The 19-kDa antigen of Mycobacterium tuberculosis is a major adhesin that

binds the mannose receptor of THP-1 monocytic cells and promotes

phagocytosis of mycobacteria

Hugo Diaz-Silvestre, Patricia Espinosa-Cueto, Alejandro Sanchez-Gonzalez,

Miguel A. Esparza-Ceron, Ana Laura Pereira-Suarez, German Bernal-Fernandez,

Clara Espitia, Raul Mancilla*

Departamento de Inmunologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico,

Apartado Postal 70-228, 04510 Mexico, DF, Mexico

Received 6 October 2003; received in revised form 11 June 2005; accepted 20 June 2005

Abstract

Identification of mycobacterial adhesins is needed to understand better the pathogenesis of tuberculosis and to develop new strategies to

fight this infection. In this work, THP-1 monocytic cells were incubated with Mycobacterium tuberculosis culture filtrate proteins labelled

with biotin and a dominant 19-kDa adhesin was found. This adhesin was characterized as the glycosylated and acylated 19-kDa antigen (Rv

3763). These findings were confirmed in assays with culture filtrate proteins and cell-wall fractions from a recombinant Mycobacterium

smegmatis strain that overexpresses the 19-kDa antigen. Further, fluorescent microspheres coated with recombinant culture filtrate proteins

adhere to cells in higher numbers than microspheres coated with native M. smegmatis proteins. The binding of the 19-kDa antigen to cells

was inhibited with mannose receptor competitor sugars, Ca2C chelators and with a monoclonal antibody to the human mannose receptor.

Phagocytosis assays showed high-level binding of bacilli to THP-1 cells that was inhibited with a-methyl-mannoside, mannan, EDTA and

mAbs to the mannose receptor and to the 19-kDa M. tuberculosis antigen. Immunoprecipitation, cell-surface ELISA and immunostaining

confirmed the expression of the mannose receptor by THP-1 cells. In conclusion, here we show that the macrophage mannose receptor,

considered a pathogen pattern recognition receptor, may interact with mannose residues of mycobacterial glycoproteins that could promote

the phagocytosis of mycobacteria.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Mycobacterium tuberculosis; 19-kDa antigen; Mannose receptor; Immunoprecipitation; Macrophages; Phagocytosis

1. Introduction

Mycobacterium tuberculosis is an intracellular pathogen

that infects preferentially the lungs. The initial event in the

pathogenesis of tuberculosis (TB) is the adherence of bacilli

to host alveolar macrophages. For this, bacilli interact with a

variety of macrophage surface receptors including comp-

lement receptors CR1, CR3, and CR4 [1–3] and the mannose

receptor [2,4,5]. Receptors for the surfactant protein A [6,7],

0882-4010/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micpath.2005.06.002

* Corresponding author. Tel.: C52 55 5622 3868; fax: C52 55 5622

3369.

E-mail address: mancilla@servidor.unam.mx (R. Mancilla).

the CD14 glycoprotein [8] and scavenger receptors [9] have

also been implicated in cell infection. Little is known on the

role of mycobacterial ligands (adhesins) in the adherence of

bacilli to macrophages. LAM, a cell-wall lipoglycan,

interacts with macrophages promoting phagocytosis of

bacilli [5,10,11]. There are few reports describing myco-

bacterial proteins with an adhesin function. Non-invasive

E. coli transfected with M. tuberculosis DNA encoding a

50-kDa peptide acquires the ability to invade epithelial cells

[12]. It has been reported that macrophages ingest microbe-

ads coated with the antigen 85 complex [13] and that the

mycobacterial heparin-binding-hemaglutinin binds epi-

thelial cells and macrophages [14]. The receptors involved

in the binding of these putative adhesins and their

participation in the uptake of bacilli by human macrophages

are not documented.

Microbial Pathogenesis 39 (2005) 97–107

www.elsevier.com/locate/micpath

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–10798

Considering that the identification and characterization

of mycobacterial adhesins could help to understand better

the pathogenesis of TB and to develop strategies aimed to

block initial events in the infection, we carried out the

present study. Our results show that the 19-kDa antigen of

M. tuberculosis (Rv 3763) binds THP-1 monocytic cells via

the macrophage mannose receptor (MR) promoting the

uptake of mycobacteria. These observations add importance

to the 19-kDa antigen, a mycobacterial moiety with

variegated biological effects including the induction of

immune responses [15–17], macrophage apoptosis [18] and

inhibition of Class II antigen presentation [19].

2. Results

2.1. The 19-kDa antigen of M. tuberculosis is a major

cell-wall located adhesin that binds THP-1 macrophage-

like cells

In Fig. 1, the immunoblotting analysis of cells incubated

with M. tuberculosis culture filtrate proteins (CFP) labelled

with biotin (A, lane 1) is shown. Preferential binding of a

19-kDa protein to THP-1 cells was observed (A, lanes 2–4).

Streptavidine–peroxidase failed to reveal bands in control

macrophages that were not incubated with CFP (A, lane 5).

The intensity of binding increased with the amount of biotin-

labelled proteins used in the assays (A), and with the

incubation time (not shown). These results were consistently

found with fresh 8-week-old CFP; prolonged storage greatly

diminished the intensity of binding. We used CFP labelled

with biotin since preliminary assays with anti-mycobacterial

antisera failed to reveal bound proteins by immunoblotting.

