JOURNAL OF BACTERIOLOGY, Sept. 1987, p. 4223-4227 Vol. 169, No. 90021-9193/87/094223-05$02.00/0Copyright © 1987, American Society for Microbiology

Structure of Bordetella pertussis PeptidoglycanW. JOSEPH FOLKENING,1 WALLACE NOGAMI,2 STEPHEN A. MARTIN,3t AND RAOUL S. ROSENTHAL'*Department of Microbiology and Immunologyl and Department of Surgery,2 Indiana University School of Medicine,

Indianapolis, Indiana 46223, and Department of Chemistry, Massachusetts Institute of Technology,Cambridge, Massachusetts 021393

Received 2 March 1987/Accepted 27 May 1987

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid mediumcontaining [3H]diaminopimelic acid and treated by a hot (96°C) sodium dodecyl sulfate extraction procedure.Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containingprotein (ca. 40%) and peptidoglycan (60%). Subsequent treatment with proteinase K yielded purifiedpeptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, anddiaminopimelic acid in molar ratios of 1:1:2:1:1 and <2% protein. Radiochemical analyses indicated that 3Hadded in diaminopimelic acid was present in peptidoglycan-protein complexes and purffied peptidoglycan asdiaminopimelic acid exclusively and that pertussis peptidoglycan was not 0 acetylated, consistent with it beingdegraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers andpeptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from thedistribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and IIIpeptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived peptidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrom-etry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of>95% N-acetylglucosaminvl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl-alanine.

Peptidoglycan (PG), a heteropolymer unique to bacterialcell walls, is well recognized as the principal determiner ofbacterial shape and physical integrity (8). However, al-though once regarded merely as an inert bacterial corset andas a target for penicillin action, it is now clear that PGfragments are potent modulators of inflammation and im-munity in host tissues (see reference 27 for a review). Amongthe many diverse PG-mediated biological activities areadjuvanticity (2, 4), arthropathic activity (6, 14), enhance-ment of nonspecific resistance to infection and tumors (7,13), activation of macrophages (1, 29), and (perhaps surpris-ingly) sleep-promoting activity (15, 16).During the course of studies in this laboratory on the role

ofPG in disease due to Neisseria gonorrhoeae (6, 18, 19, 22),it came to our attention through the work of Goldman et al.(11) that there were certain functional similarities betweengonococcal PG and PG derived from virulent Bordetellapertussis. Of particular interest was that each of theseorganisms released low-molecular-weight PG derivativeswhich promoted the loss of ciliated epithelial cells frommucosal surfaces (11, 18). Human fallopian tube mucosa wasemployed as the model system in the case of gonococcal PG,whereas hamster trachea epithelium was the target tissue inthe case of pertussis PG.These findings raise the intriguing hypothesis that release

of cytotoxic PG fragments might be a common denominatorfor at least some bacteria in which pathogenesis dependsinitially on colonization of ciliated mucosal surfaces. Ouroverall objective is to define the structural relationshipswhich govern these functional similarities between gonococ-cal and pertussis PG. Fulfillment of such an objective wouldbe compromised by the current lack of knowledge of the

* Corresponding author.t Present address: Department of Cell and Molecular Pharmacol-

ogy and Experimental Therapeutics, Medical University of SouthCarolina, Charleston, SC 29425.

structure of B. pertussis PG. Accordingly, the specific aim ofthe current work is to provide these structural details.

MATERIALS AND METHODS

Bacteria and media. B. pertussis Tohama phases I and III(obtained from W. E. Goldman, Washington University)were (i) stored at -70°C in a 1:1 mixture of modified liquidStainer-Scholte medium, pH 7.6 (26), containing 1 g ofglutamic acid per liter, and of a solution containing 100 g ofdehydrated skim milk (Difco) per liter and (ii) maintained byserial passage (every 3 days) at 37°C on solid Stainer-Scholtemedium supplemented with 20 g of Noble agar (Difco) perliter and 5% (vol/vol) defribrinated sheep blood. For exper-iments, bacteria were washed from agar plates and inocu-lated at a starting density of approximately 108 bacteria perml in 4-liter Erlenmeyer flasks containing 1.25 liters of liquidStainer-Scholte medium. Liquid cultures were incubated at36.5°C on a rotary shaker. Under these conditions, thegeneration times of phase I and phase III organisms wereapproximately 6 and 4 h, respectively.

