Primary structure of the wall peptidoglycan of leprosy-derived corynebacteria

Vol. 145, No. 2 JOURNAL OFBACTERIOLOGY, Feb.1981,p.775-779

0021-9193/81/020775-05$02.00/0

Primary

Structure of the Wall

Peptidoglycan of

Leprosy-Derived Corynebacteria

E.JANCZURA,lt M.LEYH-BOUILLE,'2C.COCITO,'* ANDJ. M. GHUYSEN2

Departmentof Microbiology and Genetics, Institute of Cell Pathology, University ofLouvainMedical School, B-1200 Brussels,' and Service de Microbiologie, Facultede Medecine, Universitede Liege,In8titut

deBotanique, B22, B-400 Sart Tilman,Liege,' Belgium

The cell walls isolated from axenically grown leprosy-derived corynebacteria were submitted to various chemical and enzymatic degradations. The glycan

strands of the wallpeptidoglycanare essentially composed of

N-acetylglycosa-minyl-N-acetyhnuramic aciddisaccharide units. Small amountsof N-acetylgly-cosaminyl-N-glycolyhnuramic acid (less than 10%)were alsodetected. The

mu-ramic acid residues of adjacent glycan strands are substituted by amidated

tetrapeptide units which, inturn,arecross-linked through direct linkages

extend-ingbetween theC-terminal D-alanine residue ofonetetrapeptide andthe

meso-dianinopimnelic acid residue of another tetrapeptide. Such a structure is very

similar to that of the wall peptidoglycan found in the taxonomically related microorganisms of the Corynebacterium, Mycobacterium, andNocardia groups.

The acid-fast gram-positive organism Myco-bacteriumlepraecannotmultiplyin vitro.It is found inlepers' lesions associated with another type ofnon-acid-fast, gram-positive microorga-nism, whichcanbepropagatedinaxeniccultures (1, 4),cross-reactwithlepers' and anti-mycobac-terial sera (12, 15),are morphologically related toCorynebacterium diphtheriae (1, 4), and pos-sessintheir cell envelopes components (arabi-nose,galactose,andmycolicacids) (E.Janczura, C.Abou-Zeid,C.Gailly, and C.Cocito, submit-ted forpublication)thatareknowntobespecific

to the Corynebacterium, Mycobacterium, and Nocardia group. Since the wallpeptidoglycans of these latter bacteria have been extensively studied (2, 3, 11, 13, 14), experiments were de-vised to unravel the primary structure of the wall peptidoglycan of several leprosy-derived corynebacteria (LDC) strains. The results of theseinvestigationsarepresentedinthis report.

MATERIALS AND METHODS

Bacterial strains. The LDC strains 4, 8, 15, and

19(previouslydesignatedD43, L3, DM-86, and Manga,

respectively) were isolated fromlepromatous tissues of Africanpatients(J. Delville, UniversityofLouvain) (4). Strain20(previously designatedMedalleX)was

foundby C.Reich in abiopsymade in thePhilippines (courtesyof L.Barksdale, New YorkUniversity) (1).

Thesestrainsweregrownin Dubos medium

supple-mented with horseserum (5%, vol/vol) and sodium

succinate(7mg/ml) infermentorsat37°Cand under

forced aeration.

tOnsabbaticalleave from theDepartmentofBiochemistry and Biophysics, the Polish Academy ofSciences, Warsaw,

Poland.

Isolation and purification of wall peptidogly-can.Exponentially growing bacteria were harvested, suspended in 66 mM sodium phosphate buffer (pH 7.8), and disrupted inaC02-cooledvibrating Braun disruptor, type853032(five treatments of2min each, using50gof 0.10-mmglassbeads and10g, wet weight, ofpacked cells). After dilution of the homogenates with phosphate buffer, the remaining intactcellsand cellular debris were eliminated by filtration of the

suspensionsthrougha 2G Duransinteredglassfilter

(Jena Glass; Schott, Mainz, Germany), followed by three successivecentrifugationsat1,000xgof5min each. The crudecellenvelopeswerethensedimented

by centrifugation at 23,000 x g for 15 min at40C,

suspended inphosphatebuffer, and incubated

sequen-tially (at37°C and for 16h, in each case) with pan-creatic deoxyribonuclease I (1 mg/10 g ofcell wall

material), pancreatictrypsin (150 mg/10 g), and pan-creaticchemotrypsin(25 mg/10 g).The three enzymes used in thispurificationstepwerefromServa Feinbi-ochemica, Heidelberg, Germany. The partially puri-fied cell walls were collected by centrifugation at

