Lepr Rev (2016) 87, 501– 515
TlyA protein of Mycobacterium leprae: a probable
bio-marker of active infection
HIRAWATI DEVAL*, KIRAN KATOCH*, DEVENDRA
SINGH CHAUHAN*, ANIL KUMAR TYAGI**,
RAKESH KUMAR GUPTA******, RAJ KAMAL*,
AVNISH KUMAR***, VIRENDRA SINGH YADAV*,
VISHWA MOHAN KATOCH**** , ***** &
TAHZIBA HUSSAIN*
* Division of Microbiology & Molecular Biology, National JALMA
Institute for Leprosy and other Mycobacterial Diseases, Tajganj,
Agra
** Department of Biochemistry, South Campus, Benito Jurez Marg,
Delhi University, Delhi
*** School of Life Sciences, Dr. Bhim Rao Ambedkar University,
Agra
**** Former Director General, Indian Council of Medical Research,
New Delhi
***** Regional Medical Research Centre, Bhubaneswar
****** Department of Microbiology, Ram Lal Anand College,
University of Delhi, Benito Juarez Road, New Delhi-110021
Accepted for publication 5 October 2016
Summary
The extent of pathogenicity of the mycobacterial infections depends on virulence
factors that mediate survival inside macrophages. Virulence factors are generally
believed to be specific for pathogenic species and mutated/non-functional in nonpathogenic strains. Mycobacterial TlyA can modulate the phagolysosome maturation
pathway, immediately after entry into macrophages.
Over-expression of open reading frame (ORF) ML1358 (tlyA) in tissues of leprosy
patients by partial DNA chip and real time PCR analysis during active infection
attracted our interest to explore the properties of this gene at molecular and
serological levels, to understand its role in the host. Molecular properties were studied
by cloning and expression of the corresponding gene in pASK-iba 43(þ ) expression
vector in E. coli and bioinformatics tools while sodium dodecyl sulfate
Correspondence to: Tahziba Hussain, Regional Medical Research Centre (ICMR), Chandrasekharpur,
Nandankanan Road, Bhubaneswar - 751023, Odisha, India (Tel: þ91 674 2305640; Fax: þ91 674 2301351;
e-mail: [email protected])
0305-7518/16/064053+15 $1.00
q Lepra
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H. Deval et al.
polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA were applied to
investigate the serological significance of rTlyA protein in different clinical states of
leprosy.
We observed that TlyA has a close relation among mycobacteria with specific
protein domains in slow growing intracellular adapted pathogenic species. The
presence of trans-membrane domains indicates its association to the cell membrane.
The study revealed its highly significant sero-reactivity (P value , 0·001) in borderline lepromatous (BL) patients, and those with reversal reaction (RR) and erythema
nodosum leprosum (ENL). Its role in active infection, association with the cell
membrane, presence in pathogenic species and high sero-reactivity, suggested the
tlyA gene as a strong disease progression marker.
Introduction
The continued incidence of leprosy in countries where it is endemic is thought to be a result
of the perpetuating reservoir of Mycobacterium leprae-infected contacts or people with
subclinical leprosy where the country is declared as being in a ‘leprosy eliminated state’. In
such a situation understanding every component of this pathogen can provide a novel way to
control leprosy. Hence, detailed analysis of every protein in M. leprae could reveal novel
drug targets, preventive tools such as a peptide vaccine, or it may be provide the knowledge
database to understand survival of M. leprae inside host cells.
The product of the tlyA gene has been considered to have methylation activity of 16S and
23S rRNA as well as contact-dependent haemolytic activity; it is possibly involved in
virulence (pore formation). The mycobacterial tlyA is similar to pore-forming
haemolysin/cytotoxin, tlyA, of the swine pathogen Serpulina hyodysenteriae.1 Wren et al.
