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LipC (Rv0220) is an immunogenic cell-surface esterase of Mycobacterium 1
tuberculosis 2
3
Guomiao Shen1, Krishna Singh1, Dinesh Chandra1†, Carole Serveau-Avesque4, Damien 4
Maurin4¶, Stéphane Canaan4, Rupak Singla5, Digambar Behera5, Suman Laal1,2,3*, 5
6
1Departments of Pathology and 2Microbiology, New York University Langone Medical 7
Center, New York, NY 10016, USA. 8
3New York Harbor Health Care System, New York, NY, 10010, USA. 9
4CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la 10
Lipolyse UPR 9025, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France. 11
5Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, New Delhi, India. 12
110030. 13
† Present address: Albert Einstein College of Medicine, Bronx, NY 10461, USA 14
¶ Institut de Biologie Structurale Jean-Pierre Ebel, CNRS-CEA-UJF UMR 5075, 41 rue 15
Jules Horowitz, 38027-Grenoble Cedex, France. 16
Running Title: LipC is an immunogenic cell-surface esterase 17
18
*Corresponding Author 19
Suman Laal, Ph. D 20
Veterans Affairs Medical Center 21
423 East 23rd Street, Room 18123N 22
New York, NY 10010 23
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.05541-11 IAI Accepts, published online ahead of print on 28 October 2011
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Phone: 212-263-4164 24
Fax: 212-951-6321 25
E-mail: [email protected] 26
Key words: Mycobacterium tuberculosis; Tuberculosis; Cell-surface esterase; 27
Immunogenic protein; Epitope mapping 28
29
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Abstract 30
We have earlier reported the identification of novel proteins of Mycobacterium 31
tuberculosis (M. tb) by immunoscreening of an expression library of M. tb genomic DNA 32
with sera obtained from M. tb infected rabbits at 5 weeks post-infection. In this study, we 33
report further characterization of one of these antigens, LipC (Rv0220). LipC is 34
annotated as a member of the Lip family based on the presence of the consensus motif 35
“GXSXG’ characteristic of esterases. Although predicted to be a cytoplasmic enzyme, 36
we provide evidence that LipC is a cell-surface protein that is present both in the cell-37
wall and the capsule of M. tb. Consistent with this localization, LipC elicits strong 38
humoral immune responses in both HIV- and HIV+ TB patients. The absence of anti-39
LipC antibodies in sera from PPD+ healthy subjects confirms its expression only during 40
active M. tb infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 41
of which map to the exposed surface of the modeled LipC protein. Recombinant LipC 42
(rLipC) protein also elicites pro-inflammatory cytokine and chemokine responses from 43
macrophages and pulmonary epithelial cells. rLipC can hydrolyze short chain esters 44
with the carbon chain containing 2-10 carbon atoms. Together, these studies 45
demonstrate that LipC is a novel cell-surface associated esterase of M. tb that is highly 46
immunogenic and elicits both antibodies and cytokines/chemokines. 47
48
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Introduction 49
There are an estimated 9 x106 new cases of tuberculosis (TB), and 2 x106 TB-50
related deaths every year (56). Infection with M. tuberculosis (M. tb) is initiated with the 51
inhalation of a droplet bearing bacteria, and it takes months or years to progress to 52
clinical TB. During this progression from initial infection to clinical disease, the in vivo 53
bacteria adapt to continuously changing environments by altering their gene expression 54
(31, 34, 48, 50). Studies to delineate culture-filtrate proteins of M. tb that are recognized 55
by antibodies during the natural course of disease progression demonstrated that the 56
repertoire of antigens enlarges with the progression of infection (34-36, 41). 57
Interestingly, several of the M. tb antigens (malate synthase, MPT51 and ESAT6 for 58
example) that elicit immune responses during the early stages of active infection have 59
also been demonstrated to play important roles in the host-pathogen interaction (19-21, 60
58). 61
To identify additional antigenic proteins of M. tb that are expressed during the 62
early stages of active infection, we used sera obtained from M. tb aerosol-infected 63
rabbits that were bled at 4-5 weeks post-infection, to screen an expression library of M. 64
tb genomic DNA (44). Antibodies in these sera identified several proteins known to 65
contribute to infection and survival of the M. tb (ERP, KatG and MtrA), as well as novel 66
proteins (PTRP, PE-PGRS51 and LipC (Rv0220)) (3, 26, 44, 60). Interestingly, ERP, 67
KatG and PTRP are cell-wall proteins of M. tb (3, 43, 59) and while the precise 68
localization of MtrA in M. tb is not reported, the M. leprae homolog is also cell-wall 69
protein (27). The current studies are focused on LipC. 70
LipC is annotated as a member of the Lip family based on the presence of the 71
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consensus motif “GXSXG” characteristic of esterases and members of the hydrolase 72
fold family (40). The Lip family is comprised of 24 putative carboxyl ester hydrolases. Of 73
these, studies with 3 members, LipF (Rv3487c), LipH (Rv1399c) and LipY (Rv3097c) 74
have been reported so far (5, 10, 61). The current studies demonstrate that LipC is a 75
cell-surface protein that is present both in the cell-wall and the capsule of M. tb. 76
Consistent with this localization, LipC is a strong inducer of antibodies in patients with 77
active TB and immunodominant epitopes of the protein have been mapped. LipC also 78
elicits cytokine responses from both macrophage-like THP-1 and pulmonary epithelial 79
A549 cells. Moreover, a recombinant and enzymatically active M. tb LipC was produced 80
and purified from M. smegmatis and the biochemical characterization revealed that LipC 81
hydrolyzes short chain esters. Together, these results suggest that LipC participates in 82
progression of active TB both by contributing to utilization of lipid substrates for bacterial 83
growth and replication, and by modulating immune responses. 84
Materials and Methods 85
Bioinformatics analysis. Nucleic and protein BLAST searches with the current 86
databases were performed on the National Center for Biotechnology Information 87
website (www.ncbi.nlm.nih.gov/). Prediction of theoretical molecular weight 88
(ProtParam), transmembrane helices (TMHMM), and signal peptide (SignalP) were 89
carried out with the respective software at the ExPASy Proteomics server 90
(http://ca.expasy.org/). NetOGlyc program was used to predict potential glycosylation 91
