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Original article

Molecular mimicry between Mycobacterium leprae proteins (50S ribosomalprotein L2 and Lysyl-tRNA synthetase) and myelin basic protein A possible

mechanism of nerve damage in leprosy

Q3 Itu Singh a1 Asha Ram Yadav b Keshar Kunja Mohanty a Kiran Katoch c Prashant Sharma bBishal Mishra a Deepa Bisht b UD Gupta d Utpal Sengupta a

a Department of Immunology National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR) Tajganj Agra Indiab Department of Biochemistry National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR) Tajganj Agra India

c Clinical Division National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR) Tajganj Agra Indiad Animal Experimentation Laboratory National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR) Tajganj Agra India

Received 8 August 2014 accepted 23 December 2014

Abstract

Autoantibodies against various components of host are known to occur in leprosy Nerve damage is the primary cause of disability associatedwith leprosy The aim of this study was to detect the level of autoantibodies and lympho-proliferative response against myelin basic protein(MBP) in leprosy patients (LPs) and their correlation with clinical phenotypes of LPs Further probable role of molecular mimicry in nervedamage of LPs was investigated We observed significantly high level of anti-MBP antibodies in LPs across the spectrum and a positive sig-nificant correlation between the level of anti-MBP antibodies and the number of nerves involved in LPs We report here that 4 B cell epitopes ofmyelin A1 and Mycobacterium leprae proteins 50S ribosomal L2 and lysyl tRNA synthetase are cross-reactive Further M leprae sonicatedantigen hyperimmunization was responsible for induction of autoantibody response in mice which could be adoptively transferred to naive miceFor the first time our findings suggest the role of molecular mimicry in nerve damage in leprosycopy 2015 Published by Elsevier Masson SAS on behalf of Institut Pasteur

Keywords Leprosy Autoimmunity Autoantibodies Myelin basic protein Molecular mimicry

1 Introduction

Leprosy is primarily a granulomatous disease of the pe-ripheral nerves and skin Nerve damage is the major cause ofdisability associated with leprosy Mycobacterium leprae (M

leprae) is remarkably non-toxic organism and most of thetissue damage and neural damage in leprosy is known to bedue to the hosts immune response to M leprae antigens [1] Ithas also been reported that this immune response by the hostmay lead to auto-antibody response against nerve antigens [2]and could be responsible for demyelination

Elevated level of anti-myelin basic protein (MBP) auto-antibodies has been reported in multiple sclerosis (MS) anautoimmune disease of CNS [34] and autism [5] Presence ofanti-MBP autoantibodies in MS patients showed site-specificproteolytic cleavage of the MBP molecule that might beresponsible for pathological destruction of the myelin sheath[6] Earlier it was reported that MBP is one of the constituentsin the circulating immune complexes (CIC) of lepromatous

Corresponding author Present address Stanley Browne Laboratory The

Leprosy Mission Community Hospital Nand Nagri Delhi 110093 India Tel

thorn91 9212761651 fax thorn91 562 2331755Q1

E-mail addresses itusinghgmailcom (I Singh) keshar63yahoocom

(KK Mohanty) prashant_jalmarediffmailcom (P Sharma)

bishalatulmpigmailcom (B Mishra) abd1109rediffmailcom (D Bisht)

senguptautpal2013gmailcom usengupta2002yahoocom (U Sengupta)1 Present address Stanley Browne Laboratory The Leprosy Mission

Community Hospital Nand Nagri Delhi 110093 IndiaQ2

Please cite this article in press as Singh I et al Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA

synthetase) and myelin basic protein A possible mechanism of nerve damage in leprosy Microbes and Infection (2015) httpdxdoiorg101016

jmicinf201412015

Microbes and Infection xx (2015) 1e11wwwelseviercomlocatemicinf

+ MODEL MICINF4240_proof 8 January 2015 111

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646566676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128

httpdxdoiorg101016jmicinf201412015

1286-4579copy 2015 Published by Elsevier Masson SAS on behalf of Institut Pasteur

leprosy (LL) patients and was suggested that MBP releasedafter nerve damage caused by M leprae was responsible forproduction of anti-MBP antibodies which further may lead todemyelination and nerve damage [7] Molecular mimicry maybe the underlying mechanism for production of autoantibodiesand might be responsible for pathological destruction inleprosy [89] Molecular mimicry is defined as immunologicalcross-reactivity between infectious agent and host antigenicdeterminants [10] In the recent past Vardhini et al (2004)[11] pointed out that molecular mimicry between mycobac-terial proteins (ferredoxin-NADP-reductase and a conservedmycobacterial membrane protein) with myelin P0 (a proteinthat aids in compacting myelin through homotypical in-teractions) might be responsible for demyelination Thoughthe molecular mechanism leading to axonal degeneration andor demyelination in leprosy have rarely been explored indetail the present data indicate that molecular mimicry mightresult in immune mediated degeneration of myelin thatcontribute to nerve damage leading to deformities and per-manent loss of nerve function in leprosy patients

Hence the purpose of our study was to find out presence ofanti-MBP antibodies and lymphocyte proliferative response toMBP in leprosy patients and their correlation if any with thedisease spectrum We also aimed to investigate the probablerole of molecular mimicry for such an immune responseleading to nerve damage

