

Rabbit immunization

Two New Zealand strain rabbits per peptide (previously

determined to be nonreactive to M. tuberculosis sonicate, as

assessed by western blot) were injected with 500 lg of

peptide 11027 (181ADGKPVTIATFFRPRNLGLV200) or

500 lg of peptide 11031 (242SPIDSVKASIETVGSNIG

GSVY262) [polymerized via the addition of cysteine at the

amino and carboxy termini and mixed with Freund’s

Incomplete Adjuvant (Sigma)] on days 0, 20 and 40. Final

bleeding was carried out on day 60 and sera were collected

in accordance with the Fundacion Instituto de Inmunolo-

gia’s Bioethics Committee recommendations. The Rv2560

protein peptide sequences chosen for the immunization of

rabbits were obtained using T-epitope prediction software

M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.

6360 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS

syfpeithi (15-mer T-epitope for major histocompatibility

complex class II only) downloaded from http://www.

syfpeithi.de/Scripts/MHCServer.dLl/EpitopePrediction.htm.

SDS-PAGE and immunoblotting

M. tuberculosis sonicate or membrane fraction (1 mg) was

loaded in a discontinuous SDS-PAGE system, using a 10–

20% (w ⁄ v) acrylamide gradient, and then transferred to

nitrocellulose paper using the semidry blotting technique

[34]. The filters were incubated with a 1 : 100 (v ⁄ v) dilutionof the sera obtained from rabbits immunized with polymer-

ized 11027 or 11031 peptides. Sera were diluted in NaCl ⁄Tris ⁄Tween 20 (0.02 m Tris-HCl, pH 7.5, 0.05 m NaCl, 1%

Tween 20) and 5% skimmed milk. Incubation for 1 h with

1 : 3000 (v ⁄ v) alkaline phosphatase-conjugated anti-rabbit

IgG (ICN Biomedicals, Costa Mesa, CA, USA) followed

five NaCl ⁄Tris ⁄Tween 20 washes. The reaction was devel-

oped with Nitro Blue tetrazolium ⁄ 5-bromo-4-chloroindol-

2-yl phosphate (KPL, Gaithersburg, MD, USA).

Immunoelectron microscopy

Immunoelectron microscopy studies were carried out on a

Philips CM 10 transmission electron microscope (Philips,

Suresne, Hauts-de-Seine, France). Briefly, a wet pellet

(50 lL) of M. tuberculosis H37Rv was fixed with a 4%

paraformaldehyde)0.5% glutaraldehyde solution for 2 h at

4 �C. After fixation, the pellet was dehydrated in graded

ethanol and then embedded in LR-white hard-grade acrylic

resin (Sigma) for 4 days at 4 �C. Thin slices (400 nm) were

cut and mounted on 300 mesh nickel grids. The slices were

incubated in a saturated sodium metaperiodate solution for

1 h at 20–22 �C for antigen retrieval [35]. The grids were

then floated, section 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 (NaCl ⁄Tris) (0.05 m Tris in

isotonic saline, pH 7.6) containing 0.05% BSA, the grids

were incubated in either 1 : 10 (v ⁄ v) or pure rabbit

polyclonal serum for 1 h at 37 �C. Following an NaCl ⁄Tris ⁄ 0.025% Tween 20 wash, the grids were immersed in a

1 : 50 (v ⁄ v) dilution of 5 nm gold-labelled anti-rabbit IgG

(Sigma) for 1 h at 20–22 �C. The grids were then washed

with NaCl ⁄Tris ⁄Tween 20 and fixed in 2.5% glutaralde-

hyde, followed by 15 min of incubation in 1% uranyl ace-

tate. The grids were washed with distilled water and dried

at 20–22 �C before observation.

Cell culture

A549 human lung epithelial (ATCC CCL-185) and U937

monocyte-like (ATCC CRL-1593.2) cells were grown at

37 �C and 5% CO2 in RPMI 1640 medium (Gibco-BRL)

supplemented with 10% fetal bovine serum (Hyclone,

Logon, UT, USA). A549 cells were dislodged with 0.1%

EDTA–NaCl ⁄Pi. Cell lines were collected in 50 mL tubes,

centrifuged at 1000 g for 5 min and washed with RPMI

1640.

Peptide synthesis

Sixteen sequential 20-mer peptides, corresponding to the

Rv2560 amino acid sequence [9], were synthesized in this

study by a solid-phase multiple peptide system [36,37].