sites, and MeMo (http://www.bioinfo.tsinghua.edu.cn/~tigerchen/memo/links.html) to 92
predict potential arginine or lysine sites that may be methylated. A model of 3-D 93
structure of LipC was generated from the automatic protein structure homology 94
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modeling server using I-TASSER server (33). Rv0220 sequence was submitted against 95
the non-redundant sequence database structural classification of proteins and the PDB. 96
The molecular graphics software program PyMol (http://www.pymol.org/) was used to 97
display and draw the structure from the PDB files generated by the I-Tasser server. 98
Bacterial strains, plasmids and growth media. Mycobacterial strains were grown in 99
Middlebrook 7H9 broth (Becton Dickinson and Company (BD), Sparks, MD) 100
supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% Albumin Dextrose Saline 101
(ADS; 0.5% bovine serum albumin, fraction V (Sigma, St. Louis, MO), 0.2% dextrose; 102
and 0.085% NaCl) at 37°C under constant shaking at 110 rpm. E. coli strains were 103
grown in LB broth (DIFCO Laboratories) at 37°C with shaking (220 rpm) in the presence 104
of either hygromycin (200 µg/ml), kanamycin (50 µg/ml), ampicillin (100 µg/ml) or 105
chloramphenicol (34 μg/ml) as required. 106
Immunoscreening of λgt11 library. The λgt11 expression library of M. tb H37Rv DNA 107
from the World Health Organization (WHO) was screened with pooled sera obtained 108
from M. tb aerosol-infected rabbits at 5 weeks post aerosol infection as described 109
previously (44). The reactive λgt11 clones were purified and the inserts were sequenced 110
to identify the gene encoding the immunoreactive protein (44). 111
Expression and purification of recombinant LipC (rLipC). For expression of rLipC in 112
E. coli, a 1235 bp DNA fragment containing the entire lipC open reading frame was 113
amplified from M. tb H37Rv genomic DNA using primers F1 5’-114
CCCATATGAACCAGCGACGCG-3’ and R1 5’-CCCTCGAGTTG 115
GCCGGCGTTTAGATG-3’ (underlined sequence indicates NdeI and XhoI sites, 116
respectively) and cloned into the pCR-Blunt cloning vector (Invitrogen, Carlsbad, CA). 117
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This intermediate plasmid (pCR-Blunt-lipC) was digested with NdeI and XhoI and the 118
resulting lipC gene cloned into pET14b expression vector (Novagen, EMD Biosciences, 119
Inc. San Diego, CA) at NdeI and XhoI sites to produce inframe fusion with His-tag at the 120
N-terminus position. The open reading frame of recombinant plasmid (pET14b-lipC) 121
was verified by DNA sequencing. The recombinant plasmid (pET14b-lipC) was 122
transformed into E. coli BL21 (DE3) pLysS (Invitrogen). After IPTG induction, the 123
recombinant protein was expressed in inclusions bodies and standard procedures wirh 124
urea were followed to obtain the purified His-tagged rLipC by affinity chromatography on 125
Ni-NTA Agarose column (Qiagen, Chatsworth, CA). Endotoxins were removed by 126
washing the protein-loaded affinity column with 10 mM Tris-HCl in 6 M Urea, followed 127
by 0.5% ASB-14 in 6 M Urea. rLipC was eluted (20 mM Tris-HCl pH 7.9, 1 M Imidazole, 128
6 M Urea) and fractions containing purified rLipC pooled and dialyzed against 10 mM 129
ammonium bicarbonate (pH 8.0) with stepwise decreased concentration of urea. The 130
purified rLipC formed aggregates which were readily solubilized in 0.1% SDS. The 131
limulus amoebocyte lysate (LAL) assay was used to determine the endotoxin content in 132
the purified LipC preparation as per the manufacturer’s instructions (Bio Whittaker, 133
Walkersville, MD). Proteomic analysis of rLipc was performed by Quadrupole time-of-134
flight (Q-TOF) mass spectrometry at the NYU protein analysis facility. All studies except 135
for the assessment of enzymatic activity were performed with this rLipC. 136
Since the rLipC obtained from E. coli was enzymatically inactive, lipC was also 137
expressed in M. smegmatis to determine enzymatic activity and substrate specificity. 138
Briefly, the lipC gene was amplified from M. tb H37Rv genomic DNA with primers F2 5’-139
GCATCCATGGTACAGCGACGCGCCGCCGGGTC-3’ and R2 5’-140
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CTTATGAAGCTTAGATGACCTCTTTCGCGAACTG-3’, containing NcoI and HindIII 141
restriction sites, respectively. The amplified lipC fragment was double digested (with 142
NcoI and HindIII) and after purification, the fragment was cloned into pMyNt vector (a 143
shuttle vector containing a TEV-cleavable N-terminal 6 His-tag, an acetamide inducible 144
promoter and a hygromycin resistance gene provided by EMBL-Hamburg). The 145
resulting recombinant plasmid (pMyNt-lipC) was verified by DNA sequencing and 146
electroporated into M. smegmatis mc2155. The bacteria expressing rLipC were cultured 147
until an OD600 value of 3 was reached, induced for 16 h by adding 0.2% (w/v) acetamide 148
and harvested by centrifugation. The bacterial pellet was suspended in 10 mM Tris 149
buffer (pH 8.0) containing 300 mM NaCl and 1% N-laurylsarcosine (sarkosyl) and 150
broken in a French Press. The His-tagged rLipC was purified by Ni-NTA agarose 151
column based affinity chromatography and eluted with 10 mM Tris buffer ( pH 8.0) 152
containing 300 mM NaCl and 100 mM Imidazol. The fractions containing rLipC were 153
pooled and further purified by superdex 200 column equilibrated with 10 mM Tris buffer 154
(pH 8.0) containing 300 mM NaCl. 155
Localization of LipC. Purified rLipC from E. coli suspended in incomplete Freund’s 156
adjuvant (IFA; Sigma) was used to immunize a New Zealand white rabbit to obtain 157
polyclonal antibodies (16). Immunoglobulin G (IgG) from normal rabbit serum or serum 158
from the immunized rabbit was purified by Protein A-sepharose 4B columns (Amersham 159
Bio-sciences). M. tb H37Rv sub-cellular protein fractions (cytoplasm (Cyt), total cell-wall 160
(TCW), SDS-extracted cell-wall (SDS-CW), culture-filtrate (CF)) (NIH/NIAID TB 161
Research Materials contract, CSU) and M. tb capsular material (kindly provided by Dr. 162
Mary Jackson, CSU) were separated by SDS-PAGE (5.0 µg/lane) and the western blots 163
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probed with rabbit anti-LipC IgG (1:1000) or normal rabbit IgG, followed by alkaline 164
phosphatase (AP)-conjugated anti-rabbit IgG (1:2000) and BCIP-NBT substrate (KPL 165
Inc, Gaithersburg, MD). Previous studies have demonstrated that M. tb malate synthase 166
is present in all the subcellular fractions of M. tb (19). Identical western blots of all 167
subcellular fractions were probed with anti-malate synthase IgG (1:1000) to ensure 168