Preparation of purified PG, PG-protein complexes, andlow-molecular-weight PG fragments. B. pertussis was grownto the late-exponential phase (approximately 109 bacteria perml) in liquid medium containing 0.2 to 0.4 ,uCi of [G-3H]diaminopimelic acid (DAP; Amersham Corp., ArlingtonHeights, Ill.) per ml. Bacteria were concentrated about100-fold by centrifugation and treated with 10% (wt/vol)ice-cold trichloroacetic acid for at least 30 min. The tri-chloroacetic acid-insoluble residue was washed and treatedwith 40 g of sodium dodecyl sulfate (SDS) per liter at 96°Cfor 1 h in 0.05 M sodium acetate buffer (pH 5.1), aspreviously employed by us to isolate purified intact PG fromN. gonorrhoeae (22).. The SDS-insoluble residue was either(i) washed extensively and lyophilized to obtain a prepara-tion termed PG-protein complexes or (ii) treated overnightwith proteinase K (50 ,ug/ml in 0.05 M Tris hydrochloride,

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TABLE 1. Amino acid analysis of PG-protein, PG, anddisaccharide peptide monomers from B. pertussis

Molar ratiob

Phase I Phase IIIAmino acida

Disaccharide

PG-protein PG PG-protein PG peptidemonomer

Asp 0.45 0.04 0.52 0.04 0.05Thr 0.26 0.03 0.31 0.03 <0.02Ser 0.30 <0.02 0.32 <0.02 <0.02Muramic acid 0.34 0.90 0.29 1.10 0.72Glu 1.00 1.00 1.00 1.00 1.00Pro 0.22 <0.02 0.28 <0.02 <0.02Gly 0.51 0.07 0,57 0.08 0.07Ala 1.62 1.96 1.45 1.99 1.97Val 0.26 <0.02 0.41 <0.02 <0.02Glucosamine 0.40 1.04 0.29 1.17 0.97Met <0.02 <0.02 <0.02 <0.02 <0.02DAP 0.73 1.14 0.30 0.94 1.17Ile 0.25 <0.02 0.23 <0.02 <0.02Leu 0.47 <0.02 0.51 <0.02 <0.02Tyr 0.08 <0.02 0.14 <0.02 <0.02Phe 0.19 0.02 0.20 0.03 <0.02His 0.13 0.03 0.11 <0.02 <0.02Lys 0.24 <0.02 0.26 <0.02 <0.02Arg 0.31 0.02 0.34 0.02 <0.02

a Amounts of cysteine, tryptophan, glutamine, and asparagine could not bedetermined accurately due to their degradation during acid hydrolysis.

bRelative to glutamic acid.

pH 7.4, containing 0.2% SDS; 22), washed extensively, andlyophilized to obtain purified intact PG.To obtain purified disaccharide peptide monomers and

other low-molecular-weight PG fragments, purified intactpertussis PG was digested completely with N-acetyl-muramidase from Streptomyces globisporus (muramidaseSG; Miles Laboratories, Inc., Elkhart, Ind.) as described forgonococcal PG (23). The muramidase-soluble monomers andpeptide-cross-linked dimers and higher oligomers were sep-arated as described previously (24, 28) for gonococcal PGfragments by gel filtration on connected columns of Sepha-dex G-50 and G-25 or by molecular-sieve high-performanceliquid chromatography (HPLC) on connected columns ofTSK SW3000 and TSK SW2000 (Varian, Palo Alto, Calif.).PG standards obtained from N. gonorrhoeae were preparedas described previously (18, 21, 24, 25, 28).