Biotinylation of antigens, antibodies, hormones, and other

proteins has proven useful in tracing studies. Biotin binds

Fig. 1. Immunoblotting of THP-1 cells incubated with M. tuberculosis

culture filtrate proteins (CFP) labeled with biotin (A). Blots were

developed with streptavidin labeled with peroxidase. Lane 1, CFP used

in the assay. THP-1 cells (5!105) were incubated for 1 h with 25 (lane 2),

50 (lane 3), and 100 (lane 4) mg CFP. Control macrophages not incubated

with CFP (lane 5). (B) Binding assay with an M. tuberculosis cell-wall

fraction (lane 1). THP-1 cells were incubated with 20 mg cell-wall fraction

(lane 2). Control macrophages that were not incubated with the cell-wall

fraction (lane 3). All lanes were loaded with 5 mg protein. Blots in panel B

were developed with the mAb IT-19, specific for the 19-kDa

M. tuberculosis antigen.

lysine residues that are present in most proteins, without

significantly altering their biological properties [20].

To determine the identity and topographical location of

the 19-kDa adhesin, a cell-wall fraction obtained from

M. tuberculosis bacilli was incubated with THP-1 cells and

the mAb IT-19 was used to reveal immunoblots. This

antibody was raised against an M. tuberculosis 19-kDa

antigen [21], that was later characterized as a glycolipo-

protein by Garbe et al. [15]. The mAb IT-19 recognized the

19-kDa band in both the cell-wall fraction used in the assay

(B, lane 1) and in cells which were incubated with such

fraction (B, lane 2). There was no band reactivity in control

macrophages that were not incubated with the cell-wall

fraction (B, lane 3). In preliminary studies, similar binding

of the 19-kDa antigen to human monocyte-derived

macrophages was observed (not shown).

2.2. M. smegmatis transfected with the plasmid p16R1

overexpresses the 19-kDa M. tuberculosis antigen,

which binds THP-1 monocytic cells

To confirm the above observations, we obtained a

recombinant M. smegmatis strain transfected with the

plasmid p16R1 that overexpresses the 19-kDa antigen

(a kind gift of Y. Zhang, MRC Tuberculosis and Related

Infections Unit, Hammersmith Hospital, London, UK). The

analysis of this strain is shown in Fig. 2. Immunoblotting of

recombinant M. smegmatis CFP with the mAb IT-19

showed a thick 19-kDa band (lane 1) which was not

demonstrated in CFP from native M. smegmatis (lane 2).

Mannose residues in the recombinant 19-kDa antigen were

demonstrated by affinity blotting with Con A labelled with

peroxidase (lane 3). Labelling of native CFP with Con

A-peroxidase, revealed, together with high molecular

weight bands, a thin 19-kDa reactive band (lane 4), that

Fig. 2. Analysis of the 19-kDa antigen produced by recombinant

M. smegmatis transformed with the plasmid p16Rl. Immunoblotting of

culture filtrate proteins (CFP) of recombinant (lane 1) and native

M. smegmatis (lane 2) developed with the mAb IT-19. CFP from

recombinant (lane 3) and native (lane 4) M. smegmatis reacted with Con

A labeled with peroxidase. Lane 5, recombinant M. smegmatis CFP

digested with a-D-mannosidase. Lane 6, undigested CFP. Lanes 5 and 6

were developed with the mAb IT-19. Each lane was loaded with 5 mg CFP.

Fig. 3. Cell binding assay with THP-1 cells and culture filtrate proteins (CFP) from recombinant M. smegmatis that overexpresses the 19-kDa antigen (A) and

with CFP from native M. smegmatis (B). THP-1 cells (5!105) were incubated with 1 (lanes 1), 10 (lanes 2), and 50 (lanes 3) mg CFP. Lanes 4, control

macrophages that were not incubated with CFP. All blots were developed with the mAb IT-19. Lanes were loaded with 5 mg CFP.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.1 1 10 20 50

O.D

. 405

nm

***

*

native M. smegmatis

recombinant M. smegmatis

Culture filtrate proteins (µg)

Fig. 4. Cell-Surface ELISA to analyze the cell binding of the 19-kDa

antigen to THP-1 cells; 1!105 cells were incubated for 1 h with 0.1, 1, 10,

20, and 50 mg CFP from recombinant or native M. smegmatis. Binding was

analyzed with the mAb IT-19 and a secondary anti-mouse IgG antibody

labeled with alkaline phosphatase. Control wells were incubated with an

unrelated lgG1 isotype antibody. Statistically significant differences in

optical density (OD) are shown. The data represent the mean valuesGSD

from three experiments with duplicate samples. *P!0.05; ***P!0.001.

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–107 99

was not recognized by the mAb IT-19 (lane 2). Digestion of

M. smegmatis CFP with a-D-mannosidase resulted in a

19-kDa antigen with lower apparent molecular mass

(lane 5). Binding assays with recombinant M. smegmatis

CFP revealed dose-dependent binding of the 19-kDa antigen

to THP-1 cells (Fig. 3A). There was no cell binding with

native M. smegmatis CFP (Fig. 3B). To confirm the

immunoblotting, results we carried out a cell-surface

ELISA assay, which detects primarily plasma membrane

bound ligands [22]. Dose-dependent binding was demon-

strated with the mAb IT-19 when cells were incubated with

recombinant M. smegmatis CFP but not with native

M. smegmatis CFP (Fig. 4). The differences in OD were

highly significant in assays using 50 mg CFP.