4-Methylbenzhydrylamine resin (0.7 meqÆg)1), t-Boc amino

acids and low–high cleavage techniques were used [38]. Pep-

tide identity and purity were analysed by MALDI-TOF MS

and analytical reverse phase HPLC. An extra tyrosine resi-

due was added to any peptide C-terminus that did not

contain it to enable radiolabelling.

Peptide radiolabelling

Radiolabelling with 125I was performed according to previ-

ously described techniques [24,25,39,40], in which chlor-

amine-T (2.25 mgÆmL)1) and 3.2 lL Na125I (100 mCiÆmL)1)

were added to 5 lL peptide solution (1 lgÆlL)1); 15 lLsodium bisulfite (2.75 mgÆmL)1) and 50 lL NaI (0.16 m)

were added after 5 min of reaction at 18 �C. The radiola-

belled peptide was then separated from reaction by-prod-

ucts on a Sephadex G-10 column (Pharmacia Biotech,

St Albans, UK) (80 · 5.0 mm).

Binding assay

A549 and U937 cells (1 · 106) were incubated with increas-

ing quantities of each Rv2560 m. tuberculosis putative mem-

brane protein 125I-radiolabelled peptide (100–2000 nm) at

100 lL total volume for 90 min at 4 �C, in the presence or

absence of 40 lm unlabelled peptide, to determine the bind-

ing specificity. After incubation, unbound peptide was

removed from the cells by sedimentation through a dioctyl-

phthalate–dibutylphthalate cushion (d ¼ 1.015 gÆmL)1) and

centrifuged at 9000 g for 2 min [18,19]. The assay was car-

ried out in triplicate in identical conditions; bound and free

peptides were determined by measuring the cell-associated

radioactivity on a gamma counter (Gamma Counter

Cobra II, Packard Instrument Co., Meriden, CT, USA);

the average results of the assays performed in triplicate are

reported and shown graphically in Fig. 4.

Saturation assay

A549 and U937 cells (1 · 106) were incubated with increas-

ing concentrations (100–2000 nm) of radiolabelled peptide

at 120 lL total volume for 90 min at 4 �C, in the presence

or absence of 40 lm unlabelled peptide, to determine the

binding specificity. After incubation, unbound peptide was

D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization

FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6361

removed from the cells by sedimentation through a dioctyl-

phthalate–dibutylphthalate cushion (d ¼ 1.015 gÆmL)1) and

centrifuged at 9000 g for 2 min. As before, each assay was

performed in triplicate; bound and free peptides were deter-

mined by measuring the cell-associated radioactivity on a

gamma counter. Triplicate assay data were averaged. The

curves obtained were analysed and the dissociation con-

stants were determined by the Hill equation [18,19].

Analogue peptide competition binding assay

HABP 11024 glycine scanning analogues were synthesized

to identify critical residues for binding to U937 cells. For

the competition binding assays, 1 · 106 U937 cells were

incubated with increasing quantities (16 and 400 nm) of

each unlabelled analogue peptide or original unlabelled

peptide in the presence of native 125I-labelled peptide. After

90 min of incubation at 4 �C, the mixture was centrifuged

at 9000 g for 2 min on a dioctylphthalate–dibutylphthalate

cushion (d ¼ 1.015 gÆmL)1). Radioactivity bound to

U937 cells was determined by gamma counter measurement

[17].

Cross-linking assays

U937 cells (2 · 106) were subjected to a conventional bind-

ing assay (90 min of incubation at 4 �C) with HABP 11024.

Following incubation, the cells were washed with NaCl ⁄Pi

and cross-linked with 25 lm bis(sulfosuccinymidyl suberate)

(Pierce, Rockford, IL, USA) for 20 min at 4 �C. The reac-

tion was stopped with 40 nm Tris ⁄HCl (pH 7.4), and washed

again with NaCl ⁄Pi. The cells were then treated with lysis

buffer (5% SDS, 10 nm iodoacetamide, 1% Triton X-100,

100 mm EDTA, 10 mm phenylmethanesulfonyl fluoride).

The membrane proteins so obtained were solubilized in

Laemmli buffer and separated by SDS-PAGE. Proteins

cross-linked with radiolabelled peptides were exposed on a

Bio-Rad Imaging Screen K (Bio-Rad Molecular Imager FX;

Bio-Rad Quantity One Quantitation Software; Bio-Rad,

Hercules, CA, USA) for 7 days.