2 Research design

21 Proteins

MBP from bovine brain was procured from SigmaeAldrichUSA Non-irradiated M leprae bacilli derived from Armadilloliver was procured fromColorado State University Fort CollinsColorado USA (under WHO contract number NIH-NO1-AI-25469 Leprosy Research Support)M leprae sonicated antigen(MLSA) was prepared by sonication of bacilli according to thepublished protocol [12] The protein concentration of MLSAwas assayed by Bradfords method (Sigma USA) [13]

22 Study subjects

221 Patients and healthy controlsOne hundred twenty four clinically diagnosed (based on

cardinal signs) leprosy patients attending the outpatientdepartment of National JALMA Institute for Leprosy andOther Mycobacterial Diseases (NJIL amp OMD) ICMR Agrawere recruited in the study Leprosy patients were classifiedaccording to the classification laid by Ridley and Jopling scale[14] which included 20 borderline tuberculoid (BT) 21borderline borderline (BB) 23 borderline lepromatous (BL)29 lepromatous leprosy (LL) 21 type 1 reaction (T1R) 10erythema nodosum leprosum (ENL) Blood samples from 43healthy controls (HC) individuals with no signs and symp-toms of leprosy and any other illness were taken as controlThis study was approved by the Institutional Ethical Com-mittee and all participants consented to the study

222 Experimental animalsOutbred female New Zealand white rabbits (weighing

about 1 kg and 3e5 months old) and inbred strains of femaleBALBc mice (6e8 weeks weighing 20 plusmn 2 gms) were ob-tained from the Laboratory Animal Division of Central DrugResearch Institute (CDRI) Lucknow All animals weremaintained under standard conditions in the Department ofAnimal Experimentation of NJIL amp OMD Agra

All animal experiments included in this manuscript wereapproved by Institutional Animal Ethical Committee and wefollowed the guidelines laid down by Animal Research EthicsBoard at our institute

23 Production of polyclonal rabbit antibodies to MLSAand MBP

Rabbits were hyperimmunized with 250 mg of proteinconcentration of MLSA as well as 250 mg of host protein MBPto produce polyclonal antibodies against these proteins Boththe proteins were emulsified in Freunds Incomplete Adjuvant(IFA) (Sigma USA) and 02 ml emulsion was injected intra-dermally (id) The booster doses were given weekly up to 8thweek Control group of rabbits were administered with theemulsion of normal saline with IFA at the same time intervalsBlood samples (5 ml each time) were drawn from marginal earvein before the booster doses with the antigen emulsion

24 MLSA-hyperimmunized mice

Mice of the control (n frac14 10) and experimental (n frac14 15)group were hyperimmunized with saline and 25 mg of proteinconcentration of MLSA emulsion respectively as previouslydescribed [8]

25 Protocol for transfer of auto-reaction by adoptivetransfer

The adoptive transfer experiment was carried out as describedin Singh et al 2012 [8] Briefly adoptive transfer was done inthree groups of mice by inoculating 100 ml (1X107 cellsml) ofcell suspension In the first control group ofmice (nfrac14 5) mixtureof suspensions of cells of splenocytes and lymph nodes cells inisotonic saline obtained fromnaivemicewere intravenously (iv)administered in tail vein Second experimental group (n frac14 5)were similarly iv administered with immune cells (whole cells)acquired from MLSA hyperimmunized mice Third group ofmice (n frac14 5) were iv inoculated with nylon wool separated Tcells [15] obtained from MLSA hyperimmunized mice

26 Estimation of auto-antibodies against myelin basicprotein by ELISA

261 Human seraELISAwas performed by using the protocol with some mod-

ifications as described earlier [8] Briefly each well of microtitreplates (NuncDenmark)was coatedwith 5mgml ofMBP (SigmaUSA) for 18 h at 4 CWells were blocked with 3BSA (Sigma

2 I Singh et al Microbes and Infection xx (2015) 1e11

Please cite this article in press as Singh I et al Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA

synthetase) and myelin basic protein A possible mechanism of nerve damage in leprosy Microbes and Infection (2015) httpdxdoiorg101016

jmicinf201412015

MICINF4240_proof 8 January 2015 211

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

USA) in PBS for 1 h Serum (1100) was added in duplicate wellsfollowedby incubationat37 Cfor2hAfter3washings110000dilutedanti-human IgGperoxidase conjugated (SigmaUSA)wasadded into each well After 1 h incubation plates were washed 3times and 100 ml of substrate solution [orthophenylene diamine(OPD) tabletof05mgmland02mlmlH2O2]wasaddedandwaskeptat roomtemperature for30min indarkReactionwas stoppedbyadding50mlwell of 3MH2SO4 (stop solution) and absorbancewas recordedat492nMusingSpectramax-M2Reader (MolecularDevices Sunnyvale CA USA)

262 Experimental animal seraWe quantified the level of autoantibodies against MBP in

MLSA hyper-immunized rabbits and mice seraplasma usingabove mentioned ELISA protocol with minor differences asfollows e

For rabbit serum peroxidase conjugated anti-rabbit IgGwas used as secondary antibody

