In the present study, we characterized the forma-tion of the complex between the ESAT-6 family protein Rv3619c and its pairing partner Rv3620c, using isothermal titration calorimetry ITC
Trang 1and Rv3620c from Mycobacterium tuberculosis H37Rv
Anjum Mahmood1, Shubhra Srivastava1, Sarita Tripathi1, Mairaj Ahmed Ansari2, Mohammad Owais2and Ashish Arora1
1 Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow, India
2 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India
Keywords
binding constant;
Mycobacterium tuberculosis; Rv3619c;
thermal unfolding; vaccine
Correspondence
A Arora, Molecular and Structural Biology
Division, Central Drug Research Institute,
Lucknow 226001, India
Fax: +91 522 223405
Tel: +91 522 2612411 ext: 4329
E-mail: ashishcdri@yahoo.com
(Received 5 September 2010, revised 1
November 2010, accepted 9 November
2010)
doi:10.1111/j.1742-4658.2010.07958.x
Rv3619c and Rv3620c are the secretory, antigenic proteins of the
ESAT-6⁄ CFP-10 family of Mycobacterium tuberculosis H37Rv In this article, we show that Rv3619c interacts with Rv3620c to form a 1 : 1 heterodimeric complex with a dissociation constant (Kd) of 4.8· 10)7M The thermal unfolding of the heterodimer was completely reversible, with a Tm of
48C The comparative thermodynamics and thermal unfolding analysis of the Rv3619c–Rv3620c dimer, the ESAT-6–CFP-10 dimer and another ESAT family heterodimer, Rv0287–Rv0288, revealed that the binding strength and stability of Rv3619c–Rv3620c are relatively lower than those
of the other two pairs Molecular modeling and docking studies predict the structure of Rv3619c–Rv3620c to be similar to that of ESAT-6–CFP-10 Spectroscopic studies revealed that, in an acidic environment, Rv3619c and Rv3620c lose their secondary structure and interact weakly to form a com-plex with a lower helical content, indicating that Rv3619c–Rv3620c is destabilized at low pH These results, combined with those of previous studies, suggest that unfolding of the proteins is required for dissociation
of the complex and membrane binding In the presence of membrane mimetics, the a-helical contents of Rv3619c and Rv3620 increased by 42% and 35%, respectively In mice, the immune response against Rv3619c pro-tein is characterized by increased levels of interferon-c, interleukin-12 and IgG2a, indicating a dominant Th1 response, which is mandatory for protec-tion against mycobacterial infecprotec-tion This study therefore emphasizes the potential of Rv3619c as a subunit vaccine candidate
Structured digital abstract
l MINT-8056093 : Rv0288 (uniprotkb: P0A568 ) and Rv0287 (uniprotkb: O53692 ) bind ( MI:0407 )
by isothermal titration calorimetry ( MI:0065 )
l MINT-8055978 : Rv3620c (uniprotkb: O07932 ) and Rv3619c (uniprotkb: P96364 ) bind ( MI:0407 ) by circular dichroism ( MI:0016 )
l MINT-8055964 : Rv3620c (uniprotkb: O07932 ) and Rv3619c (uniprotkb: P96364 ) bind ( MI:0407 ) by isothermal titration calorimetry ( MI:0065 )
Abbreviations
ASA, accessible surface area; BCG, bacille Calmette–Gue´rin; DMPC, dimyristoylphosphatidylcholine; DPC, dodecylphosphocholine;
HRP, horseradish peroxidase; IFN, interferon; IL, interleukin; ITC, isothermal titration calorimetry; MRE, mean residual ellipticity;
MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; RD, region of deletion; SI, stimulation index; TFE, trifluoroethanol.