Mycobacterium culture and staining

for invasion assay

M. tuberculosis H37Rv bacilli were collected during the log-

arithmic phase, washed and suspended in NaCl ⁄Pi. The sus-

pension was briefly sonicated (30 W for 5 s) on a Branson

sonifier (VWR Scientific, Boston, MA, USA); the suspen-

sion turbidity was adjusted to 3 · 108 colony-forming

unitsÆmL)1 according to McFarland’s pattern. M. tuber-

culosis was labelled with SYBR Safe (Invitrogen, Eugene,

OR, USA) at 20 · final concentration at 37 �C for 20 min

in the dark with constant shaking, washed twice and centri-

fuged at 12 000 g for 20 min; excess dye was removed.

Bacteria were then suspended in RPMI 1640, and labelling

was further verified by flow cytometry and fluorescence

microscopy.

Invasion inhibition assay with HABPs

The possible biological relevance of the HABPs identified

in Rv2560 was evaluated by an assay developed by our

group [41]. U937 or A549 cells (1 · 106) suspended in com-

plete RPMI 1640 medium without antibiotics were preincu-

bated for 1 h in the presence of 50, 100 or 200 lm

HABP 11024 for U937 cells and 100)105 nm or 200 lm

HABP 11024 for A549 cells before the addition of 1 · 107

M. tuberculosis H37Rv bacilli (multiplicity of infection,

1 : 10) at a final volume of 200 lL. They were incubated at

37 �C in 5% CO2 for 2 h with constant shaking, and then

placed in 20 lgÆmL)1 amikacin-supplemented medium (ICN

Biomedicals) for 30 min to kill off the extracellular bacte-

ria, whilst the intracellular bacteria remained viable.

Two RPMI 1640 washes preceded centrifugation at 500 g

for 5 min. The cells were fixed in 1% paraformaldehyde

(Merck, Darmstadt, Germany) in RPMI 1640 at 4 �C for

1 h, washed and suspended in 50 lL RPMI 1640. The cells

were counterstained with freshly prepared 3% methylene

blue in 30% ethanol, 5 min before flow cytometry, and

incubated at 20–22 �C. The cells used to determine the

autofluorescence control were treated using the same experi-

mental conditions as those employed for the cells incubated

with bacteria. Invasion control was carried out in the pres-

ence of 30 lm colchicine (Sigma) as inhibitor, added to the

cells for 1 h at 37 �C prior to the addition of bacilli to inhi-

bit cell internalization. The inhibitors were maintained

throughout the 2 h invasion period. Student’s t-test was

used to verify possible differences between peptide treat-

ment and cell control, which had not been preincubated

with peptides or colchicine [20,26].

Flow cytometry quantification

Samples were analysed on a FACScan (Becton Dickinson)

flow cytometer. CellQuest software (Becton Dickinson) was

used for flow cytometry capture and analysis. NaCl ⁄Pi,

which had been filtered with a 0.2 lm membrane, was used

as sheath fluid. Uninfected cells were discriminated from

infected cells on the basis of light characteristics on fluores-

cence channel one. Samples were run at 2000 events per sec-

ond; 40 000 events were collected. The percentage of positive

SYBR safe events in the gated region was determined.

CD spectroscopy

CD was carried out for HABP 11024 and control peptide

11021 to determine whether there were any conformational

changes when the peptide concentration in the solution

M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.

6362 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS

was increased. CD spectra were recorded at 20 �C on a

Jasco J-810 spectropolarimeter at wavelengths ranging

from 260 to 190 nm in 1 cm cuvettes (Jasco, Tokyo,

Japan). The peptides were dissolved at 5, 25, 50, 100 and

200 lm concentration in 30% trifluoroethanol aqueous

solution. Each spectrum was obtained from the average of

three scans taken at a scan rate of 20 nmÆmin)1 with a

spectral bandwidth of 1 nm, corrected for baseline. The

results were expressed as the mean residue ellipticity [Q].

Acknowledgements

We wish to thank Dr Ramses Lopez, Rosalba Alfonso,

Marisol Ocampo, Martha Calderon and Javier Rosas

who started this work whilst working at Fundacion

Instituto de Immulogia de Colombia (FIDIC). This

work was supported by COLCIENCIAS contract no.

2008. The wholehearted assistance of Gloria P. Barrera

in immunoelectron microscopy, Marı́a H. Duran in

CD experiments and Jason Garry in translating the

manuscript is greatly appreciated.

References

1 World Health Organization (WHO) (2006) World

Health Report 2006. Global Tuberculosis Control: Sur-

veillance, Planning, Financing. WHO, Geneva.

2 Russell DG (2007) Who puts the tubercle in tuberculo-

sis? Nat Rev Microbiol 5, 39–47.

3 Snider DE Jr & Castro KG (1998) The global threat of

drug-resistant tuberculosis. N Engl J Med 338, 1689–

1690.