integrity of the preparations used. Localization of LipC on intact bacterial cells was 169
confirmed by immunoelectron microscopy. Briefly, M. tb clinical isolate CDC1551, and 170
transposon mutant of lipC in CDC1551 (CDC1551 lipC::Tn) were obtained from 171
TARGET (Tuberculosis Animal Research and Gene Evaluation Taskforce, John 172
Hopkins University (http://webhost.nts.jhu.edu/target/)). Bacteria from mid-log cultures 173
of M. tb H37Rv, M. tb CDC1551 and M. tb CDC1551 lipC::Tn were fixed and processed 174
as described earlier (43). The sections were examined under Philips CM 10 TEM 175
electron microscope at the NYU Image Core Facility. 176
Patients and control subjects. Sera from 19 PPD-, 29 PPD+ healthy subjects, 70 non-177
HIV, acid fast bacilli (AFB) sputum smear-positive TB patients (HIV-TB+) and 45 HIV+ 178
AFB smear-positive TB patients (HIV+TB+) from India, a TB endemic country, were 179
included in these studies. As per the DOTS (Directly observed therapy-short term) 180
programs guided by the WHO, diagnosis of TB in the endemic countries is based on 181
microscopic examination of sputum smears for AFB since this test is highly specific 182
(55). Cultures are too expensive and complex for TB diagnosis in high-burden settings; 183
for this reason the bacteriological confirmation of TB in patients included in these 184
studies was based on microscopy. Sera from 46 HIV+ asymptomatic patients on 185
antiretroviral therapy were also included (HIV+TB-). All the TB patients were bled prior 186
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to initiation of TB therapy (Table 1). All individuals were bled with informed consent after 187
permissions from the appropriate ethical committees had been obtained. 188
In vivo expression of LipC during active TB. The presence of antibodies to LipC in 189
serum specimens from TB patients and PPD+ subjects was tested to determine the in 190
vivo expression of LipC during active and latent TB. Western blots of rLipC (75 ng per 191
lane) were blocked with 3% BSA in PBS overnight, washed with PBST (2% Tween 80 in 192
PBS) and incubated with sera (1:50) from 6 PPD-, and 6 HIV+TB- subjects, as well as 6 193
PPD+, 6 HIV-TB+ and 6 HIV+TB+ patients at 4°C overnight. After extensive washing, 194
the blots were probed with a mixture of protein-A-AP (1:2000, Sigma) and anti-human 195
IgA-AP (1:1000, Sigma) followed by BCIP-NBT substrate (KPL Inc, Gaithersburg, MD). 196
To confirm results obtained in the above experiment, sera from 20 additional subjects 197
(10 PPD+ subjects and 10 HIV-TB+ patients) were evaluated similarly. Semi-198
quantitative analysis of the reactivity on western blots was performed using an Epson® 199
Perfection 4990 Professional Scanner (Epson, USA) and the relative intensity values 200
were determined by Image J software (http://rsbweb.nih.gov/ij/). 201
Identification of immunodominant regions of LipC. Having confirmed the in vivo 202
expression of LipC in humans during active TB, epitope mapping of LipC was performed 203
to identify the immunodominant regions. Overlapping N-terminal biotin labeled peptides 204
(20 amino acid (AA) length with 10 AA overlap, total 40) covering the entire LipC 205
sequence (PEPscreen; Sigma) were captured (2.5 µg/ml, 50 µl/well) in wells of 206
commercial streptavidin-coated ELISA plates (Roche Diagnostic, Indianapolis, IN). Each 207
peptide was probed with 50 µl of individual sera (1:40 final concentration) from 13 PPD-, 208
23 PPD+ subjects and 60 HIV-TB+ patients (39, 43). After washing, the wells were 209
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probed with a mixture of protein-A-AP (1:2000, Sigma) and anti-human IgA-AP (1:1000, 210
Sigma). p-nitrophenyl phosphate (pNpp) was used to detect the peptide bound 211
antibodies. Mean OD of the 36 healthy subjects (13 PPD- and 23 PPD+) sera plus 3 212
standard deviations (SD) was used as cut-off to determine positive responses in TB 213
patients. Twelve peptides that were recognized by antibodies in sera from > 50% of the 214
60 HIV-TB+ patients in the initial screening were retested twice and positive reactivity in 215
3/3 or 2/3 ELISA assays was considered positive. The reactivity of the 6 peptides which 216
were consistently recognized by >50% of the HIV-TB+ patients was evaluated with sera 217
from 46 HIV+TB- and 45 HIV+TB+ patients. 218
Cell viability assay. Since rLipC remains soluble only in the presence of 0.1% SDS, 219
experiments were performed to confirm that the SDS concentration being used does not 220
affect cell viability in the cytokine studies. The LipC stock (400 µg/ml) was diluted to 1 221
µg/ml in medium (final concentration of SDS= 0.00025% (~0.01 mM)) and 200 ul 222
solution added to ~4x104 A549 cells/well or 2X105 PMA differentiated THP-1 cells in 223
triplicate for 24 h. At the end of incubation, viability of cells was determined by 3-(4,5-224
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as per the 225
manufacturer’s instructions (Invitrogen). 226
Confirmation of lack of endotoxin contamination. The LAL assay showed that the 227
endotoxin in the purified rLipC preparation was <10 ng/ml. For biological confirmation of 228
absence of contamination with endotoxin, 2x105 PMA-differentiated THP-1 cells were 229
exposed to 200 µl medium containing 1 ng/ml LPS, 1 ng/ml LPS that was pre-incubated 230
with 10 ng/ml polymixin B for 1 h, 1 µg/ml LipC and 1 µg/ml LipC that was preincubated 231
with 10 ng/ml polymixin B in triplicate. Culture supernatants were collected 24 h later 232
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and used for cytokine assessment with commercially procured ELISA kits as per 233
manufacturer’s instruction (BD Biosciences Pharmingen, San Diego, CA). 234
Cytokine responses to rLipC. 2 X105 THP-1 or 4 X104 A549 cells/well (in 200 µl 235
medium) were exposed to different concentrations of rLipC protein for 24h and the 236
supernatants were harvested and assayed for IL-12, TNF-α, MCP-1 and IL-8 using the 237
commercial sandwich ELISA kits (BD Biosciences Pharmingen), as described above. 238
Enzymatic characterization of rLipC. The activity of rLipC expressed in M. smegmatis 239
was measured using p-nitrophenyl (pNP) esters family (Sigma) with carbon chain 240
lengths ranging from C2 to C16. Release of pNP was monitored at 410 nm using a 96-241
well plate spectrophotometer and quantified using a calibration curve of pNP (ε(λ=410nm)= 242
30 mM-1). Enzymatic reactions were performed in a 2.5 mM Tris buffer pH 8.0 243
containing 300 mM NaCl (with 0, 1 and 4 mM NaTDC) at 37°C over a period of 15 min 244
