Volume 61, Number 3Printed in the U.S.A.

(ISSN 0148-916X)

INTERNATIONAL JOURNAL OF LEPROSY

INTERNATIONALJOURNAL OF LEPROSY

And Other Mycobacterial Diseases

VOLUME 61, NUMBER 3^ SEPTEMBER 1993

Relationship Between Host Histones andArmadillo-Derived Mycobacterium leprael

Maria Cristina V. Pessolani, Shirley Wu Hunter, andPatrick J. Brennan'

Estimates of the global prevalence of lep-rosy have decreased dramatically over thelast decade due to the introduction of mul-tidrug therapy and the redefinition of aprevalent case as one requiring short-termmultidrug therapy. The estimated numberof cases in 1991 was 5.5 million as com-pared to the 10-12 million cases just a fewyears earlier (Is). Nevertheless, leprosy stillrepresents a major public health problemwithin the countries of Africa, Asia, andLatin America.

Despite positive trends, the developmentof a vaccine that would contribute to theeradication of leprosy remains a major goalof many research programs. Such a vaccinewould be inexpensive compared to che-motherapy, would not involve the specterof evolving drug resistance, and would beeasy to administer in geographical regionsnow proving much less amenable to pro-

' Received for publication on 2 February 1993; ac-cepted for publication in revised form on 11 May 1993.

2 M. C. V. Pessolani, Ph.D., Heiser Postdoctoral Re-search Fellow; S. W. Hunter, Ph.D., Associate Profes-sor; P. J. Brennan, Ph.D., Professor, Department ofMicrobiology, Colorado State University, Fort Collins,Colorado 80523, U.S.A.

Dr. Pessolani's permanent address: Setor Hansenia-se, Fundacao Oswald() Cruz, Rio de Janeiro, Brasil.

Dr. Hunter's present address: Paravax, Inc., 2301Research Blvd., Suite 110, Fort Collins, Colorado80526, U.S.A.

tracted chemotherapy. However, the pros-pects of a vaccine based on the whole bac-terium may have been undermined by thenegative preliminary results from the Ven-ezuela vaccine trials ( 3 ) (although these canbe explained on purely technical grounds),and the prospects ofa live recombinant vac-cine ( 19), probably the best hope, or one basedon cultivable mycobacteria, arc still in theoffing. We have taken the long-term tackthat the protein antigens of Mycobacteriumleprae still await basic molecular definitionand, once defined, each one requires ex-amination against a set of immunologicalcorrelates relevant to leprosy. A case in pointis our recent molecular and immunologicaldefinition of the 10-kDa antigen of M. lep-rae ( 7 .' 3 ), its single most dominant proteinimmunogen.

This particular strategy has been ham-pered, of course, by the inability to grow M.leprae in vitro. With the discovery in the1970s that the nine-banded armadillo issusceptible to infection with M. leprae C),tissues of infected animals have constitutedElie major source of the leprosy bacillus fora generation of biochemical and immuno-logical studies. Various procedures havebeen developed to purify the bacteria fromhost tissues, combining techniques suffi-ciently gentle to preserve the native struc-ture of the bacteria but powerful enough to

381

382^ International Journal of Leprosy^ 1993

remove host components (4 14, 17, 18, 20, 23) .One widely used purification procedure, de-vised for production of the one AI. leprae-derived candidate leprosy vaccine now intrial in Venezuela, Malawi, and South India,is that of Draper ( 23 ). This protocol involveswhat are considered gentle separation tech-niques including differential and densitygradient centrifugation, and partitioning inan aqueous two-phase system. Through re-search sponsored by the U.S.A. NationalInstitutes of Health, we have adopted thisprocedure in order to provide the scientificcommunity with both large amounts of ar-madillo-derived AI. leprae for experimentalpurposes and fundamental information onits major molecular constituents (- 7 ).

In the present study, we define a major17-kDa cell-wall-associated protein of thebacterium as host-derived histone H2b. Aprocedure that permits the solubilizationand quantification of histoproteins in AI.leprae preparations is described, and theimplication of this finding is discussed.

MATERIALS AND METHODSPurification of major cell-wall proteins of

M. leprae. Al. leprae was purified from ir-radiated spleens and livers of infected ar-madillos according to Draper with minormodifications ("). M. tuberculosis Erdmanstrain No. 107 from the Trudeau Mycobac-terial Culture Collection was grown as de-scribed (").