Trang 2Comparative genomic studies based on whole genome
DNA microarrays have led to the identification of 16
regions of deletion (RDs) in Mycobacterium bovis
bacille Calmette–Gue´rin (BCG), which is currently used
as a vaccine, with respect to Mycobacterium tuberculosis,
and five RDs with respect to M bovis The RD
encom-passing ORFs Rv3619c and Rv3620c is absent from all
vaccine strains of M bovis This region has been
classi-fied as RD9 by Behr et al and as RD8 by Gordon et al
[1,2] It is a stretch of 5516 bp encompassing seven
ORFs (Rv3617 to Rv3623) Rv3619c and Rv3620c are
the ESAT-6⁄ CFP-10 family members ORFs Rv3621c
and Rv3622c belong to the family encoding proteins
containing sequence motif Glu (PPE) and
Pro-Glu (PE), respectively The region also contains an
epox-ide hydrolase encoded by Rv3617, which may be
involved in detoxification, catabolism and regulation of
signaling molecules [3] Rv3618 and Rv3623 encode a
probable monooxygenase and lipoprotein, respectively
Rv3619c and Rv3620c are secretory proteins of 94
and 98 amino acids, respectively, reported in culture
filtrates of M tuberculosis [4–6] In silico studies have
predicted their presence in Mycobacterium leprae,
Mycobacterium aviumand Mycobacterium marinum [7]
They belong to the ESAT-6 family, which comprises
23 members However, they share only 20% sequence
identity with ESAT-6 and CFP-10 Within the ESAT-6
family, Rv3619c and Rv3620c are constituted within
a subfamily comprising Rv1037c⁄ Rv1038c, Rv1197 ⁄
Rv1198, Rv1792⁄ Rv1793 and Rv2346c ⁄ Rv2347c [8]
The members within this subfamily share > 90%
amino acid sequence identity with Rv3619c⁄ Rv3620c
Overlapping synthetic peptide studies have
demon-strated that the Rv3619c⁄ Rv3620c subfamily consists
of potent T-cell antigens [9]
Detailed studies of ESAT-6 and CFP-10 have
revealed that they interact strongly to form a 1 : 1
heterodimeric stable complex with a four-helix bundle
in which each protein bears a central WXG motif
[10,11] Similar results have been obtained with
Rv0287–Rv0288 [12] However, deviations from the
basic prototype structure of the ESAT-6–CFP-10
com-plex have also been reported Recent crystallographic
studies have suggested that Rv3019c–Rv3020c (ESAT-6⁄
CFP-10 homologs) exists as a heterotetramer Rv3020c
contains histidine in place of tryptophan in the WXG
motif, which induces the formation of a small helix
that joins the N-terminal and C-terminal domains [13]
The ESAT-6⁄ CFP-10 homologs in Staphylococcus
aureus (SaEsxA and SaEsxB) do not form a
hetero-dimer complex; rather, they homohetero-dimerize The crystal
structure of the homodimer of SaEsxA has been deter-mined Furthermore, sequence analysis has predicted that SaEsxB (CFP-10 homolog) will have a structure similar to that of SaEsxA; on the basis of this, it has been suggested that the two proteins may work inde-pendently [14] These variations among complexes indi-cate that different homologs and paralogs of the ESX family may have different structural properties that may lead to further functional dissimilarities There-fore, a separate detailed analysis for each pair is required, structurally as well as functionally
One of the major functions associated with ESAT-6
is its cytolytic activity Hsu et al have shown the cytolysis of host cells by ESAT-6 secreted by intracel-lular mycobacteria [15] We have previously shown that ESAT-6 adopts significant helical structure in the presence of dimyristoylphosphatidylcholine (DMPC) vesicles and dodecylphosphocholine (DPC) micelles, indicating membrane binding Furthermore, only ESAT-6, and not CFP-10 or the complex, was found
to interact with lipid membranes [16] Moreover, Smith
et al have shown that ESAT-6 induces pore formation
in M marinum in a dose-dependent manner, enabling the bacterium to escape from the vacuole to the host cell cytosol [17] The membrane-binding ability was related to disruption of the complex by Jonge et al [18] These authors demonstrated that, in the acidic phagosomal environment, the 1 : 1 ESAT-6–CFP-10 complex dissociated to free ESAT-6 to exert its cyto-lytic activity However, no studies have been per-formed on other ESAT-6⁄ CFP-10 paralogs to determine whether other members have similar mem-brane-destabilizing functions
The ESAT-6 family is a potential source of T-cell antigens, which can be exploited for the development
of suitable vaccines against mycobacteria ESAT-6 and CFP-10 are the most widely studied proteins of this family ESAT-6 and CFP-10 activate the Th1 response which is marked by T-cell proliferation and interferon (IFN)-c release [19,20] Subunit-based or DNA-based ESAT-6 vaccines have been prepared, and their protec-tive efficacy has been evaluated The first DNA-based vaccine involving ESAT-6 was reported by Kamath
et al [21] Although this vaccine provided a significant level of protection against mycobacteria, its potency was found to be lower than that of BCG Brandt et al prepared the first single protein subunit tuberculosis vaccine, using ESAT-6 with dioctadecylammonium bromide and monophosphoryl lipid A, that conferred protection similar to that provided by BCG [22] A recombinant chimeric fusion protein of ESAT-6 and