also showed the presence of tlyA homologues in M. tuberculosis, M. leprae and other
mycobacteria, and its absence in M. smegmatis, M. vaccae, M. kansasii, M. chelonae and
M. phlei.2 Cytolysin plays an important role in processes such as escape from the immune
response, intracellular multiplication and cellular spread, within eukaryotic cells.3,4
Previously tlyA was noted in other bacteria where it is associated with membrane bound
cytolysins as an important virulence factor. Soluble cytolysin of Listeria monocytogens,
listeriolysin O, is utilized by the pathogen to grow in intracellular macrophages.5,6 Despite
earlier reports7,8 on its importance in other pathogenic species of genus Mycobacterium, there
are few reports on role of the TlyA protein with regard to pathogenicity and virulence in
M. leprae.
In M. leprae, it was predicted that the tlyA gene is a part of an operon containing at least
three other genes: the first being tlyA (ML1358), the second being ppnk (ML1359) and the
third being RecN (ML1360), homologous to E.coli recN.2,8 Rahman et al. reported that
purified Rv1694 (tlyA of M. tuberculosis) exhibits a possible hemolytic and RNA methylation
activity in vitro; its amino terminus can bind with the target cell while at the same time the
carboxy terminus can anchor with the host bacterial cell wall, thus facilitating successful
entry of bacilli into the host.7 It is possible that the haemolytic activity might be relevant to
intra-cellular compartments such as phagosomes rather than the cell lysis of erythrocytes and
may have a potential role after successful entry into macrophages by M. tuberculosis.
The over-expression of tlyA (ML1358) was observed in skin biopsies of multibacillary
and reactional (ENL and RR) cases of leprosy by microarray and real-time PCR based studies
TlyA protein of M. leprae
503
(unpublished data, Indian Patents filed No-2012/DEL/2006 and 884/DEL/2007). To explore
the role of tlyA during infection, we characterised the TlyA protein at molecular level by
cloning and its expression in E. coli and tested the serological activity of the protein in sera of
leprosy patients. Sequence homology and distribution of TlyA protein among mycobacteria
was done by BLAST and MSA analysis. A phylogenetic tree was drawn to show the
evolutionary pattern of TlyA among slow-growing intracellular adapted pathogenic and fastgrowing environmental non-pathogenic mycobacteria. Structural features of the protein have
been checked by 3D modeling and trans-membrane prediction. The humoral immune
response to the protein was evaluated by ELISA in different types of leprosy patients.
Methods
ETHICAL APPROVAL
The detailed plan of study was submitted to the Ethical Committee as well as the Scientific
Advisory Committee (SAC) of the JALMA Institute, which approved the assumptions for
human research.
PATIENT CONSENT
Informed written consent was obtained from all subjects enrolled in the study and blood
samples for screening were collected from adults willing to participate in the study.
BLOOD SAMPLE PROCESSING
Blood samples, 5 ml each, were collected aseptically from 80 leprosy patients, across the
spectrum, namely 20 BT/TT, 20 BL/LL, 10 RR and 10 ENL9,10 and 20 healthy controls. The
blood samples from RR and ENL patients were collected during reactions, prior to treatment,
when they came to the Outpatient Department (OPD). These patients were admitted in the
ward of this Institute and given treatment for these conditions. Sera were separated from
blood samples by centrifugation and stored at 2 208C until further use.
PLASMIDS
The cloning vector plasmid pGEMw-T Easy (Promega Madison, WI) and expression vector
pASK-iba-43 (IBA technologies, Germany) were used in the study. An ampicillin resistance
marker was present in both vectors. The expression vector contained a Tet promoter and two
affinity tags for the protein purification (6-His-tag coding sequence and Strep-tag coding
sequence). The cloning and other recombinant DNA technology experiments were carried out
in the Department of Biochemistry [specified and approved by RDT committee], Delhi
University, Delhi.
T lyA GENE AMPLIFICATION AND CLONING
DNA was isolated from skin biopsies of BL/LL patients with bacterial index value of 4þ .
Then, the entire region of tlyA gene (ML1358) was amplified using primer pairs flanked
by restriction enzyme sites with six additional base pairs at extreme 5’ end in both primers
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H. Deval et al.