in a final volume of 300 µL, containing various amount of enzyme and 1 mM substrate. 245
Results are expressed as specific activity in international unit (U/mg) corresponding to 246
1 µmole of pNP released per minute and per mg of enzyme. rLipC activity was also 247
investigated using pH-stat, fluorescent and spectrophotometric assays for various lipids, 248
as well as phospholipids, to detect lipase or phospholipase activity, respectively. 249
Statistical analysis. Comparisons between the reactivity with sera from PPD- and 250
PPD+ healthy controls, as well as TB patients were performed by calculating P values 251
with nonparametric Mann-Whitney test using GraphPad Prism version 5 software 252
(GraphPad Software Inc,. San Diego, CA). A P value of <0.01 was considered 253
statistically significant. 254
Results 255
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Identification and characteristics of LipC. The λgt11 clone identified by the serum 256
pool from M. tb infected rabbits expressed the C-terminal half (AA 231-403) of the lipC 257
(Rv0220) gene product (data not shown). Bioinformatics analysis predicts LipC to be 44 258
kDa (403 AA) protein with a pI of 10.4. Three amino acids, Ala, Arg and Val are 259
overrepresented (12.3, 11.1 and 10.5 percent respectively) in the LipC AA sequence. 260
SignalP analysis predicted no signal sequence in LipC protein. No transmembrane 261
helices are identified by the TMHMM software. In the Tuberculist data website 262
(http://tuberculist.epfl.ch/), LipC is annotated as an esterase based on the presence of 263
the carboxylesterases type-B serine active site (GXSXG) and the hydrolase fold that is 264
characteristic of lipases/esterases. The AA sequence of lipC shows <40% identity with 265
the other 23 members of the Lip family. BLAST-P identified a homologous protein 266
(~100% identity) in M. tb H37Ra, M. bovis AF2122/97, M. bovis BCG str. Pasteur 267
1173P2, M. bovis BCG str. Tokyo 172 and all M. tb clinical isolates sequenced so far. 268
The protein is absent in M. leprae TN and M. leprae Br4923. Proteins showing 70-80% 269
identity are present in the non-tuberculous mycobacteria (NTM): M. avium 270
paratuberculosis K-10, M. avium 104, M. avium ATCC 2529, M. ulcerans Agy99, M. 271
kansasii ATCC 12478, M. parascrofulaceum ATCC BAA-614, M. intracellulare ATCC 272
13950, M. marinum and M. smegmatis mc2 155. In M. abscessus, a protein showing 273
only 23% identity is found. No glycosylation sites, but 3 potential Arg residues that may 274
be methylated are predicted. 275
The LipC protein model was generated from 10 templates available in PDB which 276
are structurally closest to proteins like the brefeldin A esterase (pdb code 1jkm) or the 277
carboxylesterase from Archaeoglobus fulgidus (1JJI). This model reveals that LipC is a 278
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globular protein similar to other α/β hydrolase serine enzymes and consists of eleven 279
α−helices and a central β-sheet core containing six parallel and one anti-parallel β-280
strand (Fig. 1). The catalytic triad is composed of residues Ser237 (nucleophilic 281
residue), Asp334 (charge relay network) and His367 (proton carrier) (Fig. 1). Ser237 is 282
located within the nucleophilic elbow connecting strand β4 and helix α8 at the center of 283
the protein. 284
Expression and purification of rLipC. When expressed in E. coli, rLipC formed 285
inclusion bodies and was purified under denaturing conditions but removal of urea at the 286
final stages of purification led to aggregation and precipitation of the protein in the 287
several buffers (Ammonium bicarbonate, PBS, or Tris) that were used for refolding. The 288
rLipC protein could be kept soluble in the presence of low concentrations (0.1%) of 289
SDS. The purified recombinant protein appeared as a single ~45 kD band on SDS-290
PAGE, and was recognized by commercial anti-His antibody and anti-LipC antibodies 291
(Fig. 2). The identification of rLipC was confirmed by protein sequencing (Table 2). 292
Since the rLipC expressed in E. coli was enzymatically inactive, the protein was also 293
expressed in M. smegmatis. rLipC expressed in M. smegmatis was also recognized by 294
anti-His antibody and anti-LipC antibodies (Fig. 2). The identity of LipC expressed in M. 295
smegmatis was also confirmed by protein sequencing (data not shown). 296
LipC is a cell-surface protein of M. tb. As reported earlier, anti-malate synthase 297
antibodies showed strong reactivity with an ~81 kDa protein in the Cyt preparation of M. 298
tb (19). Lower amounts of malate synthase were present in the CW and SDS-CW 299
preparations while strong protein bands were present both in the capsule and CF 300
preparations. Anti-malate synthase IgG did not react with the rLipC protein (Fig. 3A, top 301
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panel). In contrast, anti-LipC IgG showed strong reactivity with the ~45 kDa rLipC 302
protein band. The same antibody showed weak reactivity with a doublet of proteins at 303
~45 and ~47 kDa in the Cyt preparation of M. tb. Stronger reactivity with the doublet 304
was observed in the TCW and the SDS-CW protein preparations. Only the ~47 kDa 305
protein was identified in the capsular preparation while no protein in the CF reacted with 306
these antibodies (Fig. 3A, middle panel). Purified IgG from normal rabbit serum showed 307
no reactivity with any protein in any subcellular fraction (Fig. 3A, bottom panel). The 308
surface localization of LipC was confirmed by the presence of gold particles on M. tb 309
H37Rv and M. tb CDC1551 bacterial cells (Fig. 3B, b and d) and their paucity on the 310
cell-surface of CDC1551 lipC::Tn bacterial cells (Fig. 3B c) when examined by 311
immunoelectron microscopy. No LipC was detected on the bacterial cells when IgG 312
from normal rabbit serum were used instead of anti-LipC antibodies (Fig. 3B a) 313
Expression of LipC in vivo. To determine if LipC is expressed during M. tb infection in 314
humans, reactivity of rLipC with sera from PPD-, and HIV+TB- as well as PPD+ healthy 315
subjects and TB patients was examined by western blotting. Visual examination showed 316
that while sera from non-TB subjects showed background reactivity with the rLipC, there 317
was no difference between the reactivity of sera from PPD- and HIV+TB- subjects and 318
the PPD+ subjects. In contrast, sera from most HIV-TB+ and HIV+TB+ patients showed 319
stronger reactivity (Fig. 4A). To confirm these results, sera from additional randomly 320
selected PPD+ subjects and TB patients were tested and similar results were obtained 321