Purification of the cell walls of Al. lepraeand solubilization of their proteins with so-dium dodecyl sulfate (SDS) have been de-scribed ( 7 ). To purify the major cell wallprotein (MWP), a polypeptide doublet ofapparent molecular mass 17 kDa ( 7 ), theSDS extract from cell walls was initiallypassed through a column of Extracti-gel D(Pierce Chemical Co., Rockford, Illinois,U.S.A.) to remove the detergent. The eluatewas diluted to a final concentration of 5 mgprotein/ml in 10 mM Tris-HCI, pH 7.4,containing 150 mM NaCl and 6% TritonX-1 14, and submitted to phase separationas described by Bordier ('). Proteins fromthe detergent phase were recovered by pre-cipitation with 4 volumes of chilled (-20C)acetone. The precipitate was dissolved in10 mM piperazine, pH 5.7, containing 1%octyl-glucoside, and applied to an HPLCcolumn of AX-300 (DEAE) (Rainin Instru-

ments Company, Inc., Woburn, Massachu-setts, U.S.A.) equilibrated with the samebuffer. Proteins were eluted from the col-umn with a gradient of 0-30% NaCl. Frac-tions containing the 17-kDa protein werepooled, dialyzed against 10 mM Tris-HCI,pH 7.4, containing 6 M urea and applied toa Sephacryl S-300 gel filtration column (1x 100 cm) equilibrated with the same buff-er. Fractions containing the pure proteinwere dialyzed against water and vacuum-dried.

Acid extraction to remove histones. M.leprae (50 mg) and Al. tuberculosis (Erdmanstrain) (50 mg) were submitted to an acidextraction which favors solubilization ofhistones ( 2 '). Briefly, the bacteria were re-suspended in 1 ml of 0.25 M HCl and son-icated with a probe sonicator (W-385 Son-icator, Ultrasonic Liquid Processor; HeatSystems-Ultrasonic, Inc., Farmingdale, NewYork, U.S.A.) for 2 min on ice. Cells wereremoved by centrifugation, and the super-natants were vacuum-dried. Whole Al. lep-rae cells were extracted with PBS/EDTA asdescribed ( 16 ). Histones from uninfected ar-madillo spleens were isolated using a stan-dardized procedure ( 21 ). Total histones fromcalf thymus were purchased from Boehrin-ger Mannheim (Indianapolis, Indiana,U.S.A.). Histones VII-S (enriched in H2b)and VI-S (enriched in H2a) from calf thy-mus were obtained from Sigma ChemicalCo. (St. Louis, Missouri, U.S.A.). Proteinwas estimated by the bicinchoninic acid(BCA) protein assay reagent (Pierce) usingbovine serum albumin (BSA) as standard.

Polyacrylamide gel electrophoresis.Fractions were analyzed by SDS- ( 10) or ace-tic acid-urea-PAGE ( 12 ). Acid-urea gels con-taining 2.5 M urea were cast in glass plates22 x 20 cm with a 1-mm thick spacer andprerun in 0.9 M acetic acid at 25 mA untilconstant voltage was observed. After dis-solving in 0.9 M acetic acid containing 2.5M urea and 0.02% pyronine, the sampleswere loaded on the gel and a constant volt-age of 200 volts was applied. Gels werestained with silver nitrate or 0.1% Coomas-sie brilliant blue R in 40% methanol and10% acetic acid. The amount of histone H2bpresent in M. leprae acid extracts was de-termined by quantitative densitometry ofacid-urea gels stained by Coomassie blue,using the MicroScan 1000 2-D Gel Analysis

61, 3^Pessolani, et al.: Host Histones and Armadillo Bacilli^383

FIG. 1. Electrophoresis of the major cell-wall-as-sociated protein (MWP) doublet (ca. 17 kDa) of M.leprae. SDS-PAGE was conducted on a 16% tricine geland stained with silver nitrate. Molecular weight mark-ers are shown on the left.

System (Technology Resources, Inc., Nash-ville, Tennessee, U.S.A.).