For mouse plasma fifty fold diluted mouse plasma wasused for reaction and peroxidase conjugated anti-mouse IgGwas used as secondary antibody

27 Inhibition of binding of rabbit anti-MBPautoantibodies to MBP by leprosy sera

Polystyrene microtitre plates were coated with MBP asdescribed in previous section Fifty microliter of 1500 dilutedMBP hyper-immunized rabbit serum was added to each welland incubated for 1 h at 37 C Then 50 ml of three differentleprosy patients sera were added at five different dilutions (15110 120 140 and 180) and incubated for 1 h at 37 Cfollowed by three washings 100 ml of peroxidase conjugatedanti-human IgG (110000) (Sigma USA) was added to eachwell After incubation for 1 h at 37 C plates were washed for3 times color was developed and absorbance was taken asdescribed in previous section

Fig 1 (A) Levels of antibodies against MBP in leprosy patients across the spectrum and healthy controls Dotted horizontal line represents the cut off OD value

(mean OD value of HCs thorn 2SD) Smooth line with horizontal lines represents the mean OD value with SD of each group Each dot represents the OD value at

492 nm obtained from each individual (BeG) correlation between anti-MBP antibodies level and number of nerves involved in (B) TTBT (C) BB (D) BL (E) LL

(F) T1R (G) ENL (H) Inhibition of binding between anti-MBP rabbit sera and MBP antigen with leprosy patients sera (A p value lt 00001 One way ANOVA and

post test Dunnetts multiple comparison test p value lt 00001 p value lt 0001 p value lt 005 BeG Spearmans correlation test)

3I Singh et al Microbes and Infection xx (2015) 1e11

Please cite this article in press as Singh I et al Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA

synthetase) and myelin basic protein A possible mechanism of nerve damage in leprosy Microbes and Infection (2015) httpdxdoiorg101016

jmicinf201412015

MICINF4240_proof 8 January 2015 311

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

USA) in PBS for 1 h Serum (1100) was added in duplicate wellsfollowedby incubationat37 Cfor2hAfter3washings110000dilutedanti-human IgGperoxidase conjugated (SigmaUSA)wasadded into each well After 1 h incubation plates were washed 3times and 100 ml of substrate solution [orthophenylene diamine(OPD) tabletof05mgmland02mlmlH2O2]wasaddedandwaskeptat roomtemperature for30min indarkReactionwas stoppedbyadding50mlwell of 3MH2SO4 (stop solution) and absorbancewas recordedat492nMusingSpectramax-M2Reader (MolecularDevices Sunnyvale CA USA)

262 Experimental animal seraWe quantified the level of autoantibodies against MBP in

MLSA hyper-immunized rabbits and mice seraplasma usingabove mentioned ELISA protocol with minor differences asfollows e

For rabbit serum peroxidase conjugated anti-rabbit IgGwas used as secondary antibody

For mouse plasma fifty fold diluted mouse plasma wasused for reaction and peroxidase conjugated anti-mouse IgGwas used as secondary antibody

27 Inhibition of binding of rabbit anti-MBPautoantibodies to MBP by leprosy sera

Polystyrene microtitre plates were coated with MBP asdescribed in previous section Fifty microliter of 1500 dilutedMBP hyper-immunized rabbit serum was added to each welland incubated for 1 h at 37 C Then 50 ml of three differentleprosy patients sera were added at five different dilutions (15110 120 140 and 180) and incubated for 1 h at 37 Cfollowed by three washings 100 ml of peroxidase conjugatedanti-human IgG (110000) (Sigma USA) was added to eachwell After incubation for 1 h at 37 C plates were washed for3 times color was developed and absorbance was taken asdescribed in previous section

Fig 1 (A) Levels of antibodies against MBP in leprosy patients across the spectrum and healthy controls Dotted horizontal line represents the cut off OD value

(mean OD value of HCs thorn 2SD) Smooth line with horizontal lines represents the mean OD value with SD of each group Each dot represents the OD value at

492 nm obtained from each individual (BeG) correlation between anti-MBP antibodies level and number of nerves involved in (B) TTBT (C) BB (D) BL (E) LL

(F) T1R (G) ENL (H) Inhibition of binding between anti-MBP rabbit sera and MBP antigen with leprosy patients sera (A p value lt 00001 One way ANOVA and

post test Dunnetts multiple comparison test p value lt 00001 p value lt 0001 p value lt 005 BeG Spearmans correlation test)

3I Singh et al Microbes and Infection xx (2015) 1e11

Please cite this article in press as Singh I et al Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA

synthetase) and myelin basic protein A possible mechanism of nerve damage in leprosy Microbes and Infection (2015) httpdxdoiorg101016

jmicinf201412015

MICINF4240_proof 8 January 2015 311

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

28 Effect of MBP on lymphocyte proliferation ofleprosy patients and healthy individuals

Lymphocyte proliferation assay was done with somemodifications as described earlier [8] Cultures were set intriplicate with or without 20 mgml MBP in culture mediuminto 96 well flat bottom culture plates (Nunc Denmark) andincubated for 5 days in a humidified CO2 incubator (FormaScientific Inc USA) at 37 C with 5 CO2 in air Phyto-haemagglutinin (PHA) was included in this assay as positivecontrol After 5 days cells were pulsed with 1 mCiwell of[3H] thymidine and incubated for 18 h The cells were thenharvested on filter mats using Skatron cell harvester Theradioactivity incorporated into the DNA was determined byliquid scintillation counting (LKB Wallac RackbetaFinland)