Trang 3Ag85B was found to provide better protection than
individual proteins or a mixture of them, and is
cur-rently being evaluated as a promising vaccine
candi-date [23,24] Besides ESAT-6, other members evaluated
for immune response were Rv0288, Rv3019c, and
Rv3017c [25] Rv3019c demonstrated its potential to
be used as a heterologous prime booster in conjunction
with BCG
In the present study, we characterized the
forma-tion of the complex between the ESAT-6 family
protein Rv3619c and its pairing partner Rv3620c,
using isothermal titration calorimetry (ITC) and CD
spectroscopy We also examined the structural
prop-erties of the proteins by spectroscopy and molecular
modeling Furthermore, we evaluated the immune
response of Rv3619c in free antigenic form in mice
by determining lymphocyte proliferation, cytokine
levels, and antigen-specific antibody levels Our study
provides insights into the properties of these
secre-tory proteins
Results and Discussion
Interaction of Rv3619c and Rv3620c ITC experiments were performed to determine the thermodynamic parameters governing formation of the complex between Rv3619c and Rv3620c The raw ITC data and integrated areas under each peak versus Rv3619c⁄ Rv3620c molar ratio are shown in Fig 1A The binding isotherm was fitted to a single-site binding model for determination of thermodynamic parame-ters The parameters used in fitting were the stoichiom-etry of association (n), the binding constant (Kb), and the change in enthalpy (DHb) The values of these parameters obtained from the nonlinear least-squares
fit to the binding curve are as follows: n = 1.0,
Kb= (2.05· 106) ± (3.24· 105) m)1 and DHb = )3.35 · 104± 760.1 calÆmol)1 The saturation of heat released at a molar ratio of 1.0 strongly suggests that the proteins form a 1 : 1 heterodimeric complex The
Fig 1 Thermodynamic and spectroscopic studies on Rv3619c–Rv3620c (A) ITC measurements of the interaction between Rv3619c and Rv3620c in phosphate buffer at 25 C; raw data of heat effect (lcalÆs)1) of 30 injections (10 lL each) of 0.1 m M Rv3619c into 1.43 mL of 0.01 m M Rv3620c The data points (j) were obtained by integration of heat signals plotted against the Rv3619c ⁄ Rv3620c molar ratio in the reaction cell The solid line represents a calculated curve using the best-fit parameters obtained by a nonlinear least square fit The heat of dilution was subtracted from the raw data of titration of Rv3619c with Rv3620c (B) Far-UV CD spectra of Rv3619c, Rv3620c, and the 1 : 1 complex CD spectra of 5 l M Rv3619c (j), Rv3620c (•) and the 1 : 1 complex (m) in phosphate buffer (pH 6.5, 25 C (C) Normalized transi-tion curves for temperature-induced transitransi-tion of the complex monitored in the far-UV CD region at 222 nm The thermal unfolding (j) and thermal refolding (•) profiles of the complex were plotted as fraction of protein folded versus temperature in C.
Trang 4dissociation constant of the complex (Kd= 1⁄ Kb) was
4.8· 10)7m The free energy change (DG) and entropy
change (DS) associated with complex formation were
)8.55 kcalÆmol)1 and)83.7 calÆmol)1ÆK)1, respectively,
at 25C
A comparative thermodynamic analysis of ESAT-6⁄
CFP-10 family members is shown inTable 1 Although
Rv0287–Rv0288 is well characterized [12], we performed
ITC experiments with Rv0287 and Rv0288 to generate
the thermodynamic data for their interaction The ITC
data for ESAT-6–CFP-10 have already been obtained
[16] Data analysis revealed that Rv0288–Rv0287 has
the strongest binding affinity (Kd 10 nm), followed by
ESAT-6–CFP-10 (Kd 50 nm) Rv3619c–Rv3620c
showed the weakest binding (Kd 480 nm) The
bind-ing free energy (DG) of Rv3619c–Rv3620c revealed
dif-ferences of 2.26 kcalÆmol)1 relative to Rv0287–Rv0288
and 1.4 kcalÆmol)1 relative to ESAT-6–CFP-10 This
suggests that binding of Rv0287 and Rv0288 is
energeti-cally more favored The difference in entropy values
indicates that the conformational freedom of side chains
in Rv3619c–Rv3620c is comparatively greater The
dif-ferences in binding affinity among ESAT-6⁄ CFP-10
par-alogs suggest that the binding equilibrium in loosely
bound complexes may be shifted to the reactant side,
resulting in the release of unbound proteins under
cer-tain specific conditions, such as those in the acidic
phag-osomal environment
The conformational changes associated with
com-plex formation were estimated by recording far-UV
CD spectra The CD spectra of Rv3619c, Rv3620c and
the 1 : 1 complex were recorded (Fig 1B) at 25C,
and data were analyzed by the k2d server The CD
spectra showed that the two proteins and their 1 : 1
complex adopt a predominantly a-helical
conforma-tion The a-helical contents of Rv3619c, Rv3620c and
the 1 : 1 complex were approximately 33%, 34% and
70%, respectively The secondary structure of
Rv3619c–Rv3620c was very similar to that of
ESAT-6–10 and Rv0288–Rv0287 However, unlike
CFP-10 and Rv0287, which are unstructured, Rv3620c had
a significant a-helical content, even in the uncomplexed
state The lower values of DH and DS (Table 1)
observed for Rv3619c–Rv3620c than for ESAT-6–
CFP-10 and Rv0287–Rv0288 could result from the dif-ference in the folding state of Rv3620c from that of CFP-10 and Rv0287
