PEDS Advance Access published March 16, 2012
Protein Engineering, Design & Selection pp. 1– 8, 2012
doi:10.1093/protein/gzs011
The role of Mycobacterium tuberculosis Rv3166c
protein-derived high-activity binding peptides
in inhibiting invasion of human cell lines
Marisol Ocampo1,2,4, Daniel Aristizábal-Ramı́rez1,2,
Diana M.Rodrı́guez1,2, Marina Muñoz1,2,
Hernando Curtidor1,2, Magnolia Vanegas1,2,
Manuel A.Patarroyo1,2 and Manuel E.Patarroyo1,2,3
1
Fundación Instituto de Inmunologı́a de Colombia (FIDIC), Cra. 50
No. 26– 20, Bogotá, Colombia, 2School of Medicine and Health Sciences,
Universidad del Rosario, Bogotá, Colombia and 3School of Medicine,
Universidad Nacional de Colombia, Bogotá, Colombia
4
To whom correspondence should be addressed.
E-mail: marisol.ocampo@urosario.edu.co
Received September 12, 2011; revised February 14, 2012;
accepted February 21, 2012
Edited by Padmanabhan Balaram
Given the urgent need for designing a new antituberculo-
sis vaccine conferring total protection on patients of all
ages, following the line of research adopted by our insti-
tute, this work has identified Mycobacterium tuberculosis
(Mtb) Rv3166c protein high-activity binding peptides
(HABPs) which are able to inhibit bacterial invasion of
U937 (monocyte-derived macrophages) and A549 (type II
alveolar epithelial cells) cell lines. The presence and tran-
scription of the rv3166c gene in the Mtb species complex
was confirmed by polymerase chain reaction (PCR) and
reverse transcriptase-PCR; Rv3166c expression was eval-
uated by western blot and cellular localisation confirmed
by immunoelectron microscopy. Its presence was mainly
determined on cell surface. Sixteen peptides covering its
entire length were chemically synthesised and tested for
their ability to bind to U937 and A549 cells. Two U937
HABPs were identified and three for A549, one of them
being shared by both cell lines. The four HABPs found
inhibited Mtb entry by 15.07 –94.06%. These results led
us to including Rv3166c HABPs as candidates for further
studies contributing towards the search for a multiepi-
tope, chemically synthesised, subunit-based antituberculo-
sis vaccine.
Keywords: antituberculosis vaccine/human cell line/invasion
inhibition/sub-cellular localisation/synthetic peptide
Introduction
Tuberculosis (TB) is one of the three infectious diseases
having major public health importance worldwide, the others
being malaria and acquired immunodeficiency syndrome
(AIDS). The Mycobacterium tuberculosis (Mtb) bacillus is
responsible for 8 800 000 new cases (139 cases per 100 000
inhabitants) and around 1.7 million deaths annually (WHO,
2009). Although just 5 – 10% of the people actually infected
with tuberculosis develop it, a third of the world’s population
is infected with the bacillus thereby constituting an even
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bigger problem for people co-infected with AIDS (Corbett
et al., 2003). The appearance of multidrug-resistant tubercu-
losis strains (MDR-TB) and extremely drug-resistant tubercu-
losis strains (XDR-TB) in different geographical areas
(Iseman, 1999) must be added to this problem.
Even though there is an anti-TB vaccine (the
Mycobacterium bovis bacillus Calmette-Guérin, BCG, atte-
nuated strain), meta-analysis studies have demonstrated that
it has high variability (0 – 80%) and that it is also ineffective
in adults (Fine and Vynnycky, 1998; Black et al., 2003; Fine,
2005). Although BCG generates a circulating population of
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cells in response to Mycobacterium, these are not efficient
enough to prevent rapid bacterial growth in the lungs
(Cooper, 2009), meaning that a more effective vaccine must
be designed which confers complete protection against the
disease. Global research has focused on finding new
Mtb-specific antigens which could be used as an alternative
for replacing or improving the BCG vaccine (Kaufmann
et al., 2006; Changhong et al., 2009).
The Mtb bacillus is a facultative intracellular pathogen; in-
fection begins with its entry through the respiratory airways
after aerosols have been inhaled from an individual having
the active disease. The bacillus then becomes located in the
pulmonary alveoli where it invades epithelial cells and
becomes phagocytosed by macrophages, representing the
first step in the pathogen – host interaction (Raja, 2004). It has
been demonstrated that the bacillus is able to invade other
nonphagocytic cells, such as HeLa cells and fibroblasts
(Garcia-Perez et al., 2003).