29 Characterization of cross-reactive proteins of MLSAand MBP

Denatured proteins of MLSA and MBP (30 mglane) wereelectrophorezed by SDSePAGE [16] using 10 resolving gel(Bio-Rad Laboratories USA) and blotted [17] to nitrocellu-lose membrane (NCM) (Bio-Rad Laboratories USA) Two-

dimensional PAGE isoelectric focusing (IEF) was carriedout employing the method of Gorg et al (2000) [18] IPGstrips (Bio-Rad Laboratories USA) of pH 4e7 for MLSA pH3e10 for MBP and length 7 cm were loaded with 100 mg ofprotein samples (MBPMLSA) Proteins were separated insecond dimension in vertical 10 SDSePAGE gels followedby transfer to NCM

Blotted NCM was washed with PBS and blocked with 3BSA (Sigma USA) for 1 h at RT with gentle shaking thenprobed with 150 diluted MLSA-hyperimmunized rabbitsera while NCM of separated proteins of MLSA was probedwith 150 diluted MBP-hyperimmunized rabbit sera Theseantibody-treated NCMs were incubated overnight at 4 CNCMs were then washed for 3 times with PBS containing005 Tween-20 and incubated with 110000 dilutedperoxidase conjugated anti-rabbit IgG (Sigma USA) for 1 hAfterwards NCMs were washed for 5 times with PBS-T andantigen antibody reactivity were visualized by the colordevelopment with diaminobenzedine (Sigma USA) solu-tion Image was captured by Chemidoc (Bio-Rad Labora-tories USA) Data of 1-D western blot was analyzed byusing Quantity One Software (Bio-Rad USA) while the dataof 2-D blot was analyzed by PD Quest Software (Bio-RadUSA)

Fig 2 (A) Levels of lymphoproliferation in the presence of MBP in leprosy patient across the spectrum (Box and Whiskers graph) Each bar represents the

minimum to maximum values with median as the horizontal line and SD as error bars thorn Sign in each bar represents the mean value Dotted line represents the

SI frac14 2 (p frac14 00011 One way ANOVA test) and unpaired t test (two tailed) was used to compare HC with each group of leprosy p lt 0001 p lt 005) (B)

Levels of antibodies against MBP in MLSA hyperimmunized rabbit sera at different time intervals Each dot with error bar represents the mean OD value with SD

(C) Comparison in levels of autoantibodies against MBP in plasma of MLSA hyperimmunized female BALBc mice pre-immunized mice and control mice Each

dot represents individual OD obtained from mouse plasma Solid horizontal lines with error bars represent mean OD with SD (D) Level of antibodies against MBP

in adoptively transferred naiumlve female BALBc mice Each dot represents the individual OD Horizontal line with error bars represents mean OD value with SD (B

p lt 00001 Unpaired t test (two tailed) C D p lt 00001 p lt 0001 One way ANOVA and post test Bonferroni multiple comparison test)

4 I Singh et al Microbes and Infection xx (2015) 1e11

Please cite this article in press as Singh I et al Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA

synthetase) and myelin basic protein A possible mechanism of nerve damage in leprosy Microbes and Infection (2015) httpdxdoiorg101016

jmicinf201412015

MICINF4240_proof 8 January 2015 411

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

210 Identification of cross-reactive proteins of MLSAand MBP by MALDI TOF analysis

In-gel digestion with trypsin [19] was done with slightmodification Protein spots of interest were picked up fromcoomassie-blue stained 2-D gels using spot picker (ModelInvestigator ProPic Genomic Solutions Ltd UK) In-gelautomated robotic digestion with trypsin (Promega MS grade)of these picked proteins was done by using protein digester(Model Investigator ProPrep Genomic Solutions Ltd UK)Extracted peptides were desalted and concentrated using Zip-TipC18 (Millipore USA) according to manufacturers protocoland applied to an AnchorChip (Bruker Daltonik GmgH Leip-zig Germany) with 2 ml of matrix (saturated solution of a-cyano-4- hydroxycinnamic acid (HCCA) formed in 50 ACN

and02TFA)Mass spectra of digested peptideswere acquiredby Autoflex II TOFTOF50 (Bruker Daltonik GmgH LeipzigGermany) in positive reflectron mode and in detection range of500e3000mz Mass spectra of digested peptides were analysedusing Mascot Wizard program (Matrix Science Ltd LondonUnited Kingdom httpwwwmatrixsciencecom)

Peptidemassfingerprint of cross-reactive protein ofMBPwithanti-MLSA rabbit sera was submitted to Mascot search engineand search parameters used for the identification were peptidemass toleranceplusmn30 ppm peptide charge state 1thorn andmaximummissed cleavages 1 However search parameters used for theidentification of the cross-reactive protein of MLSA (z30 kDaprotein) were peptide mass tolerance plusmn 150 ppm maximummissed cleavages 2 MSMS ion search was done by submittingdifferent peaks (masse 1179769) of the cross-reactive protein of