23,000xgfor15minat4°C,repeatedly washed with

1MNaCl and water, extractedsuccessively with

ace-tone, ether-ethanol (1:1, vol/vol),- and chloroform-methanol (2:1,vol/vol),and dried inastreamofair. Furtherpurification wasthenachieved bymildacid

treatment (10 ml of0.1 N HCl per 100 mg ofwall material, at 60°C for 12h), followed by further

de-lipidationwithchloroform-methanol (2:1,vol/vol)for

19h at200C.The wallswerecollectedby centrifuga-tion, washedwithacetone,andair-dried.

Aminoacid analyses.Afterhydrolysiswith 6N

HCl for16hat 1050C, thetotal amino groups were

estimatedbythefluorodinitrobenzenetechnique (8),

andtheamino acid residueswerequantitatively esti-matedwitha3201LKB amino acidanalyzer(equipped

withacolumn of the cationexchangeresin

Durrum-LKB andusing66mMsodium citratebuffers,pH3.22 775

and 4.20, at 51 and 72°C, respectively). The sme

technique permitted quantitative estimation offree NE6.Characterizationofthemeso andDDisomers of

diaminopimelic acid (A2pm) wasperfo by de-scending paperchromatographyof acidhydrolysates

accordingtothetechniqueof Rhuland(citedin

Jan-czuraetal.,submitted forpublication).

Sugaranalyses.Afterhydrolysiswith 3 N HCI for

3hat100°C,thetotalhelosamineswere eimatedby theElson-Morganprcedure(8).Free glucoaiine(Rf

-0.2)and acid (Rf-0.44) were

charcter-izedbychromatography of the acidhydrolysateson

Whatman no. 1 paper the solvent 1-butanol-acetic acid-water(3:1:1, vol/vol),followedbystaining

with paradimethylbenzaldehyde as previously

de-scribed(J.Falszpn-Wietzerbin,doctoralthes, Uni-versity of Parishuth, Orsay, France, 1973). After enzymatic degradation ofthe glycan moietyof the peptidoglycan, the total reducing groups were

esti-mated with thePark-Johnsonprocedure(8),andfree

and peptide-substituted

B-1,4-N-acetylglycosamyl-N-acetylmuramicacid daccharideswereestimatedwith the Morgan-Elsn procedure (8). Free diacharide

units (R1 -0.32)werealso characterizedby chroma-tography onWhatman no. 1 paper, using the same

conditionsasforglucosamine (seeabove).Inaddition,

Sharon reagent (2%NaOH in 4:6propanol-ethanol)

(Falszpan-Wieterbin,thesis)wasalsoused for

devel-opingthe chromatograms.

Analysesof I groups.TerminalNH2and COOHgroups ofpeptideswerecharacterizedand

es-timated bydinit nylation, followedbyacid

hy-drolys, andbyhydrazinolys, respectively.Formore

details,seeGhuysenetal.(8).

EtimationofD-alanlne. FreeD-alaninewas

es-timated with theD)aminoacidoxid0 tchnique (8).

The D-lactate dehydrogenase,D-aminoacidoxidase,

andcataluseusedwerefromBoehringer, Mannheiim, Germany.

Etmai ofglyeolylgroups.Afterhydrolysi

of 10mgofwallwith4 NHCIfor24 hat1000C,the

hydrolysatewasfilteredon a0.9-mlcolumn (5by0.5

cm)of Dowex 5OW-X80,and thecolumnwaswashed

with1mlof water. Part of thefractionthusobtained

wastreatedwith 2,7-dihydrozynaphthalene in 10 M

H2SO4,andthespectrophotometric mementsat

530nmwerepmed asdesribedinreference 18.