2.3. Adherence assays with fluorescent microspheres

coated with recombinant M. smegmatis CFP

To explore the potential ability of the 19-kDa antigen to

promote phagocytosis, we incubated THP-1 cells with

fluorescent microspheres coated with CFP from native and

recombinant M. smegmatis and with bovine serum albumin

(BSA). Results expressed as the average of associated

microspheres per cell showed that the number of cell-

associated microspheres was significantly higher when they

were coated with recombinant M. smegmatis CFP at 10:1

(P!0.016) and 20:1 (P!0.003) microsphere/cell (Fig. 5).

Adherence of control microspheres coated with BSA was

negligible.

2.4. The binding of the 19-kDa antigen to THP-1 cells

is inhibited with monosaccharides, calcium chelators

and a mAb to MR

To determine if the 19-kDa antigen binds cells via the

MR, we performed inhibition assays with three mono-

saccharydes of known affinity for the MR [23,24].

By immunoblotting with the IT-19 mAb, it was observed

that preincubation of cells a-methyl-mannoside, N-acetyl-

D-glucosamine and mannan resulted in dose-dependent

reduction in binding of the 19-kDa antigen (Fig. 6). Cell-

surface ELISA was used to analyse the inhibitory capacity

of EDTA, EGTA, and a mAb against the human

macrophage MR. Inhibition of 19-kDa antigen binding

to THP-1 cells was evident with 0.2 mM EDTA (44%G20), 0.2 mM EGTA (40%G36), and with 10 mg mAb to

MR (64%G30). Assays to inhibit the adherence of

microspheres coated with recombinant M. smegmatis

CFP were also carried out. When cells were pretreated

with a-methyl-mannoside at 1, 10, 100 and 1000 mM

concentrations and incubated with microspheres at a 20/1

microsphere/cell, reduction in the percentage of attached

microspheres was observed in a dose-dependent fashion

(not shown).

Fig. 5. Adherence assays with THP-1 monocytic cells and fluorescent microspheres coated with CFP from native and recombinant M. smegmatis and with BSA

as control. Cells (5!105) were incubated for 1 h with microspheres at a 1:1, 10:1 and 20:1 microsphere/cell proportion. Fluorescence microscopy to show

adherence of microspheres to cells. Significant differences in the number of cell-associated microspheres coated with native and recombinant M. smegmatis

CFP are shown. The data represent the mean valuesGSD from three experiments with duplicates samples. *P!0.05; **P!0.01.

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–107100

2.5. THP-1 cells express surface MR as shown

by immunoprecipitation and immunostaining

Since the inhibition assays indicated a role for the

macrophage MR in 19-kDa antigen binding, we performed

studies to determine its expression by THP-1 cells. Three

approaches were undertaken. First, immunoprecipitation

using whole cell membranes isolated from THP-1 cells and

a M. tuberculosis lipoprotein fraction enriched in the

19-kDa antigen. Immunoblotting of the material immuno-

precipitated with a polyclonal anti-human MR antibody is

shown in Fig. 7. In blots of proteins separated by 7.5% SDS-

PAGE under reducing conditions, the MR polyclonal

antibody detected a 180-kDa band (lane 6) consistent with

the 162–180 kDa molecular weight reported for the human

macrophage MR [25]. The 180-kDa band was not observed

when the anti-MR antibody was omitted (not shown). MR

was demonstrated in the macrophage membranes used in the

assay (lane 5). Immunoblotting of immunoprecipitated

proteins separated by 15% non-reducing SDS-PAGE

developed with the mAb IT-19 showed a19-kDa band

Fig. 6. Assay to inhibit the binding of the 19-kDa antigen to THP-1 cells with ma

mannoside (A), N-acetyl-D-glucosamine (B), and mannan (C). After 1 h treatmen

After extensive rinsing cells were processed for immunoblotting. CFP, recombin

were developed with the mAb IT-19. Lanes were loaded with 5 mg CFP.

(lane 4) that was also present in the lipoprotein fraction used

for incubation of macrophage cell membranes (lane 2).

Reducing and non-reducing conditions were used to prevent

overlapping of the 19-kDa antigen and MR with IgG chains.

As previously demonstrated [15], the 19-kDa antigen was

shown to possess mannosyl residues as revealed by blotting

with Con A labelled with peroxidase (lane 3). Control

experiments with preimmune goat serum were negative (not

shown).

To further document the expression of the MR by THP-1

cells, we performed immunostaining with a polyclonal anti-

MR antibody and a secondary rabbit anti-goat IgG antibody

labelled with peroxidase. In permeabilized cells that were

not incubated with CFP, the anti-MR antibody revealed

abundant immunoreactive intracytoplasmic granules,

mainly in perinuclear location (Fig. 8A); in these cells

membrane labelling was weak and occasional (not shown).

In contrast, in cells incubated for 1 h with 50 mg

M. smegmatis CFP before the immunostaining procedure,

intense MR cell surface labelling was observed (Fig. 8B)

and intracellular MR was scanty (not shown). In control

nnose receptor ligands. THP-1 cells (5!105) were treated with a-methyl-

t and without washing, 20 mg recombinant M. smegmatis CFP were added.

ant M. smegmatis CFP used to incubate cells. Untreated cells (–). All blots

Fig. 7. Identification of the 19-kDa antigen as a ligand for the macrophage

mannose receptor by immunoprecipitation. After incubation of THP-1 cell

membranes (250 mg) with an M. tuberculosis lipoprotein fraction rich in the

19-kDa antigen (lane 1), complexes were precipitated with a polyclonal

antibody to the macrophage mannose receptor and Protein G bound to

agarose. Immunoblotting of the M. tuberculosis lipoprotein fraction used in

the assay developed with the mAb IT-19 (lane 2) and with Con A labeled

with peroxidase (lane 3). The macrophage membranes used in the assay are

shown reacted with a polyclonal goat antibody to MR (lane 5).