Different molecules must be expressed in the host– patho-
gen interaction for pathogenesis to occur, mainly those
exposed on the bacillus surface since they perform enzymatic
and signal transduction functions, or could be acting as
receptors (Sigler and Hofer, 1997; Asselineau et al., 2002;
Nigou et al., 2003; Malen et al., 2007).
The rv3166c gene encodes a transmembrane protein con-
taining three hundred and nineteen 33.4 kDa molecular
weight amino acids. It appears as a conserved hypothetical
protein in the Tuberculist server (http://genolist.pasteur.fr/
TubercuList/).
The Rv3166c protein may have molecular functions
according to the ProKnow annotation function server such as
zinc ion binding and catalytic activity, as well as biological
functions such as electron transport, carbohydrate metabol-
ism and transcription regulation (http://www.doe-mbi.ucla.
edu/TB/PUBLIC/qs/qsearch.php?orf=Rv3166c).
It has been reported that the Mtb rv3166c gene forms
part of the group of genes which are suppressed when the
bacillus is treated with isoniazid (INH) (Fu, 2006). It has
also been found that the SigL regulatory sigma factor helps
rv3166 gene expression, among other genes which may be
involved in important cell envelope processes (Dainese
et al., 2006).
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M.Ocampo et al.

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Fig. 1. Assessment of rv3166 gene presence and transcription. (A) Gene amplification of gDNA isolated from the strains included in this study. MWM:
200 bp molecular weight marker; lanes 1– 3: PCR amplification of the rpoB gene for 1—Mtb H37Rv, 2—Mtb H37Ra and 3—Negative PCR control; lanes 4–
6 show PCR amplification of the rv3166c gene for 4—Mtb H37Rv, 5—Mtb H37Ra and 6—Negative PCR control. (B) Gene amplification of cDNA from the
samples included in this study. MWM: 200 bp molecular weight marker; lanes 1 –4 show PCR amplification of the rpoB gene from 1—Mtb H37Rv plus
synthesis, 2—Mtb H37Rv minus synthesis, 3—Mtb H37Ra plus synthesis and 4—Mtb H37Ra minus synthesis. Lanes 5 –7 show PCR amplification of the
rv3166c gene for 5—Mtb H37Rv plus synthesis, 6—Mtb H37Ra plus synthesis and 7—negative PCR control.

The Rv3166c protein was chosen for further research to Results

build on previously obtained results (Restrepo-Montoya
et al., 2009; Vizcaino et al., 2010). The rv3166c gene’s pres-
ence and transcription was evaluated in Mtb H37Rv and Mtb
H37Ra strains using polymerase chain reaction (PCR) and
reverse transcriptase-PCR (RT-PCR) assays. Rv3166c ex-
pression and subcellular localisation was experimentally veri-
fied by western blot and immunoelectron microscopy,
respectively. The Rv3166c protein was synthesised in 20-mer
long, nonoverlapped peptides which were analysed using a
robust and highly specific methodology (widely used in
Plasmodium falciparum studies in our institute) (Garcia
et al., 2006; Patarroyo and Patarroyo, 2008; Rodriguez et al.,
2008) to identify high-activity binding peptides (HABPs).
These peptides’ ability to inhibit mycobacterial entry into
macrophages derived from monocytes (U937) and alveolar
type II epithelial cells (A549) was also evaluated. Taking
into account the results obtained for Rv3166c, then some of
the Rv3166c-HABPs mentioned below may be considered in
future studies regarding their immunomodulatory nature and
become included in designing a multiepitope, subunit-based,
chemically synthesised vaccine against TB.
rv3166 gene presence and transcription
The Mtb Rv3166c protein sequence was obtained from the
Tuberculist Webserver database (http://genolist.pasteur.fr/
TubercuList/) and used as the query sequence for BLASTp
analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). This ana-
lysis revealed 100% identity with M.bovis BCG BCG_319c
and Mycobacterium leprae MLCB1779.35c hypothetical pro-
teins, 98% with a conserved Mycobacterium parascroful-
aceum hypothetical protein and 97% with Mycobacterium
kansasii MkanA1_10062 and Mycobacterium avium
MAV_4054 hypothetical proteins.