N-Group analysis of proteins. Proteinswere transferred from acid-urea gels ontoan Immobilon polyvinylidenedifluoride(PVDF) membrane (Millipore, Bedford,Massachusetts, U.S.A.) in 0.7% acetic acid/10% methanol at a constant voltage of 40volts for 1 hr using a trans-blot unit withplate electrodes (Bio-Rad Laboratories,

Richmond, California, U.S.A.). After stain-ing with Coomassie brilliant blue (0.1% in40% methanol and 1% acetic acid) for 2min, the membrane was destained with 50%methanol, and the band of interest was ex-cised. N-terminal amino-acid sequencing ofproteins was performed in a 470A GasphaseSequencer (Applied Biosystems, San Jose,California, U.S.A.) equipped for on-line borePTH-amino acid analysis.

RESULTSIn a previous study ( 7 ), cell walls were

prepared from disrupted, armadillo-de-rived M. leprae by sucrose density centrif-ugation. The distinctive band at the 35%/45% sucrose interphase was the source ofthe cell wall, and the 17-kDa protein dou-blet, MWP, was obtained from this fractionby further SDS extraction. In the presentstudy, pure MWP was prepared by combin-ing detergent phase partitioning with ion ex-change and gel filtration chromatography(Fig. 1). Direct N-terminal amino-acid se-quencing of the purified protein readily al-lowed the identification of the first 26 aminoacids (Fig. 2). Comparison of the amino-acid sequence of MWP with that of proteinsin the National Biomedical Research Foun-dation protein sequence data base showed100% homology with the correspondingN-terminal segment of human histone H2b.This finding was a surprise and, indeed, ini-tially, a disappointment, given our earlierbelief that this protein was an importantspecific component and immunogen of Al.leprac ( 7 ).

The concept of a direct association be-tween host histoproteins and Al. leprae har-vested from such host tissues was furthersubstantiated by extracting the bacteria withacid. Figure 3A shows the protein patternson an acid-urea gel of acid extracts obtainedfrom two Al. leprae preparations and arecompared with similar preparations fromnoninfected armadillo tissue and with calfhistones. A complex mixture of bacterialproteins was observed in extracts from bothM. leprae preparations. The presence of thestress proteins of Mr 71, 65, 28, 18, and 10

10^ 20

Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala-Val-Thr-Lys-Asp-Cln-Lys-Lys-Asp-Cly-Gly-Lys-Asp

FIG. 2. The N-terminal amino-acid sequence of the MWP (17 kDa) of Al. leprac.

384^ International Journal of Leprosy 1993

A B

H2b

H2a

1^2 3^4 5 6 7 4 8FIG. 3. Gel electrophoresis of acid extracts of M. leprae (A) and M. tuberculosis (B) in acetic acid-urea gels

and staining with Coomassie brilliant blue. Lanes: 1, acid extract I.1/. leprae purified from armadillo liver; 2,acid extract of Af. leprae purified from armadillo spleen; 3, armadillo histones; 4, total histones from calf thymus;5, fraction enriched in histone H2a from calf thymus; 6, fraction enriched in histone H2b from calf thymus; 7,acid extract of Al. tuberculosis; 8, acid extract of fibrous tissue plus M. leprae clumps. Arrow indicates bandsubmitted to N-terminal amino-acid sequence analysis after transferring to PVDF membrane.

kDa, and the Mr 35 kDa membrane protein(major membrane protein I; M MP I 7 ) wereconfirmed by Western blotting using mono-clonal antibodies against these proteins pro-duced in this laboratory or obtained throughthe WHO/TDR/IMMLEP program ( 5 ' 8 ).Strong bands with electrophoretic mobilityidentical to H1, H2a, H2b, and H4 werealso observed in an acid extract ofM. lepraepurified from armadillo spleen. Incidental-ly, an acid extract of Al. tuberculosis grownin culture did not show the presence ofproducts indicative of histones (Fig. 3B); asdemonstrated (lane 7), the M. tuberculosisextracts do contain proteins, but they arenot histoproteins.

In order to confirm that the bands ob-served in acid extracts of Al. leprae wereindeed histones, proteins were transferredfrom acid-urea gels to PVDF membranes,and N-terminal amino-acid sequencing wasperformed. The first ten amino acids of theM. leprae band, corresponding to H2b,

showed 100% homology with human his-tone H2b. N-terminal amino-acid sequenc-ing of the other histones was not completedsince they proved to be blocked at the N-ter-minal end.