Thermal unfolding of proteins The presence of any stable tertiary structure in Rv3619c and Rv3620c was ruled out by thermal unfolding experiments (data not shown) The two pro-teins were denatured when the temperature was increased from 25C to 80 C, following non-coopera-tive unfolding, and the structure was not regained on cooling, indicating that they lack any stable tertiary structure The complex, however, demonstrated signifi-cant resistance to denaturation before melting The unfolding started at 38C, following a cooperative pathway with a denaturation midpoint (Tm) of 48C (Fig 1C) Rv3619c–Rv3620c showed lower thermal stability than ESAT-6–CFP-10 (Tm= 54C) and Rv0287–Rv0288 (Tm= 70C) [12] This indicates that Rv3619c–Rv3620c has a smaller intermolecular hydro-phobic overlapping interface Overall, the thermal denaturation profile and ITC data suggest that Rv3619c forms a loose complex with its genomic part-ner Rv3620c, because of a smaller protein–protein interaction surface area than that in ESAT-6–CFP-10 and Rv0287–Rv0288
The thermal renaturation profile of the 1 : 1 com-plex was recorded by cooling the sample from 80C
to 25C (Fig 1C) On reversal of the temperature, the complex completely regained its secondary structure, retracing a similar path This characteristic feature was also observed for ESAT-6–CFP-10 [16] The two pro-teins complement each other to attain a folded struc-ture However, in the absence of the other partner, they lose their structure irreversibly This implies that Rv3620c, like CFP-10, functions to keep Rv3619c in a structured and soluble form under physiological conditions
Modeling and docking The secondary structure was also analyzed by molecu-lar modeling and docking experiments On the basis of
Table 1 A comparative analysis of thermodynamic parameters of ESAT–CFP-10, Rv0287–Rv0288, and Rv3619c–Rv3620c The stoichiome-try of interaction and values of K b and DH were determined by ITC DG and DS were calculated from the thermodynamic formula
DG = )RT ln K b = DH ) TDS.
d ( M ) DH (kcalÆmol)1) DS (calÆmol)1ÆK)1) DG (kcalÆmol)1)
Trang 5the solution structure of the 1 : 1 ESAT-6–CFP-10
complex, we generated molecular models of Rv3619c
and Rv3620c using i-tasser and swiss model,
respec-tively, and molecules were docked using the patch
dockserver The model Rv3619c–Rv3620c is shown in
Fig 2 We determined the accessible surface area
(ASA) for the Rv3619c–Rv3620c model and the
ESAT-6–CFP-10 solution structure by using the
dis-covery studio2.1 package The ASA for docked
Rv3619c–Rv3620c was 12 190 A2, and the buried
surface area was 1211 A2 The ASA for the ESAT-6–
CFP-10 solution structure was 11 512 A2, and the
buried surface area was 1675 A2 The buried surface
area for docked Rv3619c–Rv3620c was smaller than
that for the ESAT-6–CFP-10 solution structure, which
is in complete agreement with our ITC data
The model suggests that the two proteins interact
with 1 : 1 stoichiometry, lying antiparallel to each
other, with each one having two helixes separated by a
loop containing the WXG motif The predicted
struc-ture resembles the four-helix bundle packing of
ESAT-6–CFP-10 The crystal structure of Rv3019c–Rv3020c
suggested that replacement of tryptophan by histidine
in the WXG motif induced the formation of a small
helix in Rv3020c, joining the N-terminal and
C-termi-nal helices, conferring a tetramer structure However,
as the WXG motif in the Rv3620c is strictly
con-served (Trp45 and Gly47), it is highly unlikely that Rv3619c–Rv3620c would form a tetrameric complex like Rv3019c–Rv3020c Despite the predicted structure being similar to that of ESAT-6–CFP-10, a consider-able difference at the C-terminus of Rv3620c was noticed The C-terminal end of CFP-10 is unstruc-tured, whereas the modeling and docking results pre-dicted that Rv3620c would possess a C-terminal helix The C-terminal end of SaEsxA from S aureus also contains a folded region [14]
In order to identify the residues forming the inter-molecular contact surface of Rv3619c–Rv3620c, we generated a contact map for docked Rv3619c and Rv3620c, using the acclerys discovery studio 2.1 software package The contact map analysis predicted that Val10, Ile17, Ala21, Leu24, Ala26, Ala30, Ile31, Ile32, Val35, Leu36, Ala38, Phe41, Cys50, Phe53, Leu57, Phe61, Val63, Ile64, Ala68, Ala70, Val75, Ala77, Ala78, Met82, Val89 and Ala94 of Rv3619c and Met12, Met15, Ala16, Phe19, Val21, Ala23, Val26, Ala30, Met33, Ala35, Ala37, Ile40, Ala43, Met48, Ala49, Leu54, Met57, Met60, Phe64, Ile67, Val68, Met70, Leu71, Val74, Leu78, Val79 and Ala82
of Rv3620c are likely to form hydrophobic contact surfaces We plotted the N-terminal and C-terminal helices of Rv3619c and Rv3620c on a heptad repeat helical wheel, on the basis of optimum sequence alignment (Figs S1 and S2) Hydrophobic residues suggested by contact map analysis occupied predomi-nantly ‘a’ and’d’ positions, suggesting that they form the core of helix bundle packing This is in full agree-ment with the models suggested for ESAT-6–CFP-10 and Rv0287–Rv0288 The complex was predicted to be stabilized by formation of a single salt bridge between Glu25 of Rv3619c and Arg31 of Rv3620c