PCR experiments with mycobacterial gDNA confirmed the
presence of the rv3166 gene in both strains tested. The 360-
and 566-bp amplification fragments were obtained for rpoB
and rv3166 genes in Mtb H37Rv and H37Ra, respectively
(Fig. 1A).
The rpoB constitutive gene amplification by Plus synthe-
sis cDNA kit (EasyScript) was used as control for tran-
scription assays for each mycobacterial strain; the absence
of product in RT control synthesis confirmed that there

Page 2 of 8

Fig. 2. Rv3166c protein recognition. Western blot with rabbit pre-immune
serum and post-third inoculation against Mtb H37Rv total sonicate (lanes 1
and 2) and subcellular fractions (lanes 3 –5). Lane 1 shows pre-immune
serum against Mtb H37Rv total sonicate, lane 2 shows post-third
immunisation serum against Mtb H37Rv total sonicate, lane 3 Mtb H37Rv
membrane, lane 4 Mtb H37Rv cell wall and lane 5 shows the Mtb H37Rv
cytosolic fraction. The molecular weight marker is shown on the left-hand
side of the figure.
was no contamination with gDNA. The 566-bp amplifica-
tion fragment observed in Mtb H37Rv and H37Ra plus
synthesis cDNA indicated that the rv3166 gene was being
transcribed in both strains in 7H9 culture medium condi-
tions (Fig. 1B).
Rv3166c protein recognition
Sera obtained from rabbits inoculated with polymer peptides
were used in western blot (Fig. 2) against Mtb H37Rv sonic-
ate and subcellular fractions (wall, membrane and cytosol).
Sera were pre-adsorbed on columns with Escherichia coli
and Mycobacterium smegmatis lysates and synthetic
P.falciparum 66 (SPf66) vaccine to eliminate cross-reactivity
against bacterial, mycobacterial and polymerisation antigens.
Pre-immune serum did not recognise proteins from Mtb
H37Rv total sonicate (Fig. 2, lane 1); the serum obtained by
inoculating peptide 16 298 recognised an intense 33 kDa
band in both total sonicate and mycobacterial wall, cytosol
and membrane fractions. A 10 kDa band was also recognised
in cell wall and cytosol fractions, but with lower intensity
(Fig. 2).
Rv3166c protein localisation was corroborated by the
immunoelectron microscopy (IEM) technique. Figure 3
shows that Rv3166c is a surface protein due to the presence
of more colloidal gold particles on Mtb H37Rv surface than
inside the bacillus.
Rv3166c peptides inhibit mycobacterial entry to cells
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Fig. 3. Immunoelectron microscopy. 6000 magnification. Rv3166c
protein detection can be seen on Mtb H37Rv bacillus surface, as indicated
by the colloidal gold particles indicated by black arrows.
Rv3166c peptides’ binding activity
Taking Rv3166c localisation into account, 20 amino acid
nonoverlapped peptides covering the whole protein sequence
were synthesised. Figure 4 gives ligand-receptor-type assay
results (i.e. the profile for Rv3166c protein peptides’ binding
to U937 and A549 cells); the black bars represent the
binding percentage. Four HABPs were identified; 16 287
(21LLMLAGAALRGHLPADDGAPY40) had high binding to
the U937 cell line, 16 295 (181LITSRRQRKSAPARI
SGDRIY200) and 16 298 (241MERELSHVPGVAPQDFDTP
TY260) had high binding to the A549 cell line and 16 296
(201ESPAPSARSESLARAAEIGLY220) had high binding to
both cell lines. Further studies will be aimed at establishing
these HABPs’ receptors on target cells.
Mycobacterial entry inhibition
Inhibition of mycobacterial entry due to HABP binding is
shown in Fig. 5. Peptide 16 295 only inhibited this by 6.38%
when it was used at the highest concentration evaluated in
the assay (100 mM). The three remaining peptides had
concentration-dependent inhibition, thereby discarding a
toxic peptide effect on these cells.
Peptide 16 296 inhibited mycobacterial entry to A549
cells by 15.07 and 46.79% at 50 and 100 mM peptide con-
centration, respectively. On the contrary, 16 298 was able to
inhibit invasion of A549 cells, even at very low peptide con-
centrations such as 5 mM (46.42%), reaching 55.77% entry
inhibition at 50 mM and 65.36% at 100 mM. The 10 mM
cytochalasin control inhibited mycobacterial entry by
89.99%; this reagent is known to be an actin polymerisation
inhibitor. Otherwise, anti-16 298 serum was used as inhibitor
in assays regarding mycobacterial invasion of A549 cells.