(P1 5’-GGA TCC GAA TTC GTG GCC CGA CGT GTC CGC G-3’ (Eco RI), P2 5’-GGA
TCC CTC GAG CTG CGG GCC TTC TGC GAG-3’ (Xho I). TlyA-PCR was conducted in a
35 ml mixture with 3U of Taq DNA Polymerase (New England BioLabs) and 10 ng of M.
leprae DNA. The primer concentrations were 1 mM each and the dNTP mix concentration
was 200 mM. The PCR was initiated by a denaturation step at 948C for 5 min, followed by 30
cycles of denaturation (948C, 1 min), annealing (608C, 1 min) and extension (728C, 2 min) and
subsequently, a final extension step (728C, 10 min). The PCR product was cloned into pGEMT easy vector (Promega), and transformed into E. coli XL-1 Blue cells. The clones of tlyA
gene from pGEMw-T were further sub- cloned in pASK-iba-43 expression vector for protein
expression.11
EXPRESSION AND PURIFICATIONI OF RECOMBINANT TLYA PROTEIN
Extracted recombinant plasmids were transformed into E. coli BL21 by heat shock and were
plated on a Luria-Bertani (LB) agar containing ampicillin at 378C overnight. Overnight
incubated colonies were inoculated into a LB broth and was grown for 3 hrs up to the OD 600
of culture reaches 0·7 (late log phase), then 100 ml anhydrous tetracycline (2 mg/ml in
dimethyl formamide; final concentration, 200 mg/liter) was added and growth was continued
in a 378C shaker incubator. The total broth media was centrifuged and the bacterial cell pellet
was dissolved in a binding buffer (10 mM imidazole, 0·3 M NaCl, 0·1 M KCl, 10% glycerol,
0·5% Triton X-100, 50 mM Tris-HCl, pH 7·6). Recombinant TlyA protein (r-TlyA) was
purified from the cell-free supernatant by chromatography on a Ni-NTA agarose column.
After washing the column with 10 mM imidazole in a lysis buffer (50 mM Tris-HCl, pH 7·8,
containing 300 mM NaCl, 100 mM KCl, 10% glycerol and 0·5% Triton X-100 1% v/v), TlyA
protein was eluted with 250 mM imidazole in the lysis buffer.12 The fraction containing
rTlyA protein was dialyzed against a PBS buffer pH 7·5 and purity was examined by 12%
SDS-PAGE. The protein concentration was estimated by Bradford’s method.13,14
ESTIMATION OF MOLECULAR WEIGHT, TOTAL YIELD AND ANTIGENICITY OF
RECOMBINANT PROTEIN
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to
determine the molecular weight and total yield using a gel documentation system (Bio-RAD,
India). To evaluate antigenicity, r-TlyA gene (ML1358) was expressed in E. coli using
pASK-iba-43 expression vector. These E. coli cells were then disrupted by French press to
prepare protein lysates and further His-tagged proteins were purified by Ni-NTA columns.
The purified proteins were finally quantified by Bradford’s reagent.
DETECTION OF ANTIBODIES AGAINST RECOMBINANT TLYA PROTEIN OF
MYCOBACTERIUM LEPRAE
Sero-reactivity of the recombinant TlyA protein was analysed by indirect ELISA using a
modified protocol by Spencer et al.15 Sera from 60 leprosy patients (10 RR, 10 ENL, 20 BL/
LL & 20 BT/TT) and 20 healthy individuals were used to determine the sensitivity and
specificity of r-TlyA. Microtiter plates (96F Maxisorp, Nunc) were coated with 50 ml of
recombinant TlyA antigen (20 ng/ml in 0·2 M carbonate-bicarbonate buffer, pH 9·6) and
incubated overnight at 48C. After blocking bouts in wells with 150 ml of 2% BSA (Sigma
TlyA protein of M. leprae
505
Aldrich, India) in PBS, pH 7·4, the plate was treated with 50 ml diluted serum (1:250 in 1%
BSA in PBS-T, pH 7·4). Following 2 h of incubation at 378C (Widson), 100 ml of horseradish
peroxidase-conjugated rabbit anti-human IgG (1 mg/ml) was added at a dilution of 1:10,000
in 1% BSA in PBS, pH 7·4. After 1 h of incubation at 378C. r-TlyA and antibody binding was
visualised by the 1 x solution of substrate [o-phenylenediamine (Sigma, India) 20 mg tablet in
20 ml solution made up of 4·95 ml of 2·1% citric acid solution, 4·95 ml of 3·5% di-sodium
hydrogen phosphate solution and 30 ml H2O2, in 10 ml distilled water], incubated for 20 min
in the dark: the reaction was stopped with 50 ml of 0·5 M H2SO4 (10%), and the absorbance
was measured at 490 nm (Labsystems, ELISA reader). The plates were washed three times
with phosphate buffer saline-1% Tween 20, pH 7·4 (PBS- T) at each step. Each serum sample
was tested in triplicate. The experiments were performed independently, to ensure the
consistency of findings.