Immunoprecipitated proteins developed with the mAb IT-19 (lane 4) and

with the antibody to the mannose receptor (lane 6). Ig Hc: immunoglobulin

heavy chains; Ig Lc: immunoglobulin light chains. MR, mannose receptor.

Lane 1 was developed with an M. tuberculosis antiserum and Protein A

labelled with peroxidase.

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–107 101

coverslips in which the goat anti-MR antibody was omitted

there was no cell labelling (not shown). To study further the

cell surface upregulation of the MR in THP-1 cells

stimulated with CFP, we carried out cell-surface ELISA

using a mAb to the MR. A statistically significant increment

in MR expression was observed only in cells that were

incubated for 1 h with recombinant M. smegmatis CFP

(Fig. 8C).

Fig. 8. Immunostaining and cell-surface ELISA to demonstrate the surface express

were incubated or not for 1 h with 50 mg recombinant M. smegmatis culture filtrate

activity, fixation in 1% paraformaldehide and permeabilization with Triton X-10

coverslips which was revealed with a secondary antibody to goat IgG labeled with

with CFP. Coverslips were counterstained with Hematoxilin–Eosin. Bars represen

rinsing, cells were incubated with 10 mg mAb anti-MR or with an isotype control a

was used. Bars with different letters are significantly different (P!0.05) by a two-w

M. smegmatis culture filtrate proteins. Isotype, isotype control antibody. a-MR, m

2.6. The interaction of the 19-kDa antigen with the MR

promotes the binding/phagocytosis of recombinant

M. smegmatis by THP-1 monocytic cells

THP-1 cells were incubated for 1 h with recombinant

M. smegmatis at a 20:1 multiplicity of infection. High-

level binding of recombinant M. smegmatis bacilli to THP-

1 cells was observed (Fig. 9, Table 1) with 74. About

84%G10.46 cells being associated with 1 or more

peroxidase-labeled bacterium; in many cells labelled

bacteria were so numerous to be reliably counted. Control

coverslips in which the anti-M. tuberculosis antiserum was

omitted were completely negative (Fig. 9B). The role of

MR in the phagocytosis of recombinant M. smegmatis was

indicated by the significant inhibition observed when cells

were preincubated with recognized MR ligands. With

a-methyl-mannoside (50.76%G17.7; P%0.05) and with

mannan (79.3%G5.33; P%0.001) significant inhibition

was observed. Treatment with 10 mM EDTA to chelate

Ca2C was also significantly inhibitory (82.6%G13.55;

P%0.01). The role of MR in the binding of recombinant

M. smegmatis was further supported in inhibition assays

with a mAb to the macrophage MR that significantly

diminished the percentage of cells with associated bacilli

(82.15%G9.33). The role of the 19-kDa antigen was

demonstrated as the percentage of cells with associated

bacilli was markedly diminished when cells were

pretreated with the mAb IT-19. This is an IgG1 mouse

mAb that, as shown by immunoblot, reacts specifically

with the 19-kDa M. tuberculosis antigen expressed in

M. smegmatis and not with native M. smegmatis

components (Fig. 2). Preincubation of cells with an IgG1

isotype control had no inhibitory effects.

ion of the mannose receptor (MR) by THP-1 cells. For both procedures cells

proteins (CFP). For immunostaining, after blocking endogenous peroxidase

0, a polyclonal goat antibody to the macrophage MR was applied to the

peroxidase. (A) Cells that were not incubated with CFP; (B) cells incubated

t 50 mM. For cell surface ELISA, after incubation with CFP and extensive

ntibody. A secondary alkaline phosphatase labelled antibody to mouse IgG

ay Anova followed by Tukey’s HSD post hoc procedure. CFP, recombinant

onoclonal antibody to the mannose receptor.

Fig. 9. Light microscopy of THP-1 cells to show bound bacilli (A). Cells (5!105) were incubated with recombinant M. smegmatis at a 20/1 bacteria/cell serum-

free RPMI. After 1 h, unbound bacilli were rinsed off and the cells were fixed in 1% paraformaldehyde and treated with methanol and H2O2 to eliminate

endogenous peroxidase activity. After permeabilization of cells, bacilli were detected with an M. tuberculosis antiserum and peroxidase-labeled Protein A.

Peroxidase was developed with 3,3 0-diaminobenzidine and H2O2. The majority of cells display at least one peroxidase-labeled particle (arrows). Inset, to show

membrane-bound bacilli. In control coverslips (B) the anti-M. tuberculosis antiserum was omitted. Coverslips were counterstained with Hematoxilin–Eosin.

Bars represent 50 mM.