The residues highlighted in Fig 2, in the Rv3619c helix, represent the nonconserved, semiconserved or substituted conserved residues at the corresponding positions among the related members Rv1037c, Rv1198, Rv1793 and Rv2346c Substitution of residues
at these positions severely alters antigenic recognition
by the cell Alderson et al demonstrated that a T-cell line specific for Rv1198 failed to recognize peptides from Rv1793 and Rv3619c with amino acid substitu-tions at the 22nd and 23rd posisubstitu-tions [9]
Effect of pH on complex stability
We analyzed pH-induced conformational changes in Rv3619c, Rv3620c and the 1 : 1 complex by recording the far-UV CD spectra at different pH values The mean residual ellipticity (MRE) at 222 nm was plotted against different pH values, as shown in Fig 3A On
G22 / A22 S23 / L23
S33 / R33 T37 / A37
S39 / G39
A48 / V48
G52 / E52
I32 / V32
N Rv3620c
C Rv3620c
N Rv3619c
C Rv3619c
Fig 2 In silico modeling and docking of Rv3619c and Rv3620c.
The two proteins form a 1 : 1 complex Rv3619c is shown in blue
and Rv3620c is shown in gray The nonconserved, semiconserved
and substituted conserved regions of paralogs of Rv3619c (Rv1037,
Rv1198, Rv1793, and Rv2346c) are highlighted in blue, orange, and
yellow.
Trang 6lowering of the pH from 6.5 to 4.5, Rv3619c and
Rv3620c showed considerable loss of conformation,
although Rv3619c resisted conformational change until
pH 5.5 However, on mixing of the two proteins at
pH 4.5, the resultant spectra, shown in Fig 3B,
dem-onstrated significant loss of structure This could be
attributable to weak interactions between two unfolded
or partially folded proteins Furthermore, it indicates
that the structural unfolding of individual proteins
could be the event that results in dissociation of the
complex at acidic pH Recently, Arbing et al also
reported the pH-induced dissociation of Rv3019c–
Rv3020c (unpublished data) [13]
Effect of trifluoroethanol (TFE) and DPC micelles
on the conformation of Rv3619c and Rv3620c
Previous work suggested that ESAT-6 adopts a helical
structure, and is sequestered and induces pore formation
in the cell membrane Furthermore, only ESAT-6, and
not CFP-10, was associated with cytolytic activity
[16,26] To investigate the role of Rv3619c, Rv3620c and the 1 : 1 complex in membrane binding, CD spectros-copy experiments were performed in the presence of TFE and DPC micelles, as shown in Fig 4A In 40% TFE, Rv3619c, Rv3620c and the 1 : 1 complex adopted
a highly folded structure In 20 mm DPC, Rv3619c and Rv3620c demonstrated 42% and 35% increases, respec-tively, in helical content However, the 1 : 1 complex did not show any significant change in conformation in
20 mm DPC This implies that the two proteins bind to the membrane individually, but not after they form a complex, as also observed for ESAT-6 and CFP-10 Furthermore, we recorded the intrinsic tryptophan fluo-rescence of Rv3619c and Rv3620c in 20 mm DPC at wavelengths ranging from 300 to 400 nm (Fig 4B) The
kmax values of Rv3619c and Rv3620c shifted to lower wavelengths by 5 nm and 10 nm, respectively, and this was accompanied by enhancements of fluorescence intensity, suggesting the relocation of tryptophans in the hydrophobic environment because of membrane bind-ing We also checked the binding of the two proteins to small unilamellar vesicles of DMPC, using CD spectros-copy Our preliminary results suggested that Rv3619c, but not Rv3620c, underwent a change in helicity in the presence of DMPC small unilamellar vesicless This does not correlate with the change in CD spectra observed in the presence of DPC micelles However, interestingly, it does correlate with the binding of the fluorescent dye 8-anilinonapthalene-1-sulfonate, which was observed for Rv3619c but not for Rv3620c (data not shown)
Previous membrane-binding studies with ESAT-6 suggested that the deeper integration of the protein into phospholipids is related to its unfolding and struc-tural transition [16] We also found unfolding of Rv3619c and Rv3620c at pH 4.5 In some cases, phagosomes containing mycobacteria may advance to the phagolysosomal stage, with concomitant lowering
of the pH from 5.1 to 4.8–4.5 It has been suggested that, under such conditions, ESAT-6 could dissociate from the complex and bind the lipid membranes [17] Considering the two events together, it can be inferred that unfolding of proteins and formation of a struc-tural intermediate is associated with membrane bind-ing The acidic phagolysosome may provide an environment that stimulates dissociation of the com-plex and structural reorientation of proteins, allowing them to assemble and penetrate the membrane deeply Taking into account that mycobacteria in infected cells inhibit the fusion of lysosomes with phagosomes, Rv3619c–Rv3620c would most likely exist as a com-plex However, under certain conditions when the infected cells also contain phagolysosomes [27,28], the
1 : 1 complex may be disrupted, and Rv3619c and
Fig 3 pH-induced conformational changes in Rv3619c, Rv3620c,
and the 1 : 1 complex (A) MREs of Rv3619c (j), Rv3620c (d) and
the 1 : 1 complex (m) at 222 nm were plotted against different pH
values (B) Far UV-CD spectra of the 1 : 1 complex at pH values of
6.5 (j), 5.5 (d), and 4.5 (m).