PHYLOGENETIC AND STRUCTURAL ANALYSIS
Distance relationships of TlyA with homologous sequences within mycobacterial species,
other organisms as well as humans were studied. The protein sequence of TlyA was derived
from the Leproma web server (http://genolist.pasteur.fr/Leproma) and BLASTp was
performed through the NCBI server (http://www.ncbi.nlm.nih.gov/BLAST/).16 MSA was
done using Clustal W with the related sequences obtained from BLASTp.17,18 Phenograms
for distance relation analysis within mycobacterial species were generated using free online
programme Phylodendron (http://iubio.bio.indiana.edu/soft/molbio/java/apps/trees/) using
the Neighbour-Joining method.19 A 3D structure of the TlyA protein of M. leprae was
predicted by the protein modeling approach. The three-dimensional structure of a putative
hemolysin from Streptococcus thermophilus was used as a template for homology modeling.
The TlyA structural model was obtained from its amino acid sequence by using SWISS
MODEL [http://swissmodel.expasy.org/] and Protein Homology/analogy Recognition
Engine (PHYRE) [http://www.sbg.bio.ic.ac.uk/phyre/] prediction servers;20,21 Imperial
College Centre for Bioinformatics (UK) and Protmod (protein modeling server), Burham
Institute for Medical Research. The obtained models were classified according to identity
percentages. The VMD 18·6 (Virtual Molecular Dynamics) tool of University of Illinois, was
used for the visualizing protein modeling.
PREDICTION OF TRANS-MEMBRANE ALPHA-HELICES IN PROKARYOTIC
MEMBRANE PROTEINS
The TlyA protein sequence was retrieved from the Leproma server and submitted to the
Dense Alignment Surface (DAS) tool of the Expasy web server (http://www.expasy.org/
tools/) to get the trans-membrane domains of the protein.22,23
STATISTICAL ANALYSIS
The data were analyzed using the Stata-7 statistical software. In order to determine the
sensitivity and specificity of r-TlyA protein, Receiver-operator characteristic (ROC) curves
and the area under the curve (AUC) were used for various classes of leprosy. The performance
of ELISA (TlyA based) was estimated by comparing the AUCs as shown in Table 1.
Sensitivity and specificity were calculated at different selected cut-off values for each curve.
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H. Deval et al.
Table 1: The sensitivity and specificity of TlyA protein for the correct classification of the various groups with cut-off
levels corresponding to a calculated sensitivity using receiver operating curve analysis
Category of patients
Cut-Off values
Sensitivity
Specificity
AUC
Correct Classification
P value
0·272
0·356
0·301
0·222
90·00%
100%
70·00%
50·00%
95·00%
100%
95·00%
80·00%
0·920
1·000
0·860
0·601
93·33%
100%
82·50%
65·00%
,0·001
,0·001
,0·001
¼0·161
Reactional (RR)
Reactional (ENL)
BL/LL
BT/TT
AUC ¼ Area under ROC curve
Note: M. leprae generates an immunological imbalance between cell mediated (CMI) and humoral immune
responses in patients and therefore, leprosy has been classified into different classes and appropriate individual cutoffs were estimated in the ROC in each class of leprosy.
Results
SERO-REACTIVITY OF THE RECOMBINANT T lyA PROTEIN (ML 1358) BY ELISA
Recombinant TlyA (r-TlyA) protein was obtained successfully from cloning vector plasmid
pGEMw-T Easy (Promega Madison, WI) and expression vector pASK-iba-43 (IBA technologies,
Germany). The estimated yield of the recombinant protein was 777 ng/ml. The molecular weight
of rTlyA protein was found to be 32 kDa on 12·5% SDS-PAGE analysis (Figure 1).
The r-TlyA showed higher sero-reactivity in serum samples of ENL and RR (reactional
cases) followed by the BL/LL and BT /TT cases (Table 1 & Figure 2).