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–107102

3. Discussion and conclusions

This study was undertaken to search for M. tuberculosis

adhesins with affinity for macrophages. In screening assays

using mycobacterial CFP labelled with biotin, it was of

interest to note that among the many proteins present in the

culture filtrate, a 19-kDa protein was preferentially bound to

THP-1 macrophage-like cells, although it was poorly

expressed in the extracts used in the assays. Incubation of

macrophages with mycobacterial cell-wall fractions showed

that, as is the case for many bacterial adhesins [26], the

19-kDa adhesin was located to the cell-wall, therefore

available for interaction with host cells. These findings,

however, do not rule out the existence of other

Table 1

Binding of recombinant M. smegmatis to THP-1 cells

Treatment Phagocytosis (%) Inhibition (%)

None 74.84G10.46 –

a-mm 36.85G13.24* 50.76G17.70

Mannan 15.49G3.99** 79.30G5.33

EDTA 12.98G10.14** 82.66G13.55

mAb-19-kDa 13.36G7.02** 82.15G9.33

mAb-MR 10.05G6.15** 86.57G8.22

Isotype control 82.97G5.30 –

THP-1 cells were incubated with recombinant M. smegmatis. Cells were

pretreated with inhibitors for 1 h. At least 200 cells were randomly

examined in each coverslip to assess the percentage of cells binding at least

one bacterium. Data shown are meansGSD of three independent

experiments with triplicate coverslips. MR, mannose receptor; a-mm, a-

methyl-mannoside. *P%0.5, **P%0.05, ***P%0.001. The effects of

treating cells with saccharydes, EDTA, and with mAbs to the Mannose

Receptor and to the 19-kDa antigen of M. tuberculosis.

mycobacterial proteins with an adhesin function that could

be revealed under different experimental conditions. It has

been reported that the antigen 85 complex coupled to

microbeads binds macrophages through CR3 [13] and that

latex beads coated with the 30-kDa heparin-binding

hemaglutinin bind macrophage-like cells in both the

presence and absence of human serum [14].

After identifying the 19-kDa adhesin as the glycolipo-

protein characterized originally by Garbe et al. [15], we

undertook this study using CFP from a recombinant

M. smegmatis strain, which overexpresses the 19-kDa

antigen of M. tuberculosis. As the M. tuberculosis protein,

the recombinant expressed in M. smegmatis was shown to

possess mannosyl residues susceptible to a-D-mannosidase

digestion. At present it is known that M. tuberculosis is

capable of synthesizing glycoproteins [27–29]. Among

these is the 19-kDa antigen herein described, which in

addition to be acylated [30], possess mannosyl residues as

shown by lectin binding assays with Con A [15]. Morever,

O-glycosylation sites have been demonstrated in this protein

by site-directed mutagenesis [31]. This antigen is biologi-

cally very active; it induces strong antibody [16] and cell-

mediated immune responses [15,17] and interacts with

macrophages via Toll-like 2 receptors inducing IL-12 and

TNFa production [32] or apoptosis [18]. Recently, it has

been shown that the 19-kDa antigen can inhibit MHC-II

antigen presentation by blocking gamma interferon

signaling [19].

Our current observations show that the 19-kDa M.

tuberculosis antigen interacts with the macrophage MR

present on the surface of THP-1 cells. In the first place,

Ca2C chelators, a-methyl-mannoside, mannan and

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–107 103

N-acetyl-D-glucosamine decreased the binding of the

19-kDa antigen to cells. Moreover, a similar effect was

observed with a mAb to the human macrophage MR.

Results of the phagocytosis assays support the above notion.

Indeed, the percentage of THP-1 cells with associated

bacilli was markedly reduced in inhibition assays with

mannan and a-methyl-mannoside, recognized MR ligands

[23,24], and with EDTA. The phagocytosis of recombinant

M. smegmatis was due to the interaction of the 19-kDa

antigen with the MR on THP-1 cells; this was demonstrated

by the marked reduction in the percentage of cells with

bound bacilli observed in inhibition assays with mAbs to the

19-kDa antigen and to the MR.

To our knowledge, there are no previous reports of

M. tuberculosis glycolipoproteins with adhesin function

capable of interacting with the MR that promote phagocy-

tosis of mycobacteria. Although it has been observed that

M. tuberculosis can be ingested by the interaction of MR

with mannosyl residues of LAM, an abundant cell-wall

located mycobacterial lipoglycan [5]. In addition to its role

in homeostasis of glycoproteins [33], the MR is considered a

pattern recognition receptor important in the innate host

response to pathogens [34] that is involved in the

phagocytosis of a variety of microbes [35–38].

As far as we know, the expression of MR on THP-1

monocytes has not yet been documented although some

suggestive observations have been made. It has been

proposed that THP-1 cells ingest Malassezia furfur [39]

and M. tuberculosis [40] via MR since uptake is inhibited by

preincubation with mannan. Another study showed that

LAM and other MR ligands induce the production of

metalloproteinase-9 by THP-1 cells that was abrogated with

an mAb to MR [41]. In this study we have obtained direct

evidences of the expression of MR by THP-1 monocytic

cells. First, immunoprecipitation with a lipoprotein fraction

from M. tuberculosis and THP-1 whole cell membranes

resulted in coprecipitation of both MR and 19-kDa antigen.

Secondly, immunostaining with and anti MR antibody

revealed strong cell surface labelling and intracellular MR

deposits. Finally, the surface expression of the MR on THP-

1 cells was also demonstrated by cell-surface ELISA, a

method that detects primarily cell membrane exposed cell

molecules [22]. It is of interest that cell surface expression

of the MR requires prior stimulation with recombinant

M. smegmatis culture filtrate proteins. These findings

propose that mycobacterial products may upregulate the

surface expression of the MR in cells of the monocytic-

macrophagic lineage which would be of great interest in the

pathogenesis of mycobacterial infections. Whether

recycling or other mechanism are involved in this

phenomenon require research that is not within the scope

of the present study. It is worth to mention that cells

expressing surface MR or with ingested bacilli often

displayed abundant well-spread cytoplasm suggesting they

had been differentiated into macrophages by the addition of

mycobacterial culture filtrate proteins or bacilli. In relation

to our current findings are two recent studies which

demonstrate surface up-regulation of the human macro-

phage MR by 1 h exposure of cells to surfactant proteins A

or D [42,43]. In a study it is proposed that the newly

expressed MR comes from preformed intracellular pools

and that de novo protein synthesis is not required for this

phenomenon to occur.