The serum (1 : 100) was able to inhibit around 29% of myco-
bacterial entry to host cells. Pre-immune and anti-16 300
Page 3 of 8
M.Ocampo et al.
Fig. 4. Rv3166c peptide binding activity profile. Amino acid sequence and specific U937 and A549 cell binding activity for Rv3166c synthesised peptides.
Peptides are shown on the left-hand side, numbers indicating their position in the native protein; Tyr was added at the C-terminal end of those peptides lacking
it. The black bars on the right-hand side represent each peptide’s binding activity determined as the specific binding/total peptide added ratio. The dotted line
separates peptides having 1% binding activity (HABPs).
Fig. 5. A549 and U937 cell invasion inhibition assays. HABP 16 295,
16 296 and 16 298 inhibition of A549 cell invasion percentages are shown at
three concentrations (5, 50 and 100 mM). HABPs 16 287 and 16 296 U937
cell invasion inhibition percentages are shown at the same three
concentrations. About 10 mM cytochalasin was used as positive invasion
inhibition control in both cases.
sera were used as control as they did not inhibit mycobacter-
ial entry.
In the assay regarding inhibition of Mtb H37Rv entry to
U937 cells, the cytochalasin control prevented inhibition by
93.67% at 10 mM concentration. Peptide 16 287 progressive-
ly inhibited entry when peptide concentration was increased:
37.37% inhibition at 5 mM, 52.81% at 50 mM and 58.68% at
100 mM. By contrast, 16 296 had very high inhibition per-
centages (92.03– 94.06%) at all concentrations used; this
could have suggested a toxic effect on the cells, even though
no damage was observed in U937 cell morphology during
this assay. This peptide thus presented a different effect com-
pared with mycobacterial entry inhibition regarding each cell
line.
Scrambled HABPs (having the same amino acid compos-
ition but different sequence) were synthesised and their
Page 4 of 8
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Fig. 6. Rv3166c HABPs’ secondary structure. The results have been
expressed in terms of mean residue ellipticity [], its units being
degreescm2 dmol, according to [] ¼ l/(100lcn). Peptides 16 287 and
16 296 had an a-helix secondary structure and peptides 16 295 and 16 298
had random coil secondary structure.
inhibitory activities were also determined by following the
same methodology. Scrambled peptide 38 270 (RALGGPAG
PMLDAYALHLLAD), 16 287 analogue and 38 271 (SSRE
YPLASGEAASEAAILRP) 16 296 analogue had ,10% in-
hibitory effect at all concentrations tested.
HAPBs’ structural elements
Figure 6 shows that peptides 16 287 and 16 296 had a-helix
structural elements, characterised by a 212 nm maximum and
207 and 222 nm minima, whereas peptides 16 295 and
16 298 had a random coil configuration, characterised by a
198 nm minimum and 212 nm maximum. These results
agreed with the secondary structure prediction given by the
self-optimised prediction method with alignment (SOPMA)
for the same peptides (http://npsa-pbil.ibcp.fr/NPSA/npsa_
sopma.html).

Discussion
Despite the efforts made at preventing and controlling tuber-
culosis, this disease continues to cause millions of deaths an-
nually. In spite of the BCG vaccine being available and drug
treatments producing variable protection, these have not been
consolidated as effective methods aimed at preventing the
disease. This has shown how imperative it is to have a new
vaccine conferring total protection against tuberculosis. A
methodology for developing a new vaccine has thus been
implemented in our institute which has been based on chem-
ically synthesised Mtb surface protein peptides having specif-
ic U937 macrophage and alveolar epithelial A549 cell
binding activity (HABPs), as well as being able to inhibit
pathogen entry to such cells (Forero et al., 2005; Garcia
et al., 2005; Vera-Bravo et al., 2005; Plaza et al., 2007;
Chapeton-Montes et al., 2008; Patarroyo et al., 2008a, b;
Cifuentes et al., 2010; Rodriguez et al., 2010; Cáceres et al.,
2011; Ocampo et al., 2011).