Sero-reactivity was statistically significant in lepromatous and reactional cases (P ,
0·001) and non-significant for BT/TT cases of leprosy (P ¼ 0·182). The cut-off values are
shown for each group (RR, ENL, BL/LL, BT/TT) estimated by the ROC curve analysis (i.e.
the empirical point that maximizes sensitivity and specificity between leprosy patients and
healthy controls) (Table 2 & Figure 3).
PHYLOGENETIC AND STRUCTURAL ANALYSIS OF T lyA PROTEIN
The phylogenetic analysis of the TlyA demonstrated that the protein is conserved among the
mycobacterial species and has very close homology (80% – 90%) with other mycobacterial
1
2
3
4
5
6
7
8
9
98,400 kDa
66,000 kDa
43,000 kDa
32 kDa
29,000 kDa
20,100 kDa
14,300 kDa
Figure 1: The SDS-PAGE results indicative of 32 kDa rTlyA protein of M. leprae.
TlyA protein of M. leprae
507
0·5
Absorbance at 490 nm
0·4
0·3
0·2
0·1
0
Healthy
BT/TT
LL/BL
RR
ENL
Figure 2: The results of ELISA using the rTlyA protein as antigen with a panel of sera from leprosy patients (BT/TT,
BL/LL, RR and ENL cases). The horizontal bar indicates cut off A490 (optical density) for each case group. The
p-value for each graph was calculated by the Mann-Whitney test.
species including M. tuberculosis, M. paratuberculosis and M. ulcerans (Table 3) and nonsignificant homology with the human genome.
It was observed that TlyA is evolutionarily distributed among mycobacteria by a vertical
transfer phenomenon (Figure 4).
The specific protein domains were selected by the MSA method (Figure 5).
These domains are present in well known, slow growing, intra-cellular adapted
pathogenic species of mycobacteria (M. leprae, M. tuberculosis, M. bovis, M. avium,
M. ulcerans, M. para-tuberculosis) and are absent in non-pathogenic, fast growing,
environmental mycobacteria (M. smegmatis, M. gilvum, M. vanbaaleni) except M. abscessus
(Figure 6A, B, C).
The 3D structure was made by the PHYRE bio-informatics server for homology
modelling to predict the structure of the protein and to show the specific domain present in the
TlyA protein (Figure 7).
Trans-membrane region prediction analysis of TlyA protein by the DAS method showed
that the three stretches of seven, 13 and 21 amino-acids (trans-membrane alpha helices) were
obtained on the basis of two cut-offs (strict and loose) as plotted in Table 4.
Table 2: The Receiver operating characteristics (ROC) curves for rTlyA protein for the diagnosis of BT/TT, BL/LL,
RR and ENL cases of leprosy. Top 20 sera of higher absorbance from each group of leprosy patient were analysed
Area Under the ROC curve
Std. Error
95% confidential Interval
p value
ENL
RR
BT/TT
BL/LL
1
0
1·000–1·000
,0·0001
0·92
0·07242
0·7780–1·062
0·0002218
0·6013
0·09207
0·4208– 0·7817
0·2733
0·86
0·03894
0·7444–0·9734
,0·0001
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H. Deval et al.
ROC of LL/BL cases:ROC curve
100
75
75
Sensitivity
Sensitivity
ROC of ENL cases:ROC curve
100
50
25
50
25
0
0
0
20
40
60
80
100% - Specificity%
100
0
40
60
80
100% - Specificity%
100
ROC of BT cases:ROC curve
100
100
75
75
Sensitivity
Sensitivity
ROC of RR cases:ROC curve
20
50
25
50
25
0
0
0
20
40
60
80
100% - Specificity%
100
0
20
40
60
80
100% - Specificity%
100
Figure 3: The Receiver operating characteristics (ROC) curves for rTlyA protein for the diagnosis of BT/TT, BL/LL,
RR and ENL cases of leprosy. Top 20 sera of higher absorbance from each group of leprosy patient were analysed.