In summary, the current observations lend further

emphasis to the importance of the 19-kDa antigen as a

mycobacterial moiety of great biological activity. These

result also show, that as other intracellular pathogens, M.

tuberculosis is armed with panoply of resources to

accomplish the crucial step of invading host cells.

4. Materials and methods

4.1. Mycobacteria and mycobacterial protein preparations

M. tuberculosis strain H37Rv (ATCC 27294), obtained

from the American Type Culture Collection (Manassas,

VA), was grown for 6–8 weeks in Proskauer and Beck

synthetic medium. M. smegmatis strain (mc2155) and its

recombinant counterpart transformed by electroporation

with the plasmid p16R1 which contains a 1.8 kb SmaI

fragment that includes the structural gene encoding the

19-kDa M. tuberculosis antigen were kindly donated by

Y. Zhang (MRC Tuberculosis and Related Infections Unit,

Hammersmith Hospital, London, UK). M. smegmatis,

native and recombinant were grown for 4–5 days in

Middlebrook 7H9 medium (BBL, Becton-Dickinson,

Cockeysville, MD) supplemented with 2% glucose and

hygromycin B (50 mg/ml). CFP from M. tuberculosis and

M. smegmatis were obtained by (NH4)2SO4 precipitation of

the culture medium after elimination of bacilli by filtration

[27]. CFP were labelled with biotin as described [44]. A

cell-wall protein fraction was obtained from M. tuberculosis

bacilli as follows. After killing with 2% sodium azide,

whole bacilli were rinsed with PBS and sonicated in ice (5!60 s pulses). After sonication the cell-wall fraction was

recovered by centrifugation (20 min at 20,000 rpm). Protein

concentration was determined by the Lowry protein assay

(Bio-Rad Laboratories, Hercules, and CA).

4.2. Characterization of the recombinant M. smegmatis

19 kDa antigen

To identify the 19-kDa antigen in mycobacterial CFP,

after SDS-PAGE, nitrocellulose membranes or PVDF

membranes (Bio-Rad) were incubated for 1 h with the

mAb IT-19 diluted 1:1000 (a kind gift from the TB Research

Material and Vaccine Testing Contract, Colorado State

University). IT-19 recognizes the 19-kDa M. tuberculosis

antigen [15,21]. The presence of mannose residues was

analysed by affinity blotting with Con A labelled with

peroxidase (Zymed Laboratories, San Francisco, CA) as

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–107104

described elsewhere [27,28]. With the same purpose, CFP

from recombinant M. smegmatis were digested with a-D-

mannosidase as described previously [28]. Briefly, 10 mg

CFP were digested for 1 h at 37 8C with jack bean a-D-

mannosidase (Sigma Chemical Co., St Louis, MO) in 0.1 M

sodium acetate buffer (pH 5), using 15 U of enzyme/mg

CFP. Digestion was stopped with sample buffer and proteins

were boiled and separated by SDS-PAGE to carry out

immunoblotting analysis.

4.3. Culture of THP-1 monocytic cells and binding assays

with mycobacterial CFP

The monocytic human cell line THP-1 was obtained

from ATCC (TIB-202) and cultured in RPMI 1640

medium supplemented with 10% fetal bovine serum

(FBS), 1 mM penicillin–streptomycin, 1 mM non-essential

aminoacids, 100 mM piruvic acid, and 1 mM L-glutamine

(Gibco BRL Products, Rockville, MD). Cell viability was

assessed by trypan blue exclusion. THP-1 cells (5!105)

were incubated with different amounts of M. tuberculosis

CFP labelled with biotin or without labelling at 37 8C,

with 5% CO2 for 1 h. Similarly, THP-1 cells were

incubated with unlabeled CFP obtained from recombinant

and native M. smegmatis 7-days-old cultures. Incubation

was carried out in RPMI 1640 with 10% FBS, 0.02%

NaN3, 0.006 PMSF, and 0.25 mM EDTA. After incubation,

the medium was carefully retired and wells were rinsed

several times by gentle pippeting with PBS and by 5 min

centrifugation in PBS at 1500 rpm in the cold. Cells were

suspended in sample buffer (19 mM EDTA, 1% glycerol,

0.1% SDS, 0.5 mM Tris–HCI pH 6.8, and 2-mercap-

toethanol) and resolved by 15% SDS-PAGE. After transfer

to nitrocellulose and blocking with 3% BSA in PBS,

membranes were incubated for 1 h with the IT-19 mAb

diluted 1:1000. Thereafter, a secondary peroxidase-labeled

goat antibody to mouse IgG was applied diluted 1:2000

(Zymed). Peroxidase was developed with 3,3 0-diamino-

benzidine and H2O2 or with a chemiluminiscence kit

(Pierce Biotechnology, Rockford, IL). For control, a mAb

to mouse b-actin was used in some immunoblots

(Chemicon International, Temecula, CA).