As one reflects on the history of the pu-rification of M. leprae from host tissue, con-tamination with host DNA always has beena problem, probably responsible, for in-stance, for the interaction and clumping ofbacteria with tissue components (P. Draper,personal communication). The inclusion inthe Draper protocol of a step in which prep-arations of M. leprae are treated withDNAasc was meant to obviate this prob-lem. To determine that the histoproteinsnow observed in preparations of Al. lepraewere derived from host chromatin contam-ination, clumps of what are considered fi-brous tissue material C11131 bacteria and whichare a common product of the Draper pu-rification steps (P. Draper, personal com-munication) were extracted with acid and

61, 3^Pessolani, et al.: Host Histones and Armadillo Bacilli^385

1 2 3 4 5FIG. 4. Evidence for a tenacious association be-

tween histoproteins and .11. leprae. Two batches of M.leprae obtained from infected spleens were extractedfirst with PBS/EDTA and then with acid (A) or directlywith acid (B). The solubilized proteins were run on a15% polyacrylamide gel and stained with Coomassieblue. Lane 1, M. leprae batch No. 1; lane 211. lepraebatch No. 2; lane 3, total histones from calf thymus.Molecular weight markers are indicated on the left.

analyzed by acid-urea gel electrophoresis(Fig. 3B). The similarity in protein contentof one of these extracts and a standard his-tone preparation pointed convincingly to thechromatin nature of the fibrous tissue ma-terial long associated with Al. leprae.

In order to obtain information on the fre-quency as well as the degree of histone con-tent of preparations, nine different batchesof Al. leprae (purified from either infectedarmadillo livers or spleens) were extracted,and acid-urea gels of the acid extracts wereanalyzed by quantitative densitometry (TheTable). All preparations showed the pres-ence of histone H2b, with absolute amountsvarying from 0.6 to 4.8 g/mg of bacteria.Bacteria obtained from livers showed fewerhistones than those obtained from spleens.An important point is that there was no

THE TABLE. Estimation of the degree ofassociation of histone H2b with M. lepraepreparations.

Batchno.

Origin oftissue

Purity ofM. leprae

(%)..

Content ofH2b (g/mg

bacteria)h

I Liver 95 1.02 95 1.03 95 0.64 90 0.85 Spleen 95 2.46 90 0.77 90 1.88 85 4.89 80 0.9

Purity based on the acid-fast aniline blue stain.h The amount of H2b in M. leprae preparations was

determined by densitometric analysis of acid extractson acid-urea gels.

apparent correlation between the amount ofhistone H2b present in a preparation andone's estimate of bacterial purity based onsoluble blue staining through the Ziehl-Neelsen reagent ( 22 ).

Arising from years of experience in thepurification of Al. leprae from armadillosources, we had dropped the dextran-poly-ethyleneglycol biphasic step of the originalDraper protocol (23 ) and replaced it by theinclusion of at least two further Percoll den-sity gradient centrifugation steps. Thismodification permitted an increase in bac-terial yields without any apparent reductionin purity. To determine if the inclusion ofthe aqueous two-phase step could reducethe extent of histone association with Al.leprae, materials prepared with and withoutthe biphase step were compared. In two in-dependent experiments, no significant dif-ference was observed in the content of Al.leprae prepared by the two methods. Somerevelation on the tenacity of the associationbetween AL leprae and the host histones wasobtained by the observation that when ba-cilli were first extracted with PBS/EDTA, astep which, incidentally, solubilizes the ex-creted 30-31-kDa protein complex of AI.leprae (16), none of the histones were re-moved. Subsequent application of a sim-plified acid extraction step to the resultingpallet whereby the bacteria were treated withacid at room temperature for 10 min withgentle agitation, omitting sonication, re-sulted in extracts in which histones wereessentially the only proteins (Fig. 4). In two

386^ International Journal of Leprosy^ 1993

such preparations, employing Al. lepraefrom spleens, the recovery of histones wasof the order of 20 pg and 100 pg of proteins/mg of bacteria.