(Fig. 4B). When the intensity of rLipC protein probed with anti-His antibodies was 322
considered as 100 units, the relative intensity obtained with individual sera from PPD-, 323
HIV+TB- and PPD+ subjects ranged from 1-14 units, and there was no difference 324
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between sera from the 3 subgroups of subjects (P>0.01). In contrast, reactivity with the 325
6 HIV-TB+ sera ranged from 7-60 units (median reactivity = 28.5 units) and for the 6 326
HIV+TB+ sera, it ranged from 33-75 units (median reactivity = 62.5 units). The 327
difference in intensity between the non-TB subjects and TB patients was statistically 328
significant (P<0.01). Using mean intensity of PPD-, PPD+ and HIV+TB- sera +3 SD as 329
cut off, sera from 11/12 TB patients in the first experiment (Fig. 4A and 4C) and from 330
8/10 TB patients in the second experiment (Fig. 4B and 4D) were positive for the 331
presence of anti-LipC antibodies. Thus, sera from ~85% of the TB patients had anti-332
LipC antibodies. Reactivity of a subset of the control and TB sera was evaluated with 333
rLipC expressed in M. smegmatis; the reactivity was similar to that observed with the 334
rLipC expressed in E. coli (data not shown). 335
Epitope mapping of LipC. When the reactivity of the 40 overlapping peptides of LipC 336
was tested with sera from 13 PPD- and 23 PPD+ subjects, there was no difference in 337
the reactivity of sera from these two types of subjects with any of the peptides. In 338
contrast, sera from a vast majority (57/60; 95%) of the HIV-TB+ patients had antibodies 339
against at least one LipC peptide (Fig. 5A); most sera (56/60, 93%) had antibodies to >1 340
peptide. As expected, there was a wide variation in the recognition of individual peptides 341
with the HIV-TB+ sera, in which different peptides were recognized by 0-70% of the 342
patients (Fig. 5A). Twelve peptides were recognized by >50% of the HIV-TB+ sera (52-343
70%). These peptides were re-tested twice for reactivity with the same sera. For 6 344
peptides (LipC3, LipC6, LipC24, LipC26, LipC34, LipC39) (Fig 5B and Table 3), the 345
reactivity was maintained (52-65%). In contrast, the reactivity with the remaining 6 346
peptides was lower during the subsequent assays (32-43%). For the 6 peptides which 347
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were consistently recognized by >50% of the HIV-TB+ sera, there was no difference 348
between the reactivity with sera from PPD+ and PPD- subjects (P=0.29-0.74) while 349
reactivity of all 6 peptides was significantly higher (P<0.001) with sera from patients. 350
The combined reactivity with the 6 peptides was ~83% in HIV-TB+ patients. These 6 351
peptides were also reproducibly recognized individually with >50% of the HIV+TB+ sera 352
(Fig. 5C) and the combined reactivity with the peptides was ~82%, while the vast 353
majority of HIV+TB- patients lacked antibodies to any of these peptides (Fig. 5C). 354
Mapping of the 6 immunodominant peptides on the model of the 3-D structure of LipC 355
showed that 5 peptides (LipC3, LipC6, LipC26, LipC34, LipC39) were totally exposed to 356
the solvent while LipC24 was buried in the folded conformation of the protein (Fig. 1). 357
Cytokine responses to LipC. The cytokine responses elicited by rLipC from THP-1 358
and A549 were also evaluated. The viability of both cell-types was unaffected by the low 359
concentration of SDS (0.00025% SDS) present in the antigen preparation used to 360
stimulate them (Fig. 6A). THP-1 cells exposed to 1 ng/ml LPS produced ~2400 pg/ml of 361
IL-12 and ~2500 pg/ml of TNF-α. Preincubation of LPS with 10 ng/ml of polymixin B 362
completely abrogated the production of both cytokines (Fig. 6B). Cells exposed to 1 363
µg/ml rLipC expressed ~2800 pg/ml and ~2700 pg/ml IL-12 and TNF-α respectively. 364
Preincubation of rLipC with polymixin B had no effect on cytokine production confirming 365
that the observed cytokine responses were not due to contaminating endotoxin (Fig. 366
6B). 367
THP-1 cells exposed to different concentrations of rLipC showed dose-368
dependent production of IL-12, TNF-α, IL-8 and MCP-1 and in most cases, peak 369
response was attained at 0.1 µg/ml (Fig. 6C). MCP-1 was the most highly induced 370
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cytokine (~7000 pg/ml), followed by IL-8 (~3500 pg/ml), IL-12 (~3000 pg/ml) and TNF-α 371
(~2500 pg/ml) (Fig. 6C). Minimal or no cytokine levels (<400 pg/ml) were detected in 372
supernatants from unstimulated cells (Fig. 6C). 373
A549 cells stimulated with rLipC expressed IL-8 and MCP-1 in a dose dependent 374
manner, and as was observed with THP-1 cells, peak MCP-1 induction was higher 375
(~5500 pg/ml) compared to peak induction of IL-8 (~1600 pg/ml) (Fig. 6D). 376
Enzymatic Characterization of LipC. To evaluate the substrate specificity of LipC, the 377
enzymatic activity of protein expressed and purified from M. smegmatis was 378
investigated towards p-nitrophenyl esters using substrates with acyl chain length 379
ranging from C2 to C16 in presence or not of NaTDC (1 or 4 mM) (Fig. 7). The presence 380
of NaTDC enhances slightly the specific activity for substrates ranging from C2 to C10 381
compared to the buffer alone. The activity of rLipC increased with the chain length of the 382
substrate reaching a maximum of specific activity of 160 (Km = 2.89 mM, Vm = 0.48 383
µM/mn) and 110 mU/mg (Km = 2 mM, Vm = 0.69 µM/mn), with and without NaTDC, 384
respectively. 385
No activity was detected for substrates with carbon chain length greater than C10 386
whatever the conditions used. Using the pH-Stat method (12), fluorescent spectrometric 387
(52) and radioactive assays (37), no activity was detected on mono, di or triacylglycerol 388
whatever the carbon chain length, indicating that this enzyme is a strict esterase and 389
does not have any lipase activity (data not shown). Also, all attempts to detect 390
phospholipase activity failed (data not shown). 391
Discussion 392
The sequencing of the M. tb genome revealed that M. tb possesses ~ 150 genes 393
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that code for enzymes involved in fatty acid degradation (6), suggesting that M. tb 394
utilizes host lipids during growth and replication in vivo. The analysis of the genes by 395
bioinformatics methods identified a family of 24 putative carboxyl hydrolases with the 396
characteristic GXSXG motif. Twenty of the Lip family members are predicted to be 397
present in the cytoplasm of the bacterium and 4 in the periplasmic space; LipC is 398