Discussion
A tlyA homologue is present in many pathogenic bacteria and is involved in adhesion of the
pathogen to host cells or tissues. TlyA promotes virulence properties in many pathogens and
functions as a pore-forming hemolysin in Serpulina hyodysenteriae and Helicobacter
pylori.7,24 – 26 Moreover, M. tuberculosis and M. leprae and many other pathogenic
mycobacterial species also possess the tlyA gene. Although, M. tuberculosis and M. leprae
evolved from a common ancestor, M. leprae possesses fewer genes.27 Genes conserved
between the two species are hence considered important for pathogenicity and virulence.
Table 3: The organisms which show maximum homology with the amino acid sequence of TlyA
Gene Identity
Name
Function
Sequence Identity (at protein level)
ML 1358
TlyA
Contact dependent hemolytic activity,
pore formation, involved in virulence
M. ulcerans - 81%
M. tuberculosis - 78·4%
M. paratuberculosis - 79%
M. avium - 78%
Serpulina hyodysenteriae - 34·9%
H. sapiens - Not Significant
TlyA protein of M. leprae
100
39
44
509
Mleprae JALMA
Mleprae TN
Mulcerans Agy 99
Mbovis AF2122/97
95
100 Mtuberculosis H37Rv
Mparatuberculosis K10
100
Mavium 2285
Msmegmatis MC2155
Mgilvum Spyr1
72
99
Mvanbaalenii PYR1
Mabscessus PAP127
0.05
Figure 4: The phylogenetic tree showing the distance relationship analysis of TlyA within mycobacteria.
Rahman et al. reported that TlyA (Rv1694) of M. tuberculosis possesses hemolytic activity
by binding with and oligomerizing into host cell membranes.7 Therefore, it raised the first
question regarding the stage of TlyA to play a beneficial role for the bacterium. Secondly,
with regard to incidence of the disease, the tlyA gene was found to be up regulated. These
reasons together suggest that controlling the expression of TlyA is of prime importance to the
bacterium. This is logical, as most pathogenic bacteria control the expression of their
virulence factors rather tightly.
Analysis of expression of hypothetically virulence-associated genes of M. leprae was
done directly in human biopsies using an indigenously designed partial DNA chip of M.
leprae at our laboratory (Indian Patent application no. 2012/DEL/2006 and 884/DEL/2007).
TlyA (ML 1358) was the one found to be consistently over-expressed during active infection.
For the first time, we have characterised M. leprae TlyA protein (a pore forming cytotoxin or
hemolysin protein) at the molecular level and established the serological importance in
leprosy patients for understanding its role during infection. We have cloned the tlyA gene in
expression vector pASK iba-43 in E. coli, as a fusion protein of 32 kDa. Cossu et al. reported
infection of THP-1 macrophages with M. avium subsp. paratuberculosis where tlyA was
5-fold up-regulated. The tlyA of M. avium subsp. paratuberculosis has 79% identity with
M. leprae tlyA, predicted to act as a virulence factor after entry of the mycobacterium into
host cells while the regulation and expression of these genes are linked to the environment.28
The sero-reactivity of the rTlyA protein as shown in Table 1 revealed that its role is
significant in various clinical conditions (ENL/RR) of leprosy. The mean OD was 0·1821 in
healthy controls, 0·3882 in ENL, 0·3094 in RR and 0·3040 in BL/LL cases, respectively. The
sensitivity and specificity were found to be maximum for ENL cases (100%); thereby
suggesting that it can detect ENL cases. The mean OD of BL/LL cases differs significantly
[P , 0·001, cut-off ¼ 0·3005, sensitivity - 70% & specificity - 95%] from BT/TT cases.
rTlyA responded least to BT/TT cases (P ¼ 0·160, cut-off - 0·2220). However, 65% BT/TT
cases could be correctly classified. This protein showed high sero-reactivity for RR and ENL
cases followed by BL/LL cases and it showed lowest sero-reactivity to the BT/TT cases of
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H. Deval et al.
Figure 5: The Multiple sequence alignment (MSA) of TlyA among mycobacteria at amino acid level: The deduced
amino acid sequences of TlyA homologues from M. gilvum, M. vanbaaleni, M. smegmatis, M. bovis, M. tuberculosis,
M. ulcerans, M. paratuberculosis, M. avium and M. abscessus were aligned by Clustal W. An asterisk indicates that
the amino acids are identical in all ten sequences; a dot indicates that they are identical in four of the sequences, and a
colon indicates that although fewer than four sequences are identical, there are conserved amino acids in all seven
sequences. Arrows shows the specific protein domains in pathogenic mycobacteria.