Fig 3 Reactivity of anti-MLSA rabbit sera with MBP (AeD) anti-MBP rabbit sera with MLSA (EeH) (A) Protein profile of MBP on SDS-PAGE gel stained with

coomassie-blue (B) Western blotting pattern of reactivity of anti-MLSA rabbit sera with MBP Lane 1 Molecular weight marker Lane 2 MBP (C) Protein profile

of MBP on 2-D gel stained with coomassie-blue (D) Western blotting pattern of reactivity of anti-MLSA rabbit sera with MBP (E) Protein profile of MLSA on

SDS-PAGE gel stained with coomassie blue (F) Western blotting pattern of reactivity of anti-MBP rabbit sera with MLSA Lane 1 Molecular weight marker Lane

2 MLSA (G) Protein profile of MLSA on 2-D gel stained with coomassie blue (H) Western blotting pattern of reactivity of anti-MBP rabbit sera with MLSA

Table 1

Cross-reactive host proteins identified by matrix-assisted laser desorptionionization-time-of-flight mass spectrometry (MALDI-TOFTOF-MSMS) analysis

Cross-reactive protein Protein identified Accession

number

Mascot

score

Nominal

mass

pI Sequence

coverageSequence

coverage for PMF results

MBP cross-reacted with

anti-MLSA rabbit sera

Myelin basic protein (MBP) (Myelin A1 protein)

(20 kDa microtubule-stabilizing protein) - Bos

taurus (Bovine)

P02687 104 18312 1128 46

MLSA cross-reacted with

anti-MBP rabbit sera

50S ribosomal protein L2 e M leprae O32984 55 30593 1141 23

Lysyl-tRNA synthetase (EC 6116)

(LysineetRNA ligase) (LysRS) e M leprae 1179769

P46861 16 56614 491 VFELNRVFR

5I Singh et al Microbes and Infection xx (2015) 1e11

Please cite this article in press as Singh I et al Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA

synthetase) and myelin basic protein A possible mechanism of nerve damage in leprosy Microbes and Infection (2015) httpdxdoiorg101016

jmicinf201412015

MICINF4240_proof 8 January 2015 511

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

MLSA (z51 kDa protein) with anti-MBP rabbit sera to Mascotsearch engine and the search parameters used for the identifica-tion were peptide mass tolerance plusmn 100 ppm fragment masstolerance plusmn 05 Da maximum missed cleavages 1

211 Prediction of mimicking B cell epitopes fromMALDI identified proteins of host and M leprae

B cell epitopes of all MALDI identified cross-reactive pro-teins were predicted using BCPREDS server 10 (httpailabcsiastateedubcpredsindexhtml) BCPREDS server used aapprediction method Predicted B cell epitope length was of 20amino acids and classifier specificity used was 75 [20]

212 Three-dimensional modeling of identified proteins

Three dimensional (3-D) structure of mimicking proteins ofM leprae and MBP was formed by submitting the sequence toCPH models 32 server (wwwcbsdtudkservicesCPHmodels) [21] Modelled structure was visualized and analysed byVMD viewer (wwwksuiuceduResearchvmd) [22]

213 Statistical analysis

Data were analyzed using GraphPad prism software version50 (GraphPad La Jolla CA) ELISA data was presented asmean plusmn 2SD P value lt005 was considered to be statisticallysignificant Individual test used for the analysis of data of eachexperiment is mentioned in respective figure or table legend

3 Results

31 Levels of antibodies against MBP in sera of leprosypatients

Highest mean OD value for anti-MBP antibodies wasobserved in the sera of patients with T1R (0355plusmn 027) andwasfollowed by ENL (0344 plusmn 026) LL (0309 plusmn 023) BL(0309 plusmn 028) BB (0224 plusmn 021) and TTBT (0208 plusmn 015)Mean OD value of T1R is significantly higher than TTBT(plt 005) Anti-MBP antibody level was significantly higher inlepromatous pole of leprosy and in reactions than HC (Fig 1A)

Highest sero-positivity for anti-MBP antibodies was alsofound in T1R (619) followed by ENL (50) LL (413) BL(304) BB (238) BT (25) and HC (0) Percentage pos-itivity of T1Rwas significantly higher than BT group (pfrac14 002)

32 Correlation of anti-MBP antibodies level andnumber of nerves involved in different groups of leprosypatients

Spearmans correlation test showed anti-MBP antibodieslevel to be significantly correlated with number of nervesinvolved for TTBT (R2 frac14 0748 p lt 00001) BB (R2 frac140643 p frac14 0001) BL (R2 frac14 0540 p frac14 002) LL (R2 frac140617 p lt 00001) T1R (R2 frac14 0631 p lt 00001) and ENL(R2 frac14 0594 p frac14 002) group of leprosy (Fig 1BeG)

33 Binding of hyper-immunized rabbit anti-MBPantibodies with MBP was inhibited by leprosy patientssera in dose dependent manner

Binding of anti-MBP antibodies of hyper-immunized rabbitwas maximum inhibited at 15 dilutions of leprosy patientssera and inhibition gradually decreased with the increasingdilutions of leprosy sera (Fig 1H) Due to the unavailability ofpatients sera all Western blot experiments were conductedusing hyper-immunized rabbit sera