Analytical methods. The following procedures employedfor chemical analysis of gonococcal PG have been applied topurified pertussis PG, PG-protein complexes, and low-molecular-weight PG fragments as described previously:amino acid analysis with a Beckman model 119CL aminoacid analyzer (24), paper chromatography in butanol-aceticacid-water (4:1:5; upper phase) for 17 h (or for 65 h in someexperiments) or in 67% ethanol for 17 h (21, 24, 28),a-elimination of lactylpeptides from reducing PG fragmentsby treatment with 0.05 M NaOH (25, 30), determination ofpercent peptide cross-linking (24), and quantitation of theextent of 0 acetylation in PG fragments derived by sequen-tial treatment of intact PG by muramidase SG and Esche-richia coli endopeptidase (23, 28).

Determination by fast atom bombardment-mass spectrom-etry and tandem mass spectrometry of the molecular weightsand the complete sequences of PG monomers isolated byreversed-phase HPLC on a Vydac C18 column (Separations

Group, Hesperia, Calif.) was performed as described previ-ously (17).To determine the extent and rate of enzymatic degradation

of B. pertussis PG, [3H]DAP-labeled PG or PG-protein wastreated with (i) proteinase K in 0.05 M Tris hydrochloride(pH 7.4) containing 0.2% SDS, (ii) hen egg white lysozyme(muramidase; Boehringer Mannheim Corp., Indianapolis,Ind.) in 0.01 M phosphate buffer (pH 7.4), or (iii)muramidase SG in 0.01 M Tris-maleate-NaOH buffer plus 4mM MgCl2 (pH 7.0), and at intervals trichloroacetic acid-insoluble radioactivity was determined (22). All enzymeswere used at a concentration of S ,ug/ml.

Radioactivity determination. Radioactivity was determinedwith a model 3255 liquid scintillation spectrometer (PackardInstrument Co., Downers Grove, Ill.) interfaced with anApple II microcomputer. The data were corrected forquench and, where appropriate (e.g., dual-labeled samplescontaining 3H-labeled pertussis PG and 14C-labeled PG stan-dards), for overlap of 14C into 3H channels by calibrationcurves determined by external standardization.

RESULTS AND DISCUSSIONComposition of PG-protein and purified PG. Treatment of

B. pertussis (phase I or phase III) with trichloroacetic acidand SDS yielded an insoluble residue that was distinctlycloudy and that was composed of approximately 35 to 45%protein amino acids; the remainder consisted of the aminoacids characteristic of PG from gram-negative bacteria (Ta-ble 1). Treatment of the SDS-insoluble PG-protein com-plexes with proteinase K clarified the suspension consider-ably, drastically reduced the protein content (typically toless than 2%), and yielded purified PG containing muramicacid, glucosamine, alanine, glutamic acid, and DAP in molarratios of approximately 1:1:2:1:1 (Table 1). Amino acidanalysis of several independent lots of purified intact PGfrom phase I and phase III yielded similar results. Further-more, the presence of very small amounts of glycine andaspartate was a consistent finding in these additional prepa-rations, but the other non-PG amino acids (threonine, histi-dine, phenylalanine, and arginine) were only detectedsporadically and thus were not considered a significantcomponent of pertussis PG. No differences in compositionbetween PGs purified from phase I and phase III organismswere detected. Preliminary experiments (J. Gibson-Salyerand R. Rosenthal, unpublished observations) to characterizethe protein(s) that presumably is covalently bound to pertus-sis PG suggested that only a few proteins constituted thebulk of the non-PG material and that these included alow-molecular-weight species of about 10,000 to 15,000.

Fate of 3H added as DAP in PG-protein and PG. Subse-quent structural studies depended heavily on radiochemicalanalysis of 3H that was added to bacterial cultures as[3H]DAP and that was incorporated into purified PG, PG-protein, and PG monomers. Paper chromatography in twodifferent solvent systems of acid hydrolysates of PG-containing preparations indicated that [3H]DAP was incor-porated into PG specifically, i.e., label added as [3H]DAPwas present in PG-protein and PG as DAP, exclusively (Fig.1). Furthermore, with the labeling regimen and cultureconditions outlined above, the 3H was incorporated into thefinal purified PG at a very reasonable specific activity(greater than 106 dpm/mg). In preliminary experiments (datanot shown), we found that under these conditions of growth[3H]DAP was incorporated into PG much more efficientlythan was labeled glutamic acid, which had been used previ-ously (11).