Trang 7Rv3620c may execute their functions independently
and bind to lipid membranes Although no pH-based
structural studies have so far been performed with
ESAT-6, our study supports the hypothesis of Simeone
et al [26] that the biological activity of ESAT-6
depends on the pH of phagosomal compartments
Proteins of the ESAT-6 family are not only involved
in spreading virulence by exhibiting cytolytic activity;
they also confer protection against M tuberculosis
through the antimicrobial Th1 host immune response
ESAT-6, Rv0288 and Rv3019c are known to stimulate
T-cells to proliferate and protect against mycobacteria
[25,29,30] Th1 activation results in IFN-c and
inter-leukin (IL)-12 release, whereas Th2 activation releases
IL-4 A balanced Th1⁄ Th2 response is important for clearance of mycobacteria To determine whether Rv3619c also activates T-cells to release cytokines, we examined its role in the immune response in mice
Analysis of immune response of Rv3619c
We evaluated the immune response of Rv3619c by injecting the free antigen in NaCl⁄ Pi in Balb⁄ c mice, with NaCl⁄ Pias a control Initially, the antigen-induced lymphocyte proliferation activity of Rv3619c was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay The proliferative response of Rv3619c was demonstrated by stimulation
Fig 4 Membrane binding of Rv3619c, Rv3620c, and the 1 : 1 complex (A) Far-UV CD spectra of 5 l M Rv3619c, Rv3620c and the 1 : 1 complex in the presence of phosphate buffer (j), 40% TFE (•), and 20 m M DPC (m) are shown All of the spectra were recorded at 25 C (B) Fluorescence emission spectra of Rv3619c and Rv3620c in phosphate buffer (j) and 20 m M DPC (•).
Trang 8index (SI), as shown inFig 5A; it was determined to be
4.695 ± 0.24 pgÆmL)1 postbooster, with a statistically
significant difference (P < 0.05) from the NaCl⁄ Pi
group The significant augmentation of antigen-specific
proliferation clearly demonstrates the presence of
immunologically active lymphocytes in immunized
mice Statistically significant levels of IFN-c (832
± 30.61 pgÆmL)1; P < 0.001) and IL-12 (481 ± 5.46
pgÆmL)1; P < 0.05) were produced by spleenocytes of
mice immunized with Rv3619c (Fig 5B); IL-4, a Th2
cytokine, was also detected in immunized groups, with a
significant difference, but the level of IFN-c was very
high when compared with that of IL-4 The humoral
response was determined by measuring the
Rv3619c-specific serum IgG level (Fig 6A) Class switching
showed a predominant IgG2aresponse, as indicated by
a postbooster IgG2a⁄ IgG1ratio greater than 1 (Fig 6B) The high levels of IFN-c and IL-12 secretion, suggesting
a biased Th1-type response of Rv3619c, is consistent with the IgG2a⁄ IgG1 ratio The study clearly demon-strates that Rv3619c is a potent T-cell antigen that may provide protection against mycobacterial infection if used in combination with a suitable adjuvant or in a mixture with other antigens
Rv3619c paralogs, i.e Rv1037c, Rv1198, Rv1793 and Rv2346c and their genomic partners, share greater than 90% amino acid sequence identity Structurally, they may form similar complexes to those observed for Rv3619c, Rv3620c, and other pairs, but the immune response may vary, as they display unique epitopes The presence of similar structure and function but different immune responses has been related to the
A
B
Fig 5 Study of immune response to Rv3619c antigen Analysis of
the Rv3619c-specific cytokine profile of 6–8-week-old female
Balb ⁄ c mice immunized with 20–25 lg of antigen (A) Lymphocyte
proliferation response of Rv3619c expressed in terms of SI Each
bar represents mean ± standard deviation (B) IFN-c, IL-12 and IL-4
levels determined 2 weeks postimmunization and 2 weeks
post-booster Three mice per group were used, and the data obtained
were statistically significant different, with ***P < 0.001,
**P < 0.01, or *P < 0.05, from those obtained with NaCl ⁄ P i
A
B
Fig 6 Estimation of humoral response to Rv3619c antigen The antibody response against Rv3619c in mice (three mice per group)
is shown Serum was collected 2 weeks postimmunization and
2 weeks postbooster The antibody level was estimated by record-ing the absorbance at 490 nm (A) Total IgG content (B) IgG 2a ⁄ IgG 1 ratio, indicating the biased Th1 response.