34 Lymphoproliferation of leprosy patients in thepresence of MBP

The highest mean value of SI was obtained in N (443 plusmn 31)group of leprosy patients which was followed by T1R(372 plusmn 29) BLLL (251 plusmn 13) TTBT (239 plusmn 15) ENL(22plusmn 15) The mean values of SI in the presence ofMBPwerefound to be significantly higher in N (p frac14 0007) TTBT(p frac14 003) BLLL (p frac14 001) T1R (p frac14 002) types of leprosypatientsgroup in comparison to that of healthy controls (Fig 2A)

35 MLSA hyperimmunization induce production ofanti-MBP antibodies in rabbit

It was observed that hyperimmunization of rabbits withMLSA evoked induction of significantly higher levels of auto-antibodies against MBP (p lt 00001) in comparison to controlrabbits The highest levels of antibodies against MBP wereobserved at 35th day of immunization with MLSA (Fig 2B)

36 Levels of antibodies against MBP in MLSA-hyperimmunized mice

It was noted that MLSA hyperimmunization till 7th weekinduced significantly higher levels of anti-MBP antibodies(0071 plusmn 0018) (p lt 00001) in hyperimmunized micecompared to those of normal saline inoculated control group(0016 plusmn 0014) and pre-immunized mice (0012 plusmn 0009)(Fig 2C) However no difference was observed in betweenpre-immunized and control mice

37 Adoptive transfer of whole immune cells and nylonwool separated T cells induce autoimmune response innaiumlve female BALBc mice

After 21 days of adoptive transfer significantly higher level ofanti-MBP antibodies were observed in the plasma of T cellstransferred mice (0063 plusmn 0008) (p lt 00001) or mixture ofsplenocytes and lymph nodes cells (0053 plusmn 0005) (p lt 0001)in comparison to those of pre-immunized mice (0034 plusmn 0009)and control group mice (0032 plusmn 0007) (Fig 2D)

38 Cross-reactive proteins of host MBP and MLSA

The specific cross-reactive proteins between host MBP andMLSA was observed by 1-D western blot and 2-D westernblot It was noted that anti-MLSA rabbit sera cross-reacted atz20 kDa protein of MBP (Fig 3AeB) with one isoform of

6 I Singh et al Microbes and Infection xx (2015) 1e11

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pI z 100 (Fig 3CeD) by 1-D and 2-D western blots On theother hand anti-MBP rabbit sera cross-reacted at z51 kDa30 kDa and 22 kDa protein of MLSA (Fig 3EeF) and atz51 kDa pI 55 and 30 kDa pI 60 (Fig 3GeH)

39 Identification of cross-reactive proteins of hostMBP and MLSA

As shown in Table 1 cross-reactive host protein MBP thatreactedwith anti-MLSArabbit serawas identifiedasMBP(myelinA1 protein) 20 kDa microtubule-stabilizing protein of Bos taurus(Bovine) (Fig1S A) The cross-reactive proteins of MLSA thosereactedwith anti-MBP rabbit sera onewas identified asz30 kDaprotein 50S ribosomal protein L2 ofM leprae (Fig1S B) and thesecond one was identified with the help of MALDI-TOFTOF-MSMS ion search by submitting different peaks (mass -1179769) of the cross-reactive protein of MLSAwith anti-MBPrabbit sera to Mascot search engine as Lysyl-tRNA synthetase(LysinedtRNA ligase) ofM leprae (Fig1S C and Table 1)

Mascot Search ResultMatch to MBP_BOVIN Score 104 Expect 2e-006Myelin basic protein (MBP) (Myelin A1 protein) (20 kDa

microtubule-stabilizing protein) - B taurus (Bovine)Nominal mass (Mr) 18312 Calculated pI value 1128Number of mass values searched 30Number of mass values matched 8Sequence Coverage 46Matched peptides shown in Bold Red

Match to RL2_MYCLE Score 55 Expect 002150S ribosomal protein L2 e M lepraeNominal mass (Mr) 30593 Calculated pI value 1141Number of mass values searched 67Number of mass values matched 8Sequence Coverage 23Matched peptides shown in Bold Red

Match to SYK_MYCLE Score 16 Expect 18e thorn 002Lysyl-tRNA synthetase (EC 6116) (LysinendashtRNA ligase)

(LysRS) e M lepraeNominal mass (Mr) 56614 Calculated pI value 491Sequence Coverage 1Matched peptides shown in Bold Red

310 Prediction of B cell epitopes of cross-reactiveproteins of MLSA and MBP

As shown in Table 1S three B cell epitopes of MBP werepredicted with the help of BCPREDS Server 10 Further 8 Bcell epitopes of 50S ribosomal protein L2 (Table 2S) and 12 Bcell epitopes of Lysyl-tRNA synthetase (Lysine-tRNA ligase)of M leprae were predicted (Table 3S)

311 Phylogenetic tree of predicted B cell epitopes ofcross-reactive proteins of MBP and M leprae

Phylogenetic tree of predicted B cell epitopes of MBP and50S ribosomal proteinL2 ofM lepraewasmadewith the help ofClustal W server to find out similarity between them (Fig2S)