DISCUSSIONAlthough H2b seems to be the predom-

inant historic associated with M. leprae cellwalls, bands with electrophoretic mobilityidentical to that of other nucleosome his-tones, as well as to histone HI, were alsoobserved. In our view, two lines of evidenceindicate that the histoproteins of Al. lepraepreparations are host-derived and are notthe products of horizontal gene transfer be-tween host and parasite ("), a transfer whichapparently accounts for the presence of his-tone-like proteins in other organisms. First-ly, from present and previous evidence ( 7 ),it is obvious that these proteins are pref-erentially associated with the surface of M.leprae, unlike the histories and histone-likeproteins of other eukaryotes and prokary-otes which are clearly cytosolic. Secondly,present experiments established that chro-matin is the major source of histoproteincontamination of M. leprae preparations.The fact that histories were recovered in thedetergent phase from the Triton phase sep-aration suggests that they are present in theirdenatured forms and probably attach to thecell envelope of M. leprae by hydrophobicinteraction. On the other hand, histopro-teins are highly cationic with a pl range of11 to 12 because of the predominance oflysine and arginine residues, and are notreadily displaced by chelating agents, allpointing to ionic interaction. What mightbe the nature of the corresponding negativecharge on the surface of AI. leprae is notknown; perhaps it is lipoarabinomannanwhich does carry an overall negative charge.

The Draper protocol has been used forseveral years for the preparation of vaccine-grade M. leprae, largely on the basis of theprotective efficacy of the product against footpad infection in mice ('"). The essence ofthe protocol is the application of relativelygentle separation methods, obviating, forinstance, the drastic alkaline and proteasetreatments which are part of other proto-cols. Thus, the presence of host-derivedcomponents in preparations of M. lepraeobtained in this way is not unexpected and,in fact, has already been demonstrated (P.

Draper, personal communication). Thepresent study clearly establishes the com-position of some of these contaminants andshows that they are not trivial. The conse-quences of the injection of host componentssuch as histories into humans in the contextof the present vaccine trials are probablynot harmful. The procedures described inthis present study allow for a quantitationof the histone content of leprosy vaccinepreparations and a determination of whateffects they might have on potency.

SUMMARYA major protein previously recognized as

being primarily associated with the cell wallsof Mycobacterium leprae, major wall pro-tein (MWP), is now identified as histopro-tein H2b based on N-terminal amino-acidsequencing, electrophoretic comparisons,and several other properties. An avid as-sociation between several host/armadillo-derived histones and M. leprae was dem-onstrated. Since such armadillo-derived AI.leprae are the basis of several ongoing vac-cine trials, a simple procedure that permitsthe prompt solubilization and quantifica-tion of histones in M. leprae preparationsis described. The quantity of histones as-sociated with AI. leprae is significant, rang-ing from 0.6 to 4.8 pg of histoprotein H2bper mg of bacteria.

RESUMENUna proteina previamente considerada como pri-

mariamente asociada a las paredes cclulares del My-cobacterium leprae (Ia proteina MWP), ahora se iden-tifica como Ia histoproteina H2b con base en susecuencia de aminodcidos N-amino terminal, su corn-portamiento electrofortico, y varias otras propieda-des. Se demostrO que existe una avida asociacin entrevarias histonas derivadas de armadillo y el M. leprae.Puesto que Af. leprae derivado de armadillo es la basede varias vacunas experimentales, aqui se describe unprocedimiento simple que permite la pronta solubili-zaciOn y cuantificacin de histonas en las preparacionesde Af. leprae. La cantidad de histonas asociadas conAl. leprae es significativa y varia de 0.6 a 4.8 pg dehistoproteina H2b por mg de bacterial.

RSUM

Unc protine majeure reconnue auparavant commcetant en premier lieu associe a Ia paroi cellulaire deMycobacterium leprae la proteine majeure de la paroi(MWP), est maintenant identifies comme une histo-protine H2b sur base de la determination de Ia se-

61, 3^Pessolani, et al.: Host Histories and Armadillo Bacilli^387

qucnce des acides amines de l'extrmit azote, decomparaisons lectrophortiques et diverses autresproprites. On a dmontr ('existence dune associationtroite entre differentes histones drives du tatou-hteet M. leprac. Comme de tels A1. leprae obtenus a partirde tatous forment la base de differents essais de vac-cination en cours, un procd simple qui permet lasolubilisation rapide et la quantification des histonesdans des preparations de M. leprac est dcrite. La quan-tit& d'histones associes avec M. lepraecst significative,allant de 0.6 a 4.2 pg d'histoproteine H2b par mg debactrie.

Acknowledgment. This work was supported by acontract (NO1 Al-05074) from the National Instituteof Allergy and Infectious Diseases, Division of Micro-bial and Infectious Diseases, National Institutes ofHealth. M.C.V.P. was supported by a Post-DoctoralFellowship from the Heiser Program for Research inLeprosy and Tuberculosis. We thank Drs. Eleanor E.Storrs and Darrel D. Gwinn for ensuring a steady sup-ply of armadillo-derived products.

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