Trang 9mycobacterial strategy for escaping the host immune
recognition system [30] Studies with tuberculosis
reac-tor animals also revealed that immunodominant
epi-topes of the ESAT-6 family come from variable
regions more than homologous regions, suggesting that
mycobacteria may vary their antigenic load according
to requirements, leading to antigenic drift that enables
mycobacterial escape [31] The presence of several
pairs of ESAT family proteins within the M
tuberculo-sisgenome suggests that they might be expressed under
different physiological conditions, and are able to
sub-stitute for each other functionally; this strategy enables
them to survive longer in host cells However, at the
same time, sequence variations among the family
mem-bers provide a pool of antigens that can generate the
effector molecules that restrict mycobacterial growth
Evaluation of the formation of complexes and their
protective efficacy will help in the development of
suit-able vaccine candidates
Conclusion
The ESAT-6⁄ CFP-10 family has 23 members, consisting
of 11 pairs and one unpaired member Although these
11 pairs are likely to form complexes in a manner
simi-lar to the formation of ESAT-6–CFP-10, they may
exhi-bit subtle variations in affinity, stability and immune
response, which in turn may further define their
individ-ual functional roles A systematic study of various
com-plex-forming pairs would improve our understanding of
the evolutionary and functional relevance of the whole
ESAT family Our study further consolidates the
hypothesis that the structural unfolding of individual
proteins under conditions of acidic pH may be the key
factor triggering the dissociation of complexes
How-ever, we believe that this aspect still needs more elegant,
unambiguous, quantitative and time-resolved
character-ization The sequence variation in the ESAT-6⁄ CFP-10
family not only determines the stability of the complex,
but also provides an antigenic pool in which a change in
a single amino acid can change the host immune
response The efficacy of peptides and proteins
incorpo-rating these antigens must be evaluated so that those
conferring protection can be developed further We are
currently working in this direction
Experimental procedures
Materials
pET expression vectors were obtained from Novagen
(Darmstadt, Germany) Oligonucleotides for gene isolation
were from BIO Serve (Hyderabad, Andhra Pradesh, India)
Restriction endonucleases, T4 DNA ligase and DNA size markers were from New England Biolabs (Beverly, MA, USA) Taq polymerase and other reagents for PCR, the plasmid miniprep kit and the gel extraction kit were from Qiagen The Ni2+–nitrilotriacetic acid superflow metal-affinity chromatography matrix was from Qiagen For protein concentration, Centricon membrane were used (molecular mass cut-off 3KDa: Millipore (India) Pvt Ltd, Bangalore, India) The rest of the chemical reagents were from Sigma (New Delhi, India)
Cloning, expression and purification
Genomic DNA of M tuberculosis H37Rv was prepared as described by Kremer et al [32] The genes encoding Rv3619c and Rv3620c were PCR-amplified with oligonu-cleotide primers and pfu DNA polymerase, and cloned into pET-NH6 This cloning strategy added an additional 30 residues at the N-terminus, including the six residues of the
BL21(kDE3) Escherichia coli cells, which were grown in LB medium supplemented with ampicillin (100 lgÆmL)1) BL21(kDE3) cells containing the plasmids pET-NH6– Rv3619c and pET-NH6–Rv3620c were grown in LB
0.5 mm isopropyl thio-b-d-galactoside The Rv3619c culture was grown for a further 12–14 h at 27C, and the Rv3620c culture for 6 h at 37C All proteins were purified over a
Ni2+–nitrilotriacetic acid matrix with a standard protocol under denaturing conditions, according to the manufac-turer’s instructions, except that NaCl and guanidine hydro-chloride were excluded from the buffer The column
gel) The proteins were refolded by dialysis, with a buffer
EDTA (pH 6.5) Refolded Rv3619c and Rv3620c were dia-lyzed against buffer containing 20 mm NaH2PO4, 50 mm
pET-NH6–Rv3619c-encoded and pET-NH6–Rv3620c-pET-NH6–Rv3619c-encoded proteins con-tained 30 extra N-terminal residues with a His-tag
ITC
VP-ITC calorimeter from Microcal (Northampton, MA, USA) The calorimeter was calibrated according to the user manual of the instrument The proteins were dialyzed
NaCl (pH 6.5) Samples were degassed prior to titration at
aliquots of Rv3619c to Rv3620c The sample cell was filled with 1.43 mL of 0.01 mm Rv3620c and titrated against 0.1 mm Rv3619c Thirty injections of 10 lL each were