TlyA protein of M. leprae
511
D
C
A
B
(A)
Normal 3D structure
(B)
3D structure with
specific domains (A, B, C and D),
indicated by red colour
A
B
58
D
6
22
6
24
9
25
26
9
9
TD
LS
LE
A
LE
G
A
VT
G
D
D
21
D
D
VA
LT
V
TT
AI
(C)
C
18 19
9 2
M
RT
W
LG
TE
47
V
Q
KG
1
Figure 6: The analysis of TlyA protein 3D structure. (A) Homology derived model of TlyA prepared using Swissmode. (B) Schematic representation of TlyA protein of M. leprae with specific protein domains (A, B, C and D).
(C) The line diagram representing the specific protein domains (A, B, C, D) present in TlyA in slow growing
intracellular adapted pathogenic mycobacteria deduced from the MSA.
leprosy. The protein showed only 70% positivity in pauci-bacillary and BT/TT cases. Thus, it
is not a potential tool for sero-diagnosis of leprosy, but its link to virulence could make it a
potential drug target.29 The ROC curve analysis demonstrated that the assay was more
sensitive to ENL cases of leprosy followed by RR cases in comparison to BL/LL and BT/TT
(without reaction). High seropositivity of rTlyA in the BL/LL and the reactional (RR and
ENL) cases suggests that M. leprae is responding to the environmental stress created by the
active immune state of the body and thus protecting itself. This suggests that TlyA might be a
possible marker during active infection. Leprosy reactions are a challenging problem because
they increase morbidity due to nerve damage even after the completion of MDT. Some
reports have confirmed the presence of high levels of pro-inflammatory cytokines such as
TNF-a, IL-6 and IL-1b in the sera of ENL patients.30 During RR reactions, serum cytokines
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H. Deval et al.
“DAS” TM-segment prediction
“DAS” profile score
3
2
1
0
0
50
100
150
200
250
300
Query sequence
loose cutoff
strict cutoff
Figure 7: The analysis of TlyA for the prediction the trans-membrane domains by the DAS method. The three
stretches of 7, 13 and 21 amino-acids were obtained on the basis of two cut offs (strict and loose). There are two cutoffs indicated on the plots: a ‘strict’ one at 2·2 DAS score, and a ‘loose’ one at 1·7. The hit at 2·2 is informative in
terms of the number of matching segments, while a hit at 1·7 gives the actual location of the trans-membrane segment.
The segments reported in the ‘FT’ records of the Swiss Prot database are marked at 1·0 DAS score (‘FT lines’).
did not show a consistent pattern.31 However, the TNF-a level was elevated in RR patients,
irrespective of treatment. Considering the study of Rehman et al,26 M. tuberculosis TlyA
directly or indirectly suppresses host protective immune responses like Th1 and Th17.
Various cytokines work as growth factors (IL-12p70 and IL-23), differentiation (IL-6 and
TGF-b) and induction (IL-1b, IL-10, and TNF-a) for Th1 and Th17 responses. Similarly,
immune response suppression by M. leprae TlyA could be suggested by these findings in
reactional cases.25 TlyA can orchestrate intra-cellular survival by modulating the
phagolysosome maturation especially, during the initial stages of establishment of infection,
while other pathogenic factors may come into play at a later point of time for the successful
establishment of disease.32
Table 4: The potential trans-membrane segments obtained by the DAS curve
Potential trans-membrane segments
Start
Stop
Length
Cut off
46
150
155
52
170
167
7
21
13
1·7
1·7
2·2
These curves were obtained by pairwise comparison of the proteins in the test set in ‘each against the rest’ fashion
(Figure 7).