4 Pillay CS, Elliott E & Dennison C (2002) Endolysoso-

mal proteolysis and its regulation. Biochem J 363, 417–

429.

5 Pieters J (2001) Entry and survival of pathogenic

mycobacteria in macrophages. Microbes Infect 3,

249–255.

6 Bermudez LE & Sangari FJ (2001) Cellular and mole-

cular mechanisms of internalization of mycobacteria by

host cells. Microbes Infect 3, 37–42.

7 El-Etr SH & Cirillo JD (2001) Entry mechanisms of

mycobacteria. Front Biosci 6, D737–D747.

8 Sigler K & Hofer M (1997) Biotechnological aspects of

membrane function. Crit Rev Biotechnol 17, 69–86.

9 Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C,

Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE

3rd, et al. (1998) Deciphering the biology of Mycobacte-

rium tuberculosis from the complete genome sequence.

Nature 393, 537–544.

10 Garcia-Perez BE, Mondragon-Flores R & Luna-Herrera

J (2003) Internalization of Mycobacterium tuberculosis

by macropinocytosis in non-phagocytic cells. Microb

Pathog 35, 49–55.

11 Chakravarti DN, Fiske MJ, Fletcher LD & Zagursky

RJ (2000) Application of genomics and proteomics for

identification of bacterial gene products as potential

vaccine candidates. Vaccine 19, 601–612.

12 Gu S, Chen J, Dobos KM, Bradbury EM, Belisle JT &

Chen X (2003) Comprehensive proteomic profiling of

the membrane constituents of a Mycobacterium tubercu-

losis strain. Mol Cell Proteomics 2, 1284–1296.

13 Sinha S, Kosalai K, Arora S, Namane A, Sharma P,

Gaikwad AN, Brodin P & Cole ST (2005) Immunogenic

membrane-associated proteins of Mycobacterium tuber-

culosis revealed by proteomics. Microbiology 151, 2411–

2419.

14 Cardoso FC, Pinho JM, Azevedo V & Oliveira SC

(2006) Identification of a new Schistosoma mansoni

membrane-bound protein through bioinformatic analy-

sis. Genet Mol Res 5, 609–618.

15 Kyte J & Doolittle RF (1982) A simple method for dis-

playing the hydropathic character of a protein. J Mol

Biol 157, 105–132.

16 El-Etr SH, Subbian S, Cirillo SL & Cirillo JD (2004)

Identification of two Mycobacterium marinum loci that

affect interactions with macrophages. Infect Immun 72,

6902–6913.

17 Garcia JE, Puentes A, Suarez J, Lopez R, Vera R,

Rodriguez LE, Ocampo M, Curtidor H, Guzman F,

Urquiza M, et al. (2002) Hepatitis C virus (HCV) E1

and E2 protein regions that specifically bind to HepG2

cells. J Hepatol 36, 254–262.

18 Ocampo M, Urquiza M, Guzman F, Rodriguez LE,

Suarez J, Curtidor H, Rosas J, Diaz M & Patarroyo

ME (2000) Two MSA 2 peptides that bind to human

red blood cells are relevant to Plasmodium falciparum

merozoite invasion. J Pept Res 55, 216–223.

19 Ocampo M, Vera R, Eduardo Rodriguez L, Curtidor

H, Urquiza M, Suarez J, Garcia J, Puentes A, Lopez R,

Trujillo M, et al. (2002) Plasmodium vivax Duffy bind-

ing protein peptides specifically bind to reticulocytes.

Peptides 23, 13–22.

20 Danelishvili L, McGarvey J, Li YJ & Bermudez LE

(2003)Mycobacterium tuberculosis infection causes differ-

ent levels of apoptosis and necrosis in human macrophag-

es and alveolar epithelial cells. Cell Microbiol 5, 649–660.

21 Forero M, Puentes A, Cortes J, Castillo F, Vera R,

Rodriguez LE, Valbuena J, Ocampo M, Curtidor H,

Rosas J, et al. (2005) Identifying putative Mycobacte-

rium tuberculosis Rv2004c protein sequences that bind

specifically to U937 macrophages and A549 epithelial

cells. Protein Sci 14, 2767–2780.

22 Garcia J, Puentes A, Rodriguez L, Ocampo M, Curti-

dor H, Vera R, Lopez R, Valbuena J, Cortes J, Vanegas

M, et al. (2005) Mycobacterium tuberculosis Rv2536

protein implicated in specific binding to human cell

lines. Protein Sci 14, 2236–2245.