4.4. Cell-surface ELISA to study binding of the 19-kDa

antigen to THP-1 cells

To quantitate the binding of the 19-kDa antigen a cell-

surface ELISA was carried out [22]. Briefly, after blocking

wells for 2 h with 3% BSA in PBS, 1!105 THP-1 cells

were placed in each well. After 2 h, 1, 10, 20 or 50 mg CFP

from native or recombinant M. smegmatis were added. After

1 h incubation at 37 8C, wells were rinsed three times with

PBS containing 1% FBS and 0.1% NaN3.Thereafter, cells

were incubated with mAb IT-19 diluted 1:100 in PBS or

with an lgG1 isotype control antibody (Dako Corporation,

Carpinteria, CA) for 1 h. After rinsing, cells were incubated

with an alkaline phosphatase-conjugated goat anti-mouse

IgG (Dako) diluted 1:1000 for 1 h at 37 8C. After rinsing

extensively, 200 ml of a 1.5 mg/ml p-nitrophenyl phosphate

solution (Sigma) were added to the wells. The reaction was

stopped with 25 ml, 3 M NaOH. Optical densities at 405 nm

were read in an ELISA reader (Bio-Tek Instruments).

4.5. Adherence assays with fluorescent microspheres

coated with mycobacterial CFP

To determine whether the 19-kDa antigen may promote

phagocytosis, assays with microspheres were performed.

Fluoresceinated polystyrene 1 mm microspheres (Molecular

Probes, Inc., Eugene, OR) were coated with various

amounts of CFP from native or recombinant M. smegmatis

and with BSA, following manufacturer’s protocols. Briefly,

1.0810!1010 microspheres/ml were mixed with 500 mg

CFP from native and recombinant M. smegmatis in 1 ml

MES Buffer [2-(N-morpholino)ethanesulfonic acid (pH

6.0)]. Then, 1-ethyl-3-(3-dimethylaminopropyl)-carbodii-

mide (Molecular Probes) was added at 2.5 mg/ml final

concentration. After overnight incubation at room tempera-

ture, glycine (100 mM) was added to stop the reaction.

Microspheres were rinsed three times with PBS and kept a

4 8C in PBS containing 1% BSA to block possible

remaining active sites. Adherence assays were carried out

as follows. THP-1 cells (5!105) were removed with a

scrapper, counted, rinsed, resuspended in RPMI 1640

containing 10% FBS and placed on acid-washed glass

coverslips. Microspheres were added to cells and after 1 h

incubation at 37 8C in 5% CO2, cells were rinsed in PBS to

remove non-adherent microspheres and fixed in 10%

paraformaldehide for 10 min. To estimate the number of

adherent microspheres, coverslips were mounted on a slide

and at least 200 randomly chosen cells were examined in a

Nikon fluorescence microscope.

4.6. Assays to inhibit the binding of the 19-kDa antigen

and of microspheres to THP-1 cells

To determine if the 19-kDa antigen binds cells through

its mannose residues, inhibition assays with mannose

competitors were performed. When immunoblotting was

used to assess binding, THP-1 cells (5!105) were treated at

37 8C with 1, 10, 100 and 1000 mM a-methyl-mannoside,

10 and 50 mM N-acetyl-D-glucosamine and 5 and 10 mg

mannan. After 1 h treatment 50 mg CFP were added to cells.

After three rinses with PBS, cells were processed for

immunoblotting as described above. The binding of the

19-kDa antigen was demonstrated as described above. A

more quantitative Cell-Surface ELISA assay was used to

analyse the effects of pretreating cells with a-methyl-

mannoside, calcium chelators and a mAb to MR; 96-well

flat bottom ELISA plates were blocked as described above

and 1!105 cells were placed in each well for 2 h. Then, 1,

10, 100 and 1000 mM a-methyl-mannoside, 2 mM EDTA,

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–107 105

2 mM EGTA and 10 mg mAb to the MR were added to cells.

At the same time, 20 mg recombinant M. smegmatis CFP

were added for 1 h. Plates were rinsed three times at

1500 rpm with PBS containing 1% FBS and 0.1% NaN3.

Then, the IT-19 mAb diluted 1:100 in 3% BSA-PBS was

added to the wells. After 1 h incubation and three PBS

rinses, bound antibody was revealed with a goat anti-mouse

IgG antibody labelled with alkaline phosphatase as

described above.

Assays to block the adherence of microspheres coated

with recombinant M. smegmatis CFP to cells were also

performed. THP-1 cells (1!105) were preincubated with 1,

10, 100 and 1000 mM a-methyl-mannoside. After 1 h,

microspheres were added at a 20:1 microsphere/cell. After

extensive rinsing with PBS, coverslips were analysed by

fluorescence microscopy to count attached microspheres in

at least 200 randomly chosen cells.

4.7. Immunoprecipitation of the mannose macrophage

receptor and the 19-kDa M. tuberculosis antigen

A cell-membrane fraction was obtained following a

reported method [45]. Briefly, THP-1 cells were homogen-

ized by vortex in a lysis buffer (50 mM Tris–HCI, 150 mM

NaCI and 1 mM EDTA, pH 8) containing a cocktail of

protease inhibitors (pepstatin, leupeptin, aprotinin, quimos-

tatin, antipain and PMSF). After elimination of nuclei, cell

membranes were isolated by centrifugation at 32,000 rpm

for 1 h. The pellet was resuspended in the lysis buffer and

membrane proteins were extracted with 0.1% Triton X-100

(Sigma). Membrane proteins (250 mg) were incubated with

50 mg of a M. tuberculosis lipoprotein fraction enriched in

the 19-kDa antigen (kindly donated by TB Research

Material and Vaccine Testing Contract, Colorado State

University), with shaking, overnight at 4 8C. The incubation

buffer contained 2 mM CaCl2 and MgCl2. Immuno-

precipitation was carried out with a goat anti-MR polyclonal

antibody bound to Protein G-agarose microbeads for 3 h at

4 8C. The MR antibody was a kind gift from P. Stahl

(Washington University, St Louis, MO). Before immuno-

precipitation, microbeads were precleaned by incubation

with macrophage membranes and the lipoprotein fraction

for 1 h at 4 8C. After rinsing, microbeads were resuspended

in sample buffer and boiled. Microbeads were eliminated by

centrifugation at 12,000 rpm for 5 min and proteins were

separated by 7.5% SDS-PAGE to detect the mannose

receptor and at 15% to detect the 19-kDa antigen. After

transfer to nitrocellulose, strips were incubated for 3 h at

room temperature with the polyclonal MR antibody diluted

1:200 in PBS-Tween. After rinsing, a rabbit anti-goat IgG

antibody labelled with peroxidase was applied (Zymed).