TlyA protein of M. leprae
513
33
Conservation of the protein was observed through MSA at genus level while the specific
protein domains were found only in slow growing intra-cellular adapted pathogenic species
of mycobacteria (M. leprae, M. tuberculosis, M. bovis, M. avium M. paratuberculosis,
M. ulcerans) and were absent in fast growing non-pathogenic environmental mycobacterial
species. The presence of specific protein domains of TlyA in slow growing mycobacterial
pathogen indicates its role in facilitating the internalization of mycobacteria inside the host
cells by modulation of the phagolysosome maturation pathway immediately after entry into
macrophages.32
TlyA showed a different evolutionary pattern for highly pathogenic slow growing and
non-pathogenic fast growing mycobacterial species. M. abscessus showed a different clade
from both pathogenic and non-pathogenic mycobacteria as shown in Figure 6. The virulent
genes are believed to be specific for pathogenic bacteria, however, non-pathogenic bacteria
may also contain these virulence factors either in mutated or silent form. In the present study,
the phylogenetic tree of TlyA indicates its active presence in pathogenic mycobacteria and
further structural analysis indicated specific domains of TlyA among pathogenic species.
Virulence genes have increased translational efficiency in pathogenic bacteria compared with
non-pathogenic species. Similarly, expression was found for TlyA in our DNA chip
experiments. These findings correlate with the work of Houben et al. on another
mycobacterial gene (pknG).34
Rahman et al.26 reported that TlyA assists M. tuberculosis survival inside the mouse
infection model by inhibiting Th1 cytokines (IL-12 and IFN-g) as well as by autophagy. They
also confirmed that the deletion of the tlyA in wild type M. tuberculosis H37Rv impedes its
pathogenicity in mice. The 3D structure of the TlyA protein was drawn to show the specific
protein domains. The seven, 21 and 13 amino acid-long stretches of hydrophobic residues on
the TlyA protein were predicted by trans-membrane analysis software, which represents the
actual trans-membrane location of this protein and hence, predicts that the protein is
membrane bound. Recently, another study by Kumar et al.29 suggests that the mycobacterial
tlyA gene product is localized to the cell wall without signal sequences and the tlyA gene
appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, rather
than M. smegmatis, M. vaccae, M. kansasii, etc. The DAS tool of expasy server, predicted the
trans-membrane helices in integral membrane proteins, composed of stretches of 15– 30
predominantly hydrophobic residues.22,23 The M. leprae cell wall, cell membrane and
secreted proteins would be the first among many such proteins that evoke an antibody
response after entry into the host.29 TlyA protein could be one among many of the cell
membrane proteins to stimulate the host immune system. Overall in-depth study is needed on
TlyA protein for designing a potential target for tuberculosis and/or leprosy vaccines and
drugs in future.
Conclusions
The rTlyA gene could be an essential virulence factor, having an important role during disease
progression and survival of M leprae in human cells. TlyA protein could be a possible
bio-marker for multi-bacillary and reactional cases (ENL and RR), but is not a potential tool
for serodiagnosis of paucibacillary cases and BT/TT cases. The bio-informatics approaches
have established its conserved nature among mycobacteria. TlyA showed a different
evolutionary pattern for highly pathogenic and slow growing and other non-pathogenic
514
H. Deval et al.
mycobacterial species, except M. abscessus which showed a different origin. The specific
protein domains were found in slow growing, well adapted, intra-cellular pathogenic
mycobacteria and absent in fast growing, non-pathogenic environmental mycobacteria except
M. abscessus which is both a pathogenic and a rapidly growing mycobacterium. The transmembrane prediction analysis indicated the nature and location of TlyA as a trans-membrane
protein containing three stretches of seven, 13 and 21 amino-acids or trans-membrane alpha
helices. Thus, in future, it may become an important target to check the growth and survival
of M. leprae (viability marker) inside the macrophages. The TlyA protein could be a potential
target for designing tuberculosis and/or leprosy vaccines and therapies.
Authors’ contributions
This article is part of the Ph.D. thesis work of HD who has conceptualised the idea, designed
the study, compiled the data and wrote the article. VMK is the Guide and KK is the Co-guide
of HD. DSC, AKT, RKG were involved in molecular work and RK provided the samples
from leprosy patients; AV and TH did the immunological part of the work. VSY of the
Biostatistics division did the statistical analysis of data. TH edited the article at each stage.
Acknowledgements
Hirawati Deval received an CSIR-UGC-fellowship for his Ph.D. The authors acknowledge
the staff of OPD and Ward of this Institute and all those who helped in the study.
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