Another part of the hydrolysate was submitted to chromatographyonWhatmanno. 1paper,usingthe

solvent 1-butanol-acetic acid-water (1:1:1, vol/vol),

and detected withbromophenolblue (Fazan-Wietz-erbin,thesis).Glycolicacid hadanRfvalue of 0.6.

lhydrolase. The

endo-N-acetyl-muramidas (EC3.2.1.17)fromChalarpsis(9) (crys-talline enzyme; a giftfrom J. A. Hash, Vanderbilt

University, Nashville, Tenn.)andStreptomyces

glob-isporus (20) (a gift from K. Yokogawa, Dainippon PharmaceuticalCo., Tokyo, Japan)hydrolyzethe ,8-1,4-glycosidiclinkages between N-acetylmuramic acid

andN-acetylgucosamine intheglycan strands. The N-acetylmuramyl-L-alanine amidas from

Sbtepto-mycesalbus G(7) hydrolyses the amide linkages at

thejunction betweentheglycanstrandsandthe pep-tide unit&TheDD-cboxypeptidasefromS.albusG

(5) (crystallie enzyme) hydrolyzes the

D-AIa-(D)-meso-A2pm interpeptide linkages in the peptide moi-ety. Amidation of thecarbonyl group of meso-A2pm in aposition to thepeptide bond does notabolish but muchdecreases the enzymeactivity. For more details onthe mode of action of thesepeptidoglyean hydro-lases, see reference 6.

RISULTS

Chemical composition ofthe wail

pepti-doglycan

On the average(Table 1), thewalLs isolatedfromthefive strains of LDC eam ed contained, per mg of dry weight, 0.51 Amol of meso-A2pm, 0.67,umol of Glu, 1.17 AmolofAla, and 1 pmolofmuramicacidplusglucosamine. LL-A2pm was not detected by Rhuland's tech-nique (cited in Janczura et al., submitted for publication). On the basis of the action exerted by the

DD-carboxyWpeptidase

(seebelow), A2pm must occurin the form of the mesoisomer. The fraction of the wall material, hydrolyzed and purifiedasdescribed in the legend to Fig. 1,thus

appeared

to consist of a classical meso-A2pm-containing peptidoglycan ofchemotype I(6).

Enzymatic

hydrolysesofthe wall

pepti-doglyean.

The walls isolated from both LDC satrins8and 15 (400mg,dry

weight,

in 400 , finalvolume, of buffer) wereseparately treated withthe S.

globisporus N-acetylmuramidase

(in 20mM sodium phosphate buffer, pH 6.5, con-taining 10

jg

ofenzyme),withtheChalaropsis

N-acetylmuramidase

(in20mMsodium acetate buffer, pH 5.0, containing5 ug of

enzyme),

and with the S. albusGDD-carboxypeptidase (in10 mMTris-hydrochloride buffer,pH 8.0, contain-ing5mMMgCl2 and 30 pg of enzyme). After 20 hat370C, thetwoN-acetylmuramidases caused a maximaldecrease of theturbidities of the wall suspensions to about 10% of theirorginal values. In parallel to this, reducing groups maxilly equivalenttoabout0.9mol of ,6-1,4-N-acetylgly-cosamyl-N-acetylmuramic acid disaccharide units per mol ofmeso-A2pmwere

exposed,

indi-cating an almostcomplete hydrolysis of the

sen-TABLE

1.

Conposition

of

the

waU

peptidoglycan

of

fiveLDCstrains

Strain Wall

peptidoglyean

(nmol/mgof

prdcelawan)

Total No. Batch meso

_A2pm

Glu Ala

hezosa-m _ ~~~~~~~munes 4 560 820 1,500 1,100 8 510 686 1,159 960 15 1 520 520 972 990 2 544 552 944 1,060 19 1 450 827 1,180 850 2 504 650 1,160 1,009 20 470 730 1,300 1,010 Average 507 670 1,170 1,000

CORYNEBACTERIAL PEPTIDOGLYCAN 777 .1.5 0E .0 oo o_z.D_u I° <.'C ,E 0.54 > x x m41 I I

I0.1

450 500 550 600 650 VOLUME Iml)

FIG. 1. Sephadex filtration of the N-acetylmuram-idase- andDD-carboxypeptidase-treated cell wall of strain 15. Cell walls(100(mgJ were suspended in 25 ml of 20 mM sodium acetate buffer (pH 5.0) and incubated with 1.25 mg ofChalaropsis enzyme for 20 h at37°C. Afterelimination of the undissolved ma-terial (20 mg) by centrifugation, the solution was neutralized with1N KOH, supplemented with 10 ml of10mMTris-hydrochloride buffer (pH8.0)

contain-ing8mg of the S. albus G enzyme, and incubated for

16h at 37°C. The resulting solution was filtered in

0.1MLiCl on a combined G-50 Sephadex column (93 by 2.7 cm)-G-25 Sephadex column (103by 2.4 cm) system, where the two columns were connected in series in the indicated order (total void volume, 475 ml). Reducing groups (dotted line) andfree amino groups (solid line) were estimated in thefractions collected.Fractions A, B, and C weredesalted. Frac-tion C consisted ofdisaccharide-(amidated) tetrapep-tide monomerunits.