The 19-kDa antigen was detected with the mAb IT-19 as

described before. A chemiluminescence kit was used to

reveal the blots (Pierce Biotechnology). Control immuno-

precipitation experiments with preimmune goat serum were

carried out.

4.8. Immunostaining procedure to demonstrate the mannose

receptor on THP-1 monocytic cells

THP-1 cells cultured on glass coverslips were incubated

for 1 h with 50 mg recombinant M. smegmatis CFP or with

culture medium alone. Cells were washed extensively with

PBS and fixed with 1% paraformaldehide at room

temperature for 30 min. After washing, cells were treated

with 3% BSA in PBS to inhibit non-specific binding.

Endogenous peroxidase activity was eliminated by treating

cells for 30 min with 5% methanol and 0.1% H2O2 and

permeabilized with Triton X-100 as described above. Then,

the goat polyclonal MR antibody diluted 1:100 in PBS was

applied to the coverslips. After rinsing three times with

PBS, a secondary peroxidase-labeled rabbit antibody to goat

IgG (Zymed) was applied diluted 1:1000. Peroxidase was

revealed with 3,3 0-diaminobenzidine and H2O2. In control

slides, the MR antibody was omitted. Coverslips were

mounted on glass slides, counterstained with Haematoxylin-

Eosin and examined in a light microscope.

4.9. Cell-surface ELISA to study the surface expression

of the mannose receptor on THP-1 cells

To quantitate MR expression a cell-surface ELISA was

carried out [22]. After blocking with BSA-PBS as described

above, 1!105 THP-1 cells were placed in each well. After

2 h 50 mg CFP from recombinant M. smegmatis were added.

After 1 h incubation at 37 8C, wells were rinsed three times

with PBS containing 1% FBS and 0.1% NaN3.Thereafter,

cells were incubated with 10 mg mAb to the MR (BD

Pharmingen) diluted 1:100 in PBS or with an lgG1 isotype

control antibody (Dako Corporation, Carpinteria, CA) for

1 h. After rinsing, cells were incubated with an alkaline

phosphatase-conjugated goat anti-mouse IgG (Dako)

diluted 1:1000 for 1 h at 37 8C. After rinsing extensively

alkaline phosphatase was developed as described above and

optical densities at 405 nm were read in an ELISA reader

(Bio-Tek Instruments).

4.10. Phagocytosis assays with recombinant M. smegmatis

and THP-1 cells

THP-1 cells plated on glass coverslips were cultured in

RPMI-1640 with 10% FBS. Cells were washed and

resuspended in RPMI-1640 in the absence of FBS. An

aliquot of recombinant M. smegmatis was thawed, rinsed

three times with PBS at 12,000 rpm for 20 min. Cells were

incubated with recombinant M. smegmatis at a 20/1

bacteria/cell. Previously, the bacterial suspension was

passed 10 times through a 25-gauge needle attached to a

1 ml syringe to eliminate clumps. After 1 h infection, cells

were washed extensively with PBS, fixed with 1%

paraformaldehide and treated with methanol and H2O2 to

eliminate endogenous peroxidase. Then, cells were permea-

bilized with 0.5% Triton X-100, 1% BSA in PBS.

H. Diaz-Silvestre et al. / Microbial Pathogenesis 39 (2005) 97–107106

After washing twice with PBS, cells were incubated for 1 h

with a rabbit antiserum to M. tuberculosis H37Rv, diluted 1:

50 in PBS. This antiserum cross-reacts extensively with

recombinant M. smegmatis CFP as seen by immunoblot (not

shown). After extensive washing with PBS, Protein A

labelled with peroxidase diluted 1:1000 in PBS was applied

to the coverslips. Peroxidase was developed as above. In

inhibition assays, cells were preincubated for 1 h with

mannan (5 mg), 100 mM a-methyl-mannoside and with

EDTA (5 mg). Inihibition assays with a mAb to the human

MR (20 mg; BD Pharmingen) and the IT-19 mAb (1:100

dilution) that recognizes the M. tuberculosis 19-kDa antigen

were also performed. Cells were preincubated with mAbs

(both IgG1 isotype) for 1 h, and without removal of the

mAb the bacilli were added. Control isotype experiments

were carried out with an unrelated IgG1 mAb diluted 1:50

(Dako). Each experiment was performed in triplicate and

results are expressed as the meanGSD.

4.11. Statistical analysis

The t Student test was used to make comparisons

between groups. ELISA data were analyzed by a two-way

ANOVA with significant differences evaluated by a Tukey’s

HSD procedure. Data are reported as meanGSEM.

Significance was set at P!0.05.

Acknowledgements

We would like to thank Ismael Ramirez for skilful

technical assistance and to Dr Imelda Lopez for reviewing

the manuscript. This work was supported by grant

IN233201 from DGAPA, UNAM.

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