As shown in phylogenetic tree 2 B cell epitopes of MBPand 50S ribosomal protein L2 ofM leprae resemble closely toeach other These epitopes are VVHFFKNIVTPRTPPPSQGK(MBP85-104) and VSPWGKPEGRTRKPNKSSNK (50S ribo-somal protein L2 of M leprae247-266) (Fig2SA) andGAPKRGSGKDGHHAARTTHY (MBP48-67) and EQA-NINWGKAGRMRWKGKRP (50S ribosomal protein L2 ofM leprae200-219) resemble very closely to each other(Fig2SA)

While one B cell epitope VVHFFKNIVTPRTPPPSQGK ofMBP (MBP85-104) and NTLSAPTFVKDFPVETTPLT (Lysyl-tRNA synthetase of M leprae384-403) resemble closely to eachother (Fig2SB)

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312 Similarity between the predicted B cell epitopes ofMBP and 50S ribosomal protein L2 of M leprae orLysyl-tRNA synthetase (Lysine-tRNA ligase) ofM leprae

As shown in Fig 4A two B cell epitopes of MBP98-104 with50S ribosomal protein L2226-237 and MBP127-131 and MBP55-60with 50S ribosomal protein L2 of M leprae41-46 aremimicking epitopes While 2 B cell epitopes of MBP85-98with Lysyl tRNA synthetase ofM leprae388-401 and MBP99-104with Lysyl tRNA synthetase of M leprae472-477 (Fig 4B) aremimicking with each other

313 Three dimensional structure of 50S ribosomalprotein L2 of M leprae

Three-dimensional structure was highlighted withmimicking epitopes of MBP and 50S ribosomal protein L2 ofM leprae It was observed that 2 mimicking B cell epitopes(MBP94-104 with 50S ribosomal protein L2226-237 and MBP55-60 MBP127-131 with 50S ribosomal protein L241-46) are presenton the surface of the 3-D structure of 50S ribosomal proteinL2 of M leprae (Fig 5A)

Above mentioned 2 mimicking B cell epitopes are presenton the surface of 3-D structure of MBP (Fig 5B)

4 Discussion

In this study we demonstrated the clinical correlationbetween level of antibodies against MBP and number ofnerve involved in leprosy patients across the spectrum(Fig 1BeG) Furthermore we found presence of signifi-cantly high level of anti-MBP antibodies (Fig 1A) in leprosyspectrum as well as significant high lympho-proliferation toMBP in all groups of leprosy patients except ENL (Fig 2A)Sero-positivity for anti-MBP antibodies was found to behighest in leprosy patients sera of T1R (619) followed byENL (50) LL (4138) BL (3043) BB (2381) andTTBT (25) These results indicate that antibodies are moreassociated with chronic phase of the disease We alsodemonstrated that binding of hyperimmunized rabbit anti-MBP antibodies with MBP was inhibited by leprosy pa-tients sera in dose dependent manner (Fig 1H) Inhibitionassay highlight that both anti-MBP antibodies raised in rabbitand antibodies present in leprosy patients sera compete withsame B cell epitopes Neural damage is one of the charac-teristic features of leprosy disease Earlier it was reportedthat M leprae has a strong demyelinating effect on infectedSchwann cells (Sc) neuron culture [23] Further it was alsoshowed by Antunes et al (2006) [24] that M leprae hasdirect destructive effect on the integrity of nerve fibers inleprosy patients However our findings suggest that anti-

MBP antibodies are associated with extent of nerveinvolvement in leprosy patients Recently Mycobacteriumavium subspecies paratuberculosis peptides have been shownto be recognized by anti-MBP antibodies in MS patients[25] In MS Ponomarenko et al (2006) [6] provided amechanistic explanation of anti-MBP antibodies in patho-logical destruction of myelin sheath Presence of anti-MBPantibodies in leprosy patients may also be involved in thepathological destruction of myelin sheath It was shown byCorsico et al (1994) [7] that in LL patient circulating im-mune complexes contain MBP as an antigen Authors sug-gested that presence of MBP could be correlated withpathogenesis of leprosy since liberation of MBP after nervedamage may induce anti-MBP autoantibodies production tomyelin breakdown which could react with peripheral nerveMBP also For the first time our study provides evidence thatanti-MBP antibodies are associated with the extent of nervedamage in leprosy patients Although M leprae specificimmunosuppression is reported in LL patients but presenceof auto-reactive cells might be responsible for positive pro-liferation to MBP as observed in this study A key questionremains to be answered that whether these auto-reactive cellsare induced by mycobacterial components The experimentscarried out by hyperimmunization of rabbits and mice withMLSA evoked a high level of anti-MBP antibodies in theirseraplasma (Fig 2BeD) and suggest that M leprae com-ponents are able to induce an imbalance in the homeostaticmechanism of the host which in turn induces autoantibodyresponse