sitive glycosidic linkages. Analyses of the ter-minal amino groups present in thewall digests showedthat about20% of themeso-A2pm resi-dues hadoneamino groupfree, thus indicating an average degree of polymerization of 80% cross-linked peptideunitsfor the intactpeptide moieties ofthe wall peptidoglycans. Contraryto the endo-N-acetylmuramidases, the S. albusG DD-carboxypeptidase caused only alimited ex-tent of clarification of the wallsuspensions (20 to25%). However, aftertreatmentof theisolated walls first with theChalaropsis enzyme (at pH 5.0) and then with the S. albus G enzyme (at pH 8.0 and in the presence of 5 mM MgCl2) underthe conditionsdescribedinthelegendof Fig. 1, analyses of the degraded products re-vealed that the glycan strandshad been com-pletely degradedintodisaccharideunits and that about 50 to65% of themeso-A2pm residuesnow occurred attheN-terminalpositionofthe pep-tidemoieties. S. albus G DD-carboxypeptidase hydrolyzed the substrate with less efficiency when acting alone than when acting subse-quentlytomuramidase.

Isolation andcharacterization of the di-saccharide-peptide monomer. The isolated wallsof LDC strain10(100mg)weresubmitted

tothesequentialaction of theChalaropsisand S. albus G enzymes, and the reactionproducts were fractionated by Sephadex filtration in 0.1 MLiCl,asdescribedinthelegendofFig.1.The three fractions, A, B, and C, collected at KD values of0.12,0.22, and0.66, respectively,were

desaltedbyfiltration onSephadexG-25 in water. Fractions A, B, andCcontained 5,20,and70%, respectively, of theoriginalwallpeptidoglycan. FractionCwascomposed ofequlimolaramounts ofglucosamine,muramicacid, L-Ala, Glu,

meso-A2pm,D-Ala, and NH3,and contained1 equiva-lentof mono-NH2-meso-A2pm.Itthus consisted of disaccharide-(monoamidated)tetrapeptide units. Thisdisaccharide-peptide monomer was, inturn,submittedtotheaction of the N-acetyl-muramyl-L-alanine amidase under the condi-tionsdescribedinthelegend of Fig. 2, causinga complete separation between the disaccharide and thepeptide units. The degradation products werefilteredin water onacolumn ofSephadex G-25. Fraction D was composed of equimolar amountsofglucosamnine and muramic acid and thusconsisted of free disaccharide units. These disaccharide units comigrated by paper chro-matography with authentic fl-1,4-N-acetylgly-cosaminyl-N-acetylmuramic acid. Fraction E was composed ofequimolar amounts of

meso-A2pm, Glu, D-Ala, L-Ala (obtained by subtract-ing D-Ala from total Ala), and NH3, and

con-_1.0 ,E "U) I00 D -CE 0.1 0 _ D

L-

_{170 180} VOLUME(ml) E 190 200 1.0_lO)(A_-K X,-_ o E 00_{cr X} 0 -z j L5X 0 4 -E .j z v 01 CT@ I _

FIG. 2. Sephadex filtration of the

N-acetylmura-myl-L-alanine amidase-treated

disaccharide-(ami-dated)tetrapeptideunitsofthewallpeptidoglycan of strain15.Asample(4.5pmol) of

disaccharide-(ami-dated)tetrapeptide (fraction C of Fig. 1) was incu-batedfor6hat37°Cin4.5mlof50mMacetatebuffer (pH5.4)containing750Il ofamidase(total

hydrolyz-ing capacity, 45ueq of N-acetylmuramyl-L-alanine

linkages hydrolyzedwhenactingonthedisaccharide peptidemonomer ,8-1,4-N-acetylglycosamyl-N-acetyl-muramyl-L-Ala-D-Glu(NH2z)L-Lys-D-Ala). The

deg-radation productswerefractionated by filtration,in

water, on a Sephadex G-25 column (103 by2.4 cm;

voidvolume, 165ml). Reducing groups (dotted line) andfreeamino groups(solid line)wereestimatedih thefractionscollected.FractionD consistedof disac-charideunits, andfractionEconsistedofamidated

tetrapeptideunits. VOL. 145,1981 I, 1, I, 1,I I I ---I

tained 1equivalent ofboth N-terminal Ala and mono-NH2--meso-A2pmperpeptide unit.Ih ad-dition, Ala was the only C-terminal amino acid residue that could be detected byhydrazinolysis. Fraction E thus consisted of (amidated) tetra-peptide units. The tetratetra-peptide was submitted topaperelectrophoresis at pH 6.5 (60 V/cm for 1h). Itbehaved as a neutral compound, a prop-ertywhich was compatible with the occurrence of oneamide group.