predicted to be a cytoplasmic enzyme (40). However, the experimental approaches 399
used in this study clearly demonstrate that native LipC is primarily present in the cell-400
wall and the capsule of M. tb. This is reminiscent of the findings with 2 other Lip family 401
members, LipF and LipY, both of which are also associated with the cell-wall (28, 45). In 402
confirmation of this localization in vivo, like LipF and LipY, LipC also participates in 403
immune responses by eliciting antibodies. It is interesting that anti-LipC antibodies 404
raised by immunization with rLipC (~45 kDa) recognize the appropriate protein band as 405
well as an additional ~47kDa protein in the native cell-wall subcellular fractions but only 406
the ~47 kDa band in the capsular fraction. This suggests that the native LipC present on 407
the bacterial surface in the capsule may have post-translational modifications while the 408
cytoplasm and cell-wall have both the modified and the non-modified LipC. The nature 409
of these post-translational modifications remains to be identified. 410
Earlier studies have suggested that there may be interspecies differences in the 411
profiles of antigens recognized by antibodies (17, 18). Since LipC was originally 412
identified by antibodies in sera from M. tb infected rabbits, it was important to confirm its 413
expression during TB in humans. Thus, sera from subjects with latent or active M. tb 414
infection were evaluated for presence of anti-LipC antibodies. Earlier studies have also 415
reported differential expression of some M. tb antigens in HIV-TB+ and HIV+TB+ 416
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patients (34, 36). For this reason, sera from both HIV-TB+ and HIV+TB+ patients were 417
included. Sera from healthy subjects with no evidence of M. tb infection (PPD-) as well 418
as HIV+TB- subjects (from the US) were included as negative controls. Antibodies to 419
LipC were present in sera from ~90% of the TB patients tested (Fig. 4), whether or not 420
co-infected with HIV, indicating the in vivo expression of the proteins during active 421
infection in both types of patients. In contrast, sera from PPD+ subjects showed similar 422
reactivity as PPD- subjects demonstrating absence of in vivo expression of LipC during 423
latent TB. 424
We have demonstrated that immunodominant epitopes of antigenic proteins can 425
replace and improve upon the detection of antibodies to the parent protein (39, 43). 426
Antibodies to at least one epitope of LipC were present in ~95% of the HIV-TB+ patients 427
and absent in the vast majority of PPD+ subjects, confirming the results of the western 428
blot studies. Moreover, reactivity with just the 6 immunodominant peptides was 429
sufficient to identify a vast majority (>80%) of the HIV-TB+ patients. This level of 430
reactivity is similar to that observed with immunodominant peptides of PTRP, which is 431
also a cell-wall protein that was identified by antibodies in rabbit sera obtained at 5-432
weeks post-infection, and whose immunodominant epitopes were mapped with sera 433
from the same cohorts as have been used in this study (43). Moreover, as was 434
observed for PTRP and its immunodominant peptides, there is no difference in the 435
recognition of LipC and its peptides between HIV-TB+ and HIV+TB+ patients. Our 436
earlier studies with CFP of M. tb showed that some antigens (Malate synthase and 437
MPT51 for example) are well-recognized by antibodies from both cavitary and non-438
cavitary TB patients, demonstrating that these antigens are expressed in vivo in both 439
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types of patients (34, 54). In contrast, other antigens (38 kDa protein and MPT32 for 440
example) appear to be expressed primarily during cavitary TB since non-cavitary TB 441
patients lack antibodies to them (34). The similar immunogenicity of LipC and its 442
peptides in the smear positive HIV-TB+ patients most of who had cavitary lesions and 443
HIV+TB+ patients who were mostly non-cavitary indicates that it is expressed by M. tb 444
during replication in both non-cavitary and cavitary environments in vivo. 445
Inexpensive and rapid point-of-care (POC) tests based on detection of antibodies 446
to antigens and/or epitopes selected by careful analysis of their immunogenic potential 447
in patients and controls have been devised for other diseases (1, 13). However, recent 448
WHO-sponsored analysis of performance of currently available commercial serological 449
tests for TB diagnosis has shown that none of these tests provide accurate and reliable 450
results in either pulmonary or extrapulmonary TB patients (46, 47, 57). These 451
commercial tests are based primarily on crude mixtures of M. tb antigens or on antigens 452
that elicited antibodies in immunized animals, rather than on a careful analysis and 453
selection of antigens relevant to human TB during the natural course of disease. Our 454
systematic studies of humoral responses in TB patients have identified a panel of 455
potential candidates for devising a rapid POC test for TB, and immunodominant 456
epitopes of some of these highly immunogenic M. tb antigens have been delineated (11, 457
34, 36, 39, 43, 44). Since combinations of multiple antigens provide increased 458
sensitivity of antibody detection (11, 15, 17, 42, 54), it is likely that combinations of 459
immunodominant epitopes from multiple highly immunogenic proteins of M. tb will 460
enable development of a peptide-based rapid test for TB. One or more of the 6 epitopes 461
of LipC defined in these studies could make significant contributions to such a 462
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diagnostic test. 463
The use of peptides is economically advantageous over recombinant purified 464
proteins which are expensive to produce, difficult to purify and pose considerable 465
challenges for maintaining batch-to-batch consistency and long-term stability. Moreover, 466
use of peptides eliminates the non-immunogenic and sometimes cross-reactive portions 467
of the antigens. The use of biotin-conjugated peptides enables equal binding of 468
individual peptides to the streptavidin-coated plates, thus eliminating differences 469
between their binding capacities when being tested by ELISA. Despite equal binding, 470
some peptides that are recognized by antibodies in a high proportion of TB patients 471
during the first screen performed poorly when retested. This was also observed when 472
epitope mapping of other immunogenic proteins was performed (39, 43). In some 473