Trang 10made at intervals of 180 s The ITC data were analyzed
with origin version 7 The amount of heat produced per
injection was calculated by integration of the area under
each peak with a baseline selected by origin
CD spectroscopy
CD measurements were performed to determine the
second-ary structure of Rv3619c, Rv3620c, and the 1 : 1 complex
The experiments were performed on a Jasco
Spectropola-rimeter model J-810 The instrument was calibrated with
(+)-10-camphorsulfonic acid The protein spectra were
recorded with the protein samples in buffer containing
concentration used was in the range 5–10 lm Three scans
were averaged for each spectrum Isothermal wavelength
scans were recorded in the range 250–200 nm, with a
path-length of 2 mm, a response time of 1 s, a scan speed of
20 nmÆmin)1, and a data pitch of 0.5 The CD results were
expressed as MRE, in degree cm)2Ædmol)1, calculated as
follows:
MRE¼ ðh 100 MrÞ=ðcdNAÞ
where h is the observed ellipiticity (), c is the protein
con-centration (mgÆmL)1), d is the pathlength (cm), and NAis
the number of amino acids Percentage secondary structure
was calculated with the online k2Dserver (http://www.embl
de/~andrade/k2d/) For thermal denaturation studies, the
spectra were recorded in the temperature range 25–80C at
a speed of 1CÆmin)1 For refolding, the temperature was
reversed at the same speed The fraction of protein folded
corresponding to the MRE at 222 nm was calculated from
the equation
½hobshden=½hnathden
where hnatand hdenare the MREs at 222 nm when proteins
are in the native state at 25C, and in the denatured state
at 80C hobs
is the observed MRE
To study the effect of membrane mimetic conditions on
conformation of proteins, far-UV CD spectra were acquired
in the presence of either 40% TFE or 20 mm DPC DPC
stock (200 mm) was prepared in buffer containing 20 mm
18 500 g to remove any suspended particles DPC was added
to 5 lm protein to a final concentration of 20 mm For TFE
experiments, 5 lm protein was added to 40% TFE The
spec-tra were recorded in the wavelength range 250–200 nm and
analyzed on the k2Dserver
Protein modeling and docking
The protein sequences of Rv3619c and Rv3620c were taken
from the TB Structural Genomics Consortium (http://
www.doe-mbi.ucla.edu/TB/), and sequences were aligned
using a server (http://www.ebi.ac.uk/clustalw) Models of Rv3619c and Rv3620c were generated using online servers (http://zhang.bioinformatics.ku.edu/I-TASSER/ and http:// swissmodel.expasy.org//SWISS-MODEL.html, respectively) The modeled structures of Rv3619c and Rv3620c were
PatchDock), a geometry-based molecular docking algo-rithm The docked complex was analyzed with accelrys discovery studio2.0
Fluorescence spectroscopy
Fluorescence spectra were acquired to record the intrinsic tryptophan fluorescence changes of Rv3619c, Rv3620c and the 1 : 1 complex in the presence of 40% TFE and 20 mm
Perkin-Elmer Life Sciences LS 50B spectroluminescence meter, with a 5-mm-pathlength quartz cell Protein (1 lm) was mixed with either 40% TFE or 20 mm DPC The maxi-mum intrinsic fluorescence was monitored to record the wavelength shift The fluorescence emission spectra were recorded in the range 300–400 nm, with an excitation wave-length of 280 nm
Animals and immunization
Female Balb⁄ c mice (6–8 weeks old) were purchased from the JALMA Institute for Leprosy and Other Microbial Diseases, Agra, India Mice were maintained in the animal facility, and the techniques used for injection and bleeding of animals were performed in strict accordance with the mandates approved by the Animal Ethics Committee (CPCSEA, Gov-ernment of India) Ten mice in two groups were taken for study One group was immunized with antigen in NaCl⁄ Pi, and the other group (control) was injected with NaCl⁄ Pionly The immunization volume was 100 lL per animal, and the antigen dose was 20–25 lg per injection The mice were immunized by subcutaneous injection in the lower abdominal region Two weeks postimmunization, a single booster with same amount of antigen was given to each animal
Lymphocyte proliferation
Cells were grown in RPMI-1640 with 10% fetal bovine serum and 1% antimycotic solution for 48 h in a CO2
aseptic conditions, with 20 lg of antigen and 2 lgÆmL)1 concanavalin A Four to five hours before completion of
was added to each well in 96-well plates to attain a final concentration of 1 mgÆmL)1 One well was kept blank; that
is, before addition of the MTT, 100 lL of lysis buffer was added to the well When dark crystals appeared, 50 lL of tissue culture-grade dimethylsulfoxide was added to each well Plates were then incubated for 2 h in a CO2incubator