T1R in leprosy is known to occur even after thecompletion of the therapy [26] and the bacterial loads inthese patients are significantly lower in comparison to LLpatients Therefore the precipitating factor for the occur-rence of T1R is still remains an enigma Earlier it has beenhypothesized that cross-reactive proteins of M leprae andhost might be responsible for initiation of reaction [927]Antigenic similarity between M leprae and human nerve andskin components has been suggested as the possible mech-anism for the development of auto-reaction in leprosy pa-tients [8911] For the first time we identified using anti-MBP antibodies and anti-M leprae antibodies in 1-D aswell as 2-D western blot (Fig 3AeH) that the cross-reactiveproteins are Lysyl-tRNA synthetase (Lysine e tRNA ligase)and 50S ribosomal protein L2 of M leprae and MBP (myelinA1 protein) of host by MALDI-TOFTOF (Fig1S and Table1) The basic A1 protein is a major structural protein of themyelin and reported to be responsible for induction of EAEPeptide fragments derived from the A1 protein are them-selves immunogenic as demonstrated by their ability for in-duction of a delayed hypersensitive response [28] We alsofind cross-reactive myelin basic protein as myelin A1 protein

Fig 4 (A) Multiple sequence alignment of predicted B cell epitopes of MBP and 50S ribosomal protein L2 of M leprae Figure showing full sequence of 50S

ribosomal protein L2 of M leprae and predicted B cell epitopes of MBP (B) Multiple sequence alignment of predicted B cell epitopes of MBP and Lysyl-tRNA

synthetase (Lysine-tRNA ligase) of M leprae Figure showing full sequence of Lysine-tRNA ligase of M leprae and predicted B cell epitopes of MBP A B

Mimicking B cell epitopes are highlighted in yellow color in the sequence Blue colored fonts are B cell epitopes of M leprae and red colored fonts are similar

sequence of B cell epitopes of MBP with B cell epitopes of M leprae

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and it might be involved in the auto-reaction in leprosy pa-tients Further using bioinformatics tools 2 mimicking B cellepitopes were identified between MBP and 50S ribosomalprotein L2 of M leprae while 1 mimicking B cell epitopewas identified between MBP and Lysyl-tRNA synthetase(Fig 4) Some of the mimicking B cell epitopes of MBP(MBP98-104 MBP85-98 MBP99-104) were found to be in theimmunodominant region of MBP which is MBP85-101MBP85-101 is found to be encephalitogenic peptide [29306]Hence these mimicking B cell epitopes might be responsiblefor demyelination of nerves in leprosy patients This sug-gested that the high prevalence of anti-MBP in leprosyspectrum and western blotting reaction between both of theseproteins (ie MBP and MLSA) might be because of thepresence of common B cell epitopes Vardhini et al (2004)[11] have also pointed out the molecular mimicry enacted bymycobacterial proteins (ferredoxin-NADP-reductase and aconserved mycobacterial membrane protein) that couldmimic myelin P0 Our findings are based on the wet lab

experiments as well as bioinformatics tools while these au-thors suggested molecular mimicry on the basis of bioin-formatics tools only From our findings we suggest thatmolecular mimicry is responsible for high level of anti-MBPantibodies in leprosy spectrum and induction of anti-MBPantibodies in hyperimmunized rabbit and mice Furtheradoptive transfer experiment in present study also providesevidence for transferring autoreaction in naiumlve mice by T aswell as B cells (Fig 2D) We also have noted T cell responseto MBP in leprosy patients (Fig 2A) Recently we havereported that CD4thornCD25thornFoxP3thornTreg cells level goes downin the hyperimmunized (with MLSA) mice [8] that in turngive strong support to our hypothesis that M leprae antigensare capable in inducing homeostatic imbalance in the im-mune system of the host and is a major factor for thedevelopment of the auto-reaction Molecular mimicry shownin the present study suggests that mimicking molecules ofhost and M leprae might be responsible for high level ofautoantibody and CMI reaction in leprosy patients

Fig 5 (A) Three dimensional structure of 50S ribosomal protein L2 of M leprae (B) Three dimensional structure of host protein MBP Yellow areas showing the

mimicking B cell epitopes of MBP and 50S ribosomal protein L2 of M leprae

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synthetase) and myelin basic protein A possible mechanism of nerve damage in leprosy Microbes and Infection (2015) httpdxdoiorg101016

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Thus our study suggests the existence of molecular mimicrybetween MBP of host and M leprae proteins (50S ribosomalprotein L2 and Lysyl-tRNA synthetase) Correlation of anti-MBP antibodies with number of nerves involved and high CMIreaction to MBP showed that molecular mimicry is the mostpossible factor for the pathogenesis of nerve damage in leprosy

Conflict of interest statement

The authors declare no conflict of interest

Acknowledgments

We thank Mr PN Sharma Mr Malikhan Singh and MrMohammad Alam for technical assistance of Department ofImmunology NJILampOMD Agra India We are grateful to DrAnnamma S John and Dr Mallika Lavania of SBL TLMDelhi for their support Indian Council of Medical Research isacknowledged for Senior Research Fellowship of IS AwardNumber 805342006-ECD-I

Appendix A Supplementary data

Supplementary data related to this article can be found athttpdxdoiorg101016jmicinf201412015

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Please cite this article in press as Singh I et al Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA

synthetase) and myelin basic protein A possible mechanism of nerve damage in leprosy Microbes and Infection (2015) httpdxdoiorg101016

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