cases, solubilized peptides can be seen to gradually aggregate and precipitate during 474
storage suggesting that they undergo conformational changes in solution; for other 475
peptides it was unclear why the reactivity decreased during retesting. It is for this reason 476
that reactivity of each peptide needs to be evaluated multiple times so that only stable 477
and highly reactive peptides are finally selected for test development. 478
Antibodies in sera from a few (<10%) of the HIV+TB- subjects showed reactivity 479
with the 6 LipC peptides. Since these HIV+TB- patients were from the US, and were 480
asymptomatic and on ART, it is unlikely that they were infected with any non-481
tuberculous mycobacteria (NTM). The cross-reactivity observed with some of these sera 482
is likely due to the hypergammaglobinemia caused by HIV-infection and may be 483
eliminated during optimization of conditions during development of the rapid test. 484
As expected from B cell epitopes, 5 of the 6 immunodominant peptides mapped 485
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to the exposed surface of the modeled protein (Fig 1). The presence of antibodies to 486
LipC24, which is not surface exposed, indicates that in vivo, shedding of the bacterial 487
capsule may result in release of LipC in somewhat unfolded conformation, providing the 488
B cells with access to this region. 489
While macrophages are well known to be the host cells for M. tb, there is 490
increasing evidence for important roles for alveolar epithelial cells in TB (2, 19, 20, 23, 491
53). We therefore evaluated the ability of purified LipC protein to elicit cytokines from 492
both THP-1 and A549 cells. While MCP-1 was the most highly induced cytokine, IL-8 493
was also expressed by the two cell types (Fig. 6C and 6D). Both MCP-1 and IL-8 are 494
involved in recruitment of macrophages, neutrophils, dendritic cells and T cells, and play 495
important roles in the formation of granulomas (51). Importantly, IL-8 and MCP-1 have 496
also been demonstrated to be present in the broncho-alveolar lavage of TB patients 497
(24). LipC also induced the expression of TNF-α and IL-12 from THP1 cells. TNF-α is 498
known to play a key role in granuloma formation, induce macrophage activation, and 499
has immunoregulatory properties (51). IL-12 is known to play a crucial role in the 500
induction of IFN-γ, which activates macrophages to kill the bacteria (7). The in vivo 501
expression of LipC in rabbits at 4-5 weeks post-aerosol infection when granuloma 502
formation occurs, and the ability of LipC to stimulate proinflammatory cytokine and 503
chemokine production suggest that the precise role of LipC in granuloma formation on 504
aerosol infection with M. tb warrants investigation in animal models. In this regards, 505
earlier studies of another carboxylesterase (Rv2224c), which also anchored to the cell 506
wall, has been shown to modulate innate immune responses to M. tb in mice (25). 507
Previous studies have shown that M. tb stores energy in the form of 508
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triacylglycerol (TAG) as it goes into the latent state which is utilized for survival during 509
dormancy and reactivation when bacterial replication is resumed (9). TAG is also the 510
common form of stored energy in hibernating animals and oil seeds when the metabolic 511
activity is low (30). TAG is hydrolyzed via the glyoxylate cycle, which has been shown to 512
be used by M. tb during intra-cellular survival (29). Interestingly, in M. tb, malate 513
synthase, which participates in the glyoxylate cycle is present not only in the cytoplasm, 514
but also on the cell-wall and in the capsule (19), and antibodies to malate synthase are 515
strongly associated with clinical TB but not latent TB, suggesting enhanced expression 516
when bacteria are replicating actively in vivo. Studies have also demonstrated that LipY 517
hydrolyses TAG (10), and LipF hydrolyzes both short-chain esters and 518
phosphotydalcholine, which is major constituent of biological membranes (45, 61). Both 519
LipY and LipF also localize to the bacterial cell-wall, and elicit antibodies in patients with 520
active TB (8, 10, 28, 45, 61). Together, these data suggest that LipY, LipF and LipC 521
may be used in tandem by M. tb for utilization of host lipid substrates during active 522
replication and growth in vivo. 523
There is increasing evidence that bacterial cell-wall/surface localized enzymes 524
also multitask as antigenic proteins that play roles in modulation of immune responses 525
and/or in invasion of host cells (4, 19, 32, 38, 49). For example, besides LipC, LipY, 526
LipF, malate synthase, MPT51, Ag85B, Ag85C and Rv2224c, are all cell-wall/cell-527
surface located enzymes of M. tb that elicit humoral and/or cellular and/or inflammatory 528
immune responses (10, 19, 21, 22, 25, 54, 58). Ag85B, malate synthase and MPT51 529
are also reported to be adhesins of M. tb that bind to extracellular matrix proteins like 530
fibronectin and laminin (14, 19, 21, 58). Interestingly, the GehD lipase of 531
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Staphylococcus epidermidis is a collagen-binding protein (4); the GbpD protein of 532
Streptococcus mutans has both lipase and glucan-binding activity (38) and an 533
extracellular lipase of Pseudomonas aeruginosa participates in release of inflammatory 534
mediators from granulocytes and monocytes (32). The current studies demonstrate that 535
LipC is a cell-surface associated esterase of M. tb that is highly immunogenic and elicits 536
both antibodies and cytokines/chemokines. We have identified immunodominant 537
regions of LipC, which may be important constituents of a peptide-based sero-538
diagnostic test for TB. The role of LipC in infection and disease progression in TB merits 539
further investigation. 540
541
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Acknowledgments 542
This work was supported by the NIH/FIC AIDS International Training and Research 543
program (AITRP, grant TW001409), by the NIH/NIAID (grant AI056257) and by a VA 544
Medical Center Merit Review Award. It was also supported by a grant from the GIP ANR 545
06-JCJC-0067-01 and the FoamyTub Program ANR-09-MIEN-009-02, French Network. 546
The authors thank Flavia Camacho for editing figures. 547
548
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McKinney. 2003. Differential expression of iron-, carbon-, and oxygen-717
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749
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Figures and Tables 750