D. F. Plaza et al. M. tuberculosis Rv2560 protein characterization

FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS 6363

23 Vera-Bravo R, Torres E, Valbuena JJ, Ocampo M,

Rodriguez LE, Puentes A, Garcia JE, Curtidor H, Cor-

tes J, Vanegas M, et al. (2005) Characterising Mycobac-

terium tuberculosis Rv1510c protein and determining its

sequences that specifically bind to two target cell lines.

Biochem Biophys Res Commun 332, 771–781.

24 Hulme E (1993) Receptor–Ligand Interactions. A Practi-

cal Approach. IRL Press, Oxford.

25 Weiland GA & Molinoff PB (1981) Quantitative analy-

sis of drug–receptor interactions. I. Determination of

kinetic and equilibrium properties. Life Sci 29, 313–330.

26 Bermudez LE & Goodman J (1996) Mycobacterium

tuberculosis invades and replicates within type II alveo-

lar cells. Infect Immun 64, 1400–1406.

27 Reddy VM & Hayworth DA (2002) Interaction of

Mycobacterium tuberculosis with human respiratory epi-

thelial cells (HEp-2). Tuberculosis (Edinb) 82, 31–36.

28 Gagliardi MC, Lemassu A, Teloni R, Mariotti S, Sar-

gentini V, Pardini M, Daffe M & Nisini R (2007) Cell

wall-associated alpha-glucan is instrumental for Myco-

bacterium tuberculosis to block CD1 molecule expression

and disable the function of dendritic cell derived from

infected monocyte. Cell Microbiol 9, 2081–2092.

29 Subramanyam MB, Gnanamani M & Ramachandran S

(2006) Simple sequence proteins in prokaryotic proteo-

mes. BMC Genomics 7, 141.

30 Cheung KJ Jr, Li G, Urban TA, Goldberg JB, Griffith

A, Lu F & Burns JL (2007) Pilus-mediated epithelial cell

death in response to infection with Burkholderia ceno-

cepacia. Microbes Infect 9, 829–837.

31 Del Portillo P, Murillo LA & Patarroyo ME (1991)

Amplification of a species-specific DNA fragment of

Mycobacterium tuberculosis and its possible use in diag-

nosis. J Clin Microbiol 29, 2163–2168.

32 Katoch VM & Cox RA (1986) Step-wise isolation of

RNA and DNA from mycobacteria. Int J Lepr Other

Mycobact Dis 54, 409–415.

33 Lee H, Park HJ, Cho SN, Bai GH & Kim SJ (2000)

Species identification of mycobacteria by PCR-

restriction fragment length polymorphism of the rpoB

gene. J Clin Microbiol 38, 2966–2971.

34 Kyhse-Andersen J (1984) Electroblotting of multiple

gels: a simple apparatus without buffer tank for rapid

transfer of proteins from polyacrylamide to nitrocellu-

lose. J Biochem Biophys Methods 10, 203–209.

35 Stirling JW & Graff PS (1995) Antigen unmasking for

immunoelectron microscopy: labeling is improved by

treating with sodium ethoxide or sodium metaperiodate,

then heating on retrieval medium. J Histochem Cyto-

chem 43, 115–123.

36 Houghten RA (1985) General method for the rapid

solid-phase synthesis of large numbers of peptides: spec-

ificity of antigen–antibody interaction at the level of

individual amino acids. Proc Natl Acad Sci USA 82,

5131–5135.

37 Merrifield RB (1969) Solid-phase peptide synthesis. Adv

Enzymol Relat Areas Mol Biol 32, 221–296.

38 Tam JP, Heath WF & Merrifield RB (1983) SN 1 and SN

2 mechanisms for the deprotection of synthetic peptides

by hydrogen fluoride. Studies to minimize the tyrosine

alkylation side reaction. Int J Pept Protein Res 21, 57–65.

39 Vera Bravo R, Marin V, Garcia J, Urquiza M, Torres

E, Trujillo M, Rosas J & Patarroyo ME (2000) Amino

terminal peptides of the ring infected erythrocyte surface

antigen of Plasmodium falciparum bind specifically to

erythrocytes. Vaccine 18, 1289–1293.

40 Yamamura H (1978) Neurotransmitter Receptor Binding.

Raven Press, New York, NY.

41 Chapeton-Montes JA, Plaza DF, Barrero CA & Patar-

royo MA (2007) Quantitative flow cytometric monitor-

ing of invasion of epithelial cells by Mycobacterium

tuberculosis. Front Biosci 30, 650–656.

M. tuberculosis Rv2560 protein characterization D. F. Plaza et al.

6364 FEBS Journal 274 (2007) 6352–6364 ª 2007 The Authors Journal compilation ª 2007 FEBS