As Mtb entry to host cells is mediated by the bacillus’
surface interacting with cell membrane receptors, our studies
have been focused on this pathogen’s surface proteins. Most
of these proteins’ functions are not known, nor are their pos-
sible role in bacterial invasion of host cells or the role they
play in bacillus survival within the host cell. This study has
been focused on the Rv3166c surface protein which has been
classified as a membrane protein according to predictions
made by using bioinformatic tools. Comparative analyses
have revealed Rv3166c protein homologues in M.leprae,
M.parascrofulaceum, M.kansasii and M.avium mycobacterial
strains outside the Mtb complex (MTC); these species are
also disease-causing agents in both humans and other
mammals. Rv3166c’s high percentage of identity with other
pathogenic species different to Mtb thus indicates this pro-
tein’s possible role in virulence and infection. It has been
possible to determine the presence of the gene encoding the
Rv3166c protein in Mtb H37Rv experimentally using the la-
boratory reference strain.
The TMHMM v. 2.0 (http://www.cbs.dtu.dk/services/
TMHMM/) tool led to determining the presence of trans-
membrane regions; four transmembrane helices were pre-
dicted in the following amino acids: i12 – 34o, o49 – 71i,
i101 –123o and o161 – 183i (i/o denotes inside or outside).
The first helix was a possible signal peptide according to
TMHMM. However, it was found that Rv3166c did not
present a signal peptide when using SignalP and SecretomeP
tools (Vizcaino et al., 2010). This suggested that the protein
was being secreted via the general export pathway (Gomez
et al., 2000).
Protein expression was assessed once rv3166c gene tran-
scription had been confirmed in Mtb H37Rv. The serum
obtained by inoculating polymer peptide 16 298 recognised
an intense 33 kDa band; taking into account that the calcu-
lated molecular weight for Rv3166c is around 33.4 kDa, then
it could be suggested that the serum contained specific anti-
bodies against the protein of interest. The low molecular
weight and weak 10 kDa band could have been due to a
protein cleavage product being recognised in the cytoplasm;
homology analysis discarded the possibility that such recog-
nition was due to a cross-reaction with another mycobacterial
antigen. These results indicated that the protein was being ef-
fectively expressed in the virulent Mtb strain and
Rv3166c peptides inhibit mycobacterial entry to cells
bioinformatics predictions were corroborated, placing
Rv3166c within the mycobacterial envelope. IEM assays cor-
roborated this protein’s location on the mycobacterial surface
thereby leading to the assumption that it might interact with
host cell surface receptors. Established Rv3166c annotation
as a secreted protein (http://www.doe-mbi.ucla.edu/TB/
PUBLIC/qs/qsearch.php?orf=Rv3166c) disagrees with the
experimental results reported here, which demonstrated its
localisation on the mycobacterial envelope.
It has been found that Mtb surface proteins interact with
different receptors on host cell surface to mediate invasion
by the bacillus; some of these would be mannose receptors,
Toll-like receptors, scavenger receptors, surfactant protein A
and D receptors and complement receptors 1, 3 and 4 (Ernst,
1998; Rooyakkers and Stokes, 2005). Taking the specific
interaction between mycobacterial protein sequences and the
host cell into account, the HABPs’ ability to bind to A549
and U937 cells was determined to establish whether peptides
binding to target cells were able to inhibit Mtb H37Rv entry
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to these cells. Peptide 16 287 presented high specific U937
binding activity at the N-terminal and significant inhibition
of mycobacterial entry to these cells while 16 295 and
16 298 bound specifically to A549 cells at the C-terminal,
even though 16 295 had very low inhibition which is why it
was assumed that this sequence’s specific interaction with
A549 epithelial cells was not relevant for Mtb entry. It is
interesting that 16 296 bound to both cell lines with high ac-
tivity and inhibited mycobacterial entry to A549 cells regard-
less of concentration, whereas it had a highly inhibitory
effect on mycobacterial entry to U937 cells at the concentra-
tions assayed here.
Inhibition assays involving U937 cells with scrambled
HABPs (i.e. peptides having the same amino acid compos-
ition but different sequence) demonstrated that HABP entry
inhibition depended exclusively on their amino acid sequence
and thus on their structural configuration since their activity
became reduced when their sequences became altered.
Bearing in mind the relationship between peptide structure
and function as an immunogen, structural element content
had to be determined by circular dichroism (CD) analysis for
all Rv3166c HABPs. The results with the corresponding de-
convolution using SELCON, CONTINLL and CDSSTR soft-
ware (Sreerama and Woody, 2000) showed b-turn and
a-helix features as being these HABPs’ main structural char-
acteristics. A relationship could not be established between
each HABP’s structure and its binding or inhibitory activity.