Fig. 1 3D model of LipC. (A) Ribbon representation; (B) Molecular surface of the 3-D 751
structure of LipC. C and D show rotation of 180° of the structures depicted in A and B, 752
respectively. The catalytic triad (Ser237, Asp334 and His367) is represented in cyan 753
sticks in A and C. The localization of the immunodominant peptides of LipC identified by 754
epitope mapping, LipC3 (red), LipC6 (orange), LipC24 (blue), LipC26 (green), LipC34 755
(yellow) and LipC39 (magenta) on the 3-D model is also depicted. 756
757
Fig. 2 Expression and purification of rLipC. Lane 1, Molecular weight markers. Lane 758
2, rLipC purified from E. coli was separated on 10% SDS-PAGE and stained with 759
Coomassie blue. Lanes 3-5, Western blots of rLipC (from E. coli) were probed with anti-760
His (lane 3), rabbit anti-LipC IgG (lane 4) and normal rabbit IgG (lane 5). Lane 6, rLipC 761
purified from M. smegmatis was separated on 10% SDS-PAGE and stained with 762
Coomassie blue. Lanes 7-9, Western blots of rLipC (from M. smegmatis) were probed 763
with anti-His (lane 7), rabbit anti-LipC IgG (lane 8) and normal rabbit IgG (lane 9). 764
765
Fig. 3 Localization of LipC protein. (A) M. tb H37Rv subcellular protein fractions 766
(5 µg/lane) and rLipC (75 ng/lane) were separated on 10% SDS-PAGE and transferred 767
to nitrocellulose membrane. The blots were probed with anti-malate synthase (MS) IgG 768
(top panel), anti-LipC IgG (middle panel) and normal rabbit IgG (bottom panel). Lane 1, 769
rLipC; 2, cytoplasm; 3, total cell-wall; 4, SDS-extracted cell-wall; 5, capsule; 6, culture 770
filtrate. (B) Immunoelectron microscopy of ultrathin sections of M. tb H37Rv (a and b), 771
M. tb CDC1551 lipC::Tn mutant (c) and M. tb CDC1551 (d). The bacterial sections were 772
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probed with either rabbit normal IgG (a) or rabbit anti-LipC IgG (b, c and d). 773
774
Fig. 4 Reactivity of rLipC with sera from TB patients and control subjects. (A) 775
Western blots of rLipC (75 ng/lane) were probed with sera (1:50) from 6 PPD- (lanes 2-776
7), 6 PPD+ (lanes 8-13), 6 HIV+TB- (lanes 14-19), 6 HIV+TB+ (lanes 20-25) and 6 HIV-777
TB+ subjects (lanes 26-31). (B) Probing of rLipC (75 ng/lane) blots with sera from 10 778
additional PPD+ healthy subjects (lanes 2-11) and 10 additional HIV-TB+ patients (Lane 779
12-21). Lane 1 in both panels A and B shows reactivity of rLipC with anti-His antibody. 780
(C and D) Reactivity of rLipC with sera from each subject was measured by determining 781
intensity of the rLipC band in panel A and B by Image J software. The relative intensity 782
of rLipC bands in panel A and B calculated by considering anti-His antibody reactivity as 783
100 Unit is plotted in C and D respectively. 784
785
Fig. 5 Epitope mapping of LipC protein. (A) Reactivity of overlapping peptides of 786
LipC with sera from 60 HIV-TB+ patients and 36 PPD- or PPD+ healthy subjects was 787
tested by ELISA. The mean optical density (OD) at 405 nm plus 3 standard deviations 788
with sera from PPD- or PPD+ healthy subjects was used as cut-off to determine positive 789
reactivity. Percent of sera from PPD- and PPD+ healthy subjects (hollow bars) and HIV-790
TB+ patients (black bars) showing positive reactivity with individual peptide is plotted. 791
(B) The reactivity of 6 LipC immunodominant peptides with sera from 13 PPD- (black 792
circles), 23 PPD+ healthy subjects (hollow circles) and HIV-TB+ patients (black 793
triangles) is shown. The OD values obtained with individual serum specimens in one 794
representative experiment are plotted. (C) The OD values obtained by reactivity of 795
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individual sera from 36 PPD- or PPD+ healthy subjects (black circles), 46 HIV+TB- 796
asymptomatic subjects (hollow circles) and 45 HIV+TB+ patients (black triangles) with 6 797
immunodominant peptides of LipC from one representative experiment is depicted. The 798
dotted lines in Fig. 5B and 5C indicate the cut off (mean OD + 3SD with sera from PPD- 799
and PPD+ subjects) for determining positive reactivity with each peptide. 800
801
Fig. 6 Cytokine production by THP1 and A549 cells in response to rLipC. (A) 802
Viability of A549 and THP-1 cells at 24 h with medium alone, medium containing 803
1 µg/ml rLipC and medium containing 0.00025%SDS. The percent viability calculated by 804
considering cells exposed to medium alone as 100% is plotted. (B) Production of IL-12 805
and TNF-α by THP-1 cells exposed to LPS alone (1 ng/ml), LPS (1 ng/ml) pre-incubated 806
with polymixin B (10 ng/ml), rLipC (1 µg/ml) and rLipC (1 µg/ml) pre-incubated with 807
polymixin B (10 ng/ml) are shown. (C) Induction of IL-12, TNF-α, IL-8 and MCP-1 in 808
THP-1 cells after incubation with various concentrations of rLipC and 1 µg/ml of LPS. 809
(D) Production of IL-8 and MCP-1 by A549 cells exposed to different concentrations of 810
rLipC and 1 µg/ml LPS. 811
812
Fig. 7 Enzymatic activity of rLipC. The enzymatic activity of rLipC was measured 813
using p-nitrophenyl esters family with carbon chain length ranging from C2 to C16 as 814
substrate using various concentration of NaTDC. The mean ± SD of specific activity 815
(mU/mg) obtained with 3 independent replicates is plotted. 816
817
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Table 1. Human subjects included in the study
Infection
status Origin
No. of
subjects
AFB Sputum
Smear status
Treatment
status
HIV-TB+ India 70 Positive Untreated
HIV+TB+ India 45 Positive Untreated
HIV+TB- USA 46 ND ART
PPD+ USA 7 ND NA
India 11 ND NA
China 7 ND NA
Cameroon 2 ND NA
Japan 1 ND NA
Colombia 1 ND NA
PPD- USA 6 ND NA
India 10 ND NA
China 3 ND NA
*AFB= Acid Fast Bacilli; ART = Anti-Retroviral Therapy; ND = Not Done; NA = Not
Applicable.
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Table 2. Sequencing of rLipC by quadrupole time-off-flight (Q-TOF) mass
spectrometry
LipC peptide fragments
mapped
Number of identified
peptides LipC AA position
AAGSTGVAYIR 1 6-16
HASDFLSATAK 2 60-70
DLLTPGINEVR 3 71-81
GIVSPDDLAVEWPAPER 12 98-114
VLYGDDPAQLLDVWR 22 123-147
AIQGYAVLSR 1 173-182
FVDFLER 2 290-296
TGPTAHAIALFLNQVHR 1 377-393
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Table 3. Amino acid sequence of immunodominant LipC peptides
Name Sequence*
LipC3 ARPADYMLALSVAGGSLPVV (Red)
LipC6 TAIGVWGARHASDFLSATAK (Orange)
LipC24 IAVAGCSAGGHLSALAGLTA (Blue)
LipC26 NDPQYQAELPEGSDTSVDAV (Green)
LipC34 GSRDCVIPVEQARSFVERLR (Yellow)
LipC39 AHAIALFLNQVHRSRAQFAK (Magenta)
*The color assigned to each peptide refers to localization of the peptide as shown in Fig.
1.
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