Occurrence ofN-acetyl- and N-glycolyl-muramic acid residues. The walls of LDC strain 15 were hydrolyzed with HC1, and the hydrolysatewasfilteredonDowex 50W-X80as

describedin Materials and Methods. Reaction with dihydroxynaphthalene and

spectrophoto-metric measurements permitted detection of, 6 nmol ofglycolic acid per mgof walls.

Glycolic

acid wasalso detected by paper chromatogra-phy, but apparently in much lower amounts. Fromtheseexperimentsitwasconcludedthata major part of muramic acid occunred as the N-acetyl derivative and that about 2 to 10% oc-curred in the form of theN-glycolyl derivative.

DISCUSSION

From the data presented above, it appears that thepeptidemoietyof the wall

peptidogly-can ofLDC is ofchemotype I (6) with

meso-A2pm linkagesservingas peptide bridges. The tetrapeptide units also possess one amide sub-stituent, but its location (on the a-carboxyl group of Gluor onthatcarboxyl group of meso-A2pm which isnotinvolved in the main back-boneof thepeptide) hasnotbeen determined. Allthemycobacteria,nocardia,and corynebac-teria strains eminedsofar possessawall pep-tidoglycanwith thesametype ofpeptidemoiety, and thesequence

L-Ala-D-Gb(L)-meso-Aipm-(L)-D-Ala (at various levels of amidation) has been assigned totheir tetrapeptide units (2, 6, 13, 14, 16, 17, 19).

F romthe datapresented above,it also appears thattheglyeanmoietyofthewallpeptidoglycan ofLDC containsasmallproportionof N-glyco-lylmuramic acidtogetherwith ahighproportion ofthewidely occurring N-acetylmuramic acid. Previous works have shown thatglycolylgroups occurinthewallpeptidoglycansof mycobacte-ria,Nocardia, andMicromonospora, but not in thoseofStreptomycesorcorynebactenia(14; Fal-szpan-Weitzerbin, thesis). More recently, how-ever, this view had to be revised, as glycolyl groups were detected in small amounts in the cell walls of Corynebacterium michiganense, Corynebacteriunsepedonicum, and Corynebac-terium bovis and in much larger amounts in those ofanunclassifiedcoryneform, IAM 1311

(18). One should note that whereas the genera Mycobacterium and Nocardia are genetically homogenous (67 to 69 mol% guanine plus cyto-sine in theDNAs), the genus Corynebacterium ishighly heterogeneous (52 to 69 mol% guanine plus cytosine) and comprises at least four broad groups related toC.

diphtheriae,

Corynebacte-riumrenale, Corynebacterium genitalium, and Rhodococcus

phylus

(10, 11). The guanine plus cytosine content of the LDC has been found to be 56 to 60 mol% (P. Danhaive, P. Hoet, and C. Cocito,unpublished data).

Another conclusion of thepresent work is that thepeptidoglycan of LDC and that of Mycobac-teriumtuberculosis are very similar. In this con-nection, the observedcross-reaction between the walls of LDC and anti-mycobacterial sera (12, 15)isworthy of mention.

ACKNOWLEDGMENT8

This work was supported in part at the Universty of Louvainby theItalian R.FollereauAssociation"Gli Amici deiLebbrosa,"and theBelgian RFollereauAsociation"Les AmisduPere Damien," whichprovidedapostdoctoral fellow-shiptoE.J., andattheUniversityofLiege bytheNational Institutesof Health(PublicHealthService contract

2-RO1-AI-13364-04MBC), theFonds de laRecherche Scientifique Medicale, Bsels(contract 3.4501.79), and the Actions

Con-cert6es,EtatBelge(convention 79/84-I1).

We thank P. Osinsky (University of Louvain) for amino acidanalyses.

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