The presence and transcription of the Rv3166c
protein-encoding gene has thus been shown experimentally
in Mtb strains. This protein was expressed in normal culture
conditions and was mainly localised on mycobacterial
surface. Only 4 of the 16 peptides constituting the protein se-
quence had high specific binding ability (HABPs) regarding
infection of target cells (macrophages and alveolar epithelial
cells). The structural elements found for these peptides fit
the predictions made by bioinformatic tools. Peptides 16 287,
16 296 and 16 298 were of special interest because they sig-
nificantly inhibited mycobacterial entry to target cells. These
peptides will be used in further studies for establishing their
immunomodulating potential, taking into account that our ap-
proach is based on the search for protein fragments which
are involved in mycobacterial – target cell interaction.
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M.Ocampo et al.
An HABP’s antiserum inhibitory effect on invasion could
be associated with Rv3166c location on the surface involved
in target cell invasion, thereby confirming the results of
protein immunolocalisation. Likewise, treating Mtb H37Rv
with proteinase K (nonspecific serin protease), removing
surface proteins, has completely inhibited mycobacterial
entry to epithelial cells and macrophages in in vitro assays,
thereby showing the importance of mycobacterial surface
protein components in an interaction with (infection) target
cells. The principles underlying the proposed methodology
have been based on identifying HABPs from Mtb proteins
involved in mycobacterial entry to the host cell for inducing
an immune response which may be able to block their
binding to the host cell. This is why HABPs which are able
to inhibit mycobacterial entry to macrophages and epithelial
cells in vitro have been of such special interest as they will
be tested in assays using animal models for evaluating their
protection-inducing potential against Mtb infection.
In our experience concerning P.falciparum, sequences spe-
cifically binding to target cells and which can block invasion
could be used as templates for designing peptides which can
immunomodulate a protective response against the disease.
An immune response triggered against mycobacterial high
specific binding sequences could prevent the invasion of
target cells, either during a first encounter with the bacillus
or during the reactivation of a latent infection.
Materials and methods
Chromosomal DNA extraction and PCR amplification
The presence and transcription of the rv3166c gene was
assessed in the Mtb H37Rv (ATCC 27 294) and H37Ra
(ATCC 25 177) strains. Genomic DNA was extracted using
an Ultra Clean Microbial DNA Isolation Kit (MO Biolab),
following the manufacturer’s recommendations. Extracted
DNA was suspended and kept in 1X TE buffer.
PCR amplifications were performed in a thermocycler
(Perkin Elmer Gene Amp PCR system 9600), 100 ng of
genomic DNA from the different Mycobacterium strains
being incubated with a PCR mixture (1.5 mM MgCl2, 1 mM
dNTPs, 1 mM of each oligonucleotide primer and 1.25 U
BioTaq DNA polymerase (Bioline), 1 Taq polymerase re-
action buffer). The PCR conditions were as follows: an
initial 5-min denaturing stage at 948C followed by 35 cycles
consisting of: 30 s at 588C, 30 s at 728C and 15 s at 948C. A
final extension cycle lasted 5 min at 728C.
Extracted DNA quality was assessed by amplifying a 360
base pair (bp) fragment from the rpoB gene (encoding the
RNA polymerase B-subunit) using sense (50 -TCAAGGAG
AAGCGCTACGA-30 ) and antisense (50 -GGATGTTGATCA
GGGTCTGC-30 ) primers. The gene encoding Rv3166c in
the Mtb H37Rv strain was analysed to design a specific
primer set to verify the presence and transcription of this
gene in in vitro culture conditions. The designed primers’
sequences were: rv3166c-sense: 50 -GCTGCTGATTGCGA
TACTG-30 and rv3166c-antisense: 50 -CCTCACGGTGCTC
CTCG-30 , which amplified a 566 bp fragment. DNase- and
RNase-free water was used as a negative PCR control for all
reactions. All amplifications were visualised on 1.5% agarose
gel stained with SYBR Safe (Invitrogen, Carlsbad, CA,
USA).
Page 6 of 8
RNA extraction and RT-PCR. Total RNA was isolated from
the bacterial pellet by homogenisation in 1 ml TRIZOL
reagent (Invitrogen) following the manufacturer’s suggested
protocol, then 10 ml of the sample were treated with amplifi-
cation grade deoxyribonuclease I (Invitrogen).
Complementary DNA (cDNA) synthesis was carried out
using SuperScript III reverse transcriptase (Invitrogen) and
random hexamers following the manufacturer’s recommenda-
tions. A negative synthesis control replacing the SuperScript
III enzyme with diethylpyrocarbonate-treated water was also
included for each sample. Two micro litre cDNA were used
as template for PCR amplification following the conditions
previously described for DNA. The rpoB gene was used as
positive transcription control (Lee et al., 2000).
Peptide synthesis and radiolabelling
Sixteen 20-mer-long non-overlapped peptides were synthe-
sised, covering the entire Rv3166c protein sequence.
Peptides were synthesised in solid phase (t-Boc technique).
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Peptides were then purified by reversed phase-high-perform-
ance liquid chromatography and characterised by matrix-
assisted laser desorption/ionization-time of flight mass spec-
trometry. A Tyr residue was added at the C-terminus of those
peptides lacking it to facilitate I125 radiolabelling (Garcia
et al., 2004; Rodriguez, et al., 2008; Ocampo et al., 2011).
Purified peptides (2 nmol) were radiolabelled with 5 ml
NaI125 (100 mCi/ml, ICN) and 0.3 mmol of chloramine-T.
After incubation for 15 min, the reaction was stopped with
0.18 mmol of sodium metabisulphite. The radiolabelled
peptide was passed through a Sephadex G-10 column
(Pharmacia) to separate it from the reaction subproducts.
Cell culture
The A549 (ATCC CLL-185) and U937 (ATCC CRL-2367)
cell lines were kept in Roswell Park Memorial Institute
(RPMI) 1640 medium culture supplemented with 10% fetal
bovine serum (FBS) at 378C and 5% CO2. These two adher-
ent cell lines were detached using 0.6% trypsin and 0.2%
ethylenediaminetetraacetic acid. Cells were spun at 500 g for
5 min and suspended in RPMI 1640 medium without serum.
Binding assays
A highly specific, sensitive and robust receptor-ligand-type
binding assay was based on previous studies with U937
human alveolar macrophages and alveolar epithelial A549
cells (Forero et al., 2005; Garcia et al., 2005; Vera-Bravo
et al., 2005; Plaza et al., 2007; Cifuentes et al., 2010;
Rodriguez et al., 2010; Cáceres et al., 2011; Ocampo et al.,
2011). 1.5  106 cells were incubated with different radiola-
belled peptide concentrations (0 –950 nM) in the presence or
absence of the same nonradiolabelled peptide (40 mM) for
90 min at 48C. An aliquot of this mixture was passed
through a 60 : 40 dioctylphthalate– dibutylphthalate cushion
(1015 g/ml) and spun at 4500 g for 3 min. A gamma counter
was used for quantifying cell-associated radioactivity. All
assays were performed in triplicate. Peptides having .1%
specific binding were considered to be HABPs, in line with
previous studies performed in our laboratory (Forero et al.,
2005; Garcia et al., 2005; Vera-Bravo et al., 2005; Plaza
et al., 2007; Cifuentes et al., 2010; Rodriguez et al., 2010;
Cáceres et al., 2011; Ocampo et al., 2011).

CD assays. Rv3166c HABPs were analysed by CD to deter-
mine possible secondary structure elements present in their
sequences and to determine whether there were a structure –
function correlation. CD spectra were registered at 208C on a
Jasco J-810 spectropolarimeter at wavelengths ranging from
260 to 190 nm, in 1-cm cuvettes (Provencher and Glockner,
1981; Greenfield, 1999). Peptides were dissolved at 0.1 mM
concentration in pure water or 30% (v/v) trifluoroethanol/
water. Each spectrum was obtained by averaging three read-
ings taken at 20 nm/min, using 1 nm spectra bandwidth, cor-
rected for base deviation. The results were expressed as
mean residue ellipticity [], its units being degreescm2 
dmol, according to [] ¼ l/(100lcn), where l is mea-
sured ellipticity, l is the optical path length, c the peptide
concentration and n the number of amino acid residues in the
peptide sequence (Sreerama et al., 1999).
Rabbit immunisation and western blot
Peptide sequences chosen for immunising rabbits were
selected based on a Rv3166c sequence analysis, using
BepiPred 1.0b Server epitope prediction software available at
http://www.cbs.dtu.dk/services/BepiPred/. Synthetic polymer
peptides 16 298 (CG 241MERELSHVPGVAPQDFDTPT260
GC) and 16 300 (CG 281VSLFAEARFSPHVMNEEHRE300
GC) from the Rv3166c protein, mixed with Freund’s
Incomplete Adjuvant, were used as immunogens in New
Zealand rabbits, using a 0-, 20- and 40-day immunisation
scheme. Final bleeding took place 20 days after the third in-
oculation and polyclonal antibodies were obtained for
protein immunodetection.
The Mtb H37Rv proteins from total sonicate and cell frac-
tions (wall, membrane, cytosol and filtrate) were separated in
a sodium dedocyl sulphate – polyacrylamide gel electrophor-
esis discontinuous system, using a 10 – 20% polyacrylamide
gradient. A total of 1 mg/ml from each fraction and sonicate
were loaded onto each gel. Gels were transferred to nitrocel-
lulose membranes using the semi-dry blotting technique
(Kyhse-Andersen, 1984) and blocked (blocking solution: 1%
TBS-Tween and 5% milk). The membranes were incubated
for 1 h with a 1 : 100 dilution of each rabbit serum.
Following five 1% TBS-Tween washes they were incubated
for 1 h with a 1 : 5000 dilution of alkaline phosphatase con-
jugated with antirabbit IgG antibody. The reactions were
revealed with nitro blue tetrazolium/5-bromo-4-chloro-3-
indolyl-phosphate.
Immunoelectron microscopy
The Rv3166c protein’s subcellular localisation was deter-
mined by Hitachi Hu-12A transmission electronic micro-
scope. Fine slices (400 nm) of mycobacteria immersed in
London resin white resin were prepared. The same immuno-
blot serum was used as primary antibody and colloidal gold
particle-coupled antirabbit IgG was used as secondary anti-
body. Six per centuranyl acetate was added to provide the
samples with contrast (Plaza et al., 2007; Cifuentes et al.,
2010; Rodriguez et al., 2010; Cáceres et al., 2011; Ocampo
et al., 2011).
Invasion inhibition assays
The assay for determining HABPs’ role in mycobacterial
entry has been described previously (Bermudez and
Goodman, 1996; Chapeton-Montes et al., 2008; Rodriguez
Rv3166c peptides inhibit mycobacterial entry to cells
et al., 2010). Mycobacterium tuberculosis H37Rv was cul-
tured in a Middlebrook 7H9 medium for 12 days (mid-log
phase), then harvested and suspended in 1 phosphate buf-
fered saline. Bacteria were labelled with 2 ml 20 SYBR
Safe.
A549 and U937 cells were loaded into six-well plates with
FBS-supplemented RPMI 1640 medium (Gibco) and incu-
bated at 378C in 5% CO2 overnight. Different peptide con-
centrations of 400 ml (5, 50 and 100 mM) were added to each
well and incubated at 378C for 1 h. Each treatment was per-
formed in triplicate with its respective negative control. Cells
were incubated with RPMI 1640 medium as invasion control
and cytochalasin D (10 mM) was used as inhibition control.
About 1  109 Mtb bacilli, suspended in 200 ml RPMI
medium without antibiotics, were loaded per well.
Multiplicity of infection (MIO) was 1 : 1000. Plates were
incubated at 378C overnight. This was followed by three
washes with Hank’s buffered salt solution to remove extra-
cellular bacteria. The cell monolayer was detached and
Downloaded from http://peds.oxfordjournals.org/ by guest on March 23, 2012
infected cells were fixed with 400 ml 2% paraformaldehyde
at 48C for 1 h. Before the samples were read on the cyt-
ometer, cells were incubated for 5 min with 200 ml 0.3%
methylene blue to eliminate autofluorescence. The samples
were analysed on a FACScan cytometer (Becton Dickinson)
equipped with a 488-nm argon laser. Data were acquired
using Cellquest software (Becton Dickinson) and analysed
by using FCS Express V3 cytometry software; 5000 events
were read for each sample (Chapeton-Montes et al., 2008).
The data were statistically analysed using Student’s t-test.
Acknowledgements
We would like to thank Jason Garry for translating and reviewing this manu-
script. The Mtb H37Rv subcellular protein fractions were obtained through
the National Institute of Health (NIH) Biodefense and Emerging Infection
Research Resources Repository, National Institute of Allergy and Infectious
Diseases (NIAID).
Conflict of interest
The authors declare no conflict of interest. The authors alone
are responsible for the content and writing of this
manuscript.
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