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STRUCTURE OF PERTUSSIS PG 4225

Isolation of muramidase-derived PG fragments and deter-mination of peptide cross-linking. [3H]DAP-labeled PG wasdigested completely with muramidase SG, and the solublePG products were fractionated by gel filtration (or by mo-lecular-sieve HPLC in some experiments). This procedureserved two essential functions: (i) determination of theextent of peptide cross-linking of pertussis PG and (ii)isolation of low-molecular-weight fragments for structuralanalysis. From the distribution of un-cross-linked disaccha-ride peptide monomers and peptide-cross-linked dimers andhigher oligomers, the cross-linking of pertussis PG wasfound to be slightly less than 50% (Table 2), a relatively highvalue among PGs derived from gram-negative bacteria.There was no detectable difference in the extent of cross-linking between phase I and phase III PG.0 acetylation and anhydromuramyl fragments. Labeled

disaccharide peptide monomers were derived from purifiedPG by using muramidase SG alone or by using muramidaseSG followed by E. coli endopeptidase. The former treatmentpermitted isolation of fragments which existed in intact PGas monomer subunits. The latter dual-enzyme procedurewas employed to break peptide-cross-linking bonds (in ad-dition to glycosidic bonds) and thereby to convert insolublePG quantitatively to disaccharide peptide monomer sub-units. Such monomers would include those that existed innative PG as the various cross-linked oligomers as well asthose that existed as un-cross-linked monomers. The result-ing monomeric fragments from either enzyme regimen wereisolated and analyzed (i) to determine the distribution of0-acetylated and non-O-acetylated subunits in pertussis PGand (ii) to evaluate for the presence of nonreducing anhydro-muramic acid-containing fragments. The majority of pertus-sis PG monomers comigrated with the reducing (NaOH-sensitive) non-O-acetylated disaccharide peptide standard(Fig. 2), regardless of their derivation. In fact, we found noevidence for the presence of 0-acetylated subunits in pertus-sis PG. That is, only a small percentage of the 3H monomersderived by sequential muramidase-endopeptidase actioncomigrated with the labeled 0-acetylated monomer standard(Fig. 2), and the small fraction that did comigrate wasresistant to degradation by mild alkali. This compound wastherefore not a reducing monomer containing an O-acetylsubstituent in the number 6 position of muramic acid.

ECL

-

0

toa

3

0

0

CM

FIG. 1. Paper chromatography of DAP standard (STD), acid-hydrolyzed purified PG, and acid-hydrolyzed PG-protein in butanol-acetic acid-water (BAW; 4:1:5) upper phase (UP) or 67% ethanol(ETOH) for 17 h.

H-DAP-LABELEDXEl l MUR-MONOMERS

O-NaOH

E

la 21 4 3H-DAP-LABELEDO l_ I l MUR/ENDO-MONOMERS

0- NaOH

O*NaOH

15 NON-O-N-0-

ANHYDRO-

MONOMERMONOMER |-|

+(or - NaOH) 3H-GLCNH2-LABELED

a L0 STD0-MONOMER *-NaOH

5 / \ ttv0+ NaOH

0 10 20 30 40CM

FIG. 2. Paper chromatography in butanol-acetic acid-water(4:1:5; upper phase) for 65 h of [3H]DAP-labeled B. pertussis PGmonomers derived by muramidase alone (MUR-MONOMERS) orby sequential treatment with muramidase and endopeptidase(MUR/ENDO-MONOMERS) and of glucosamine (GLCNH2)-labeled PG monomer standards. Portions of each sample weretreated before chromatography with NaOH under conditions thatcause the degradation of reducing PG monomers, such as glucosa-mine-labeled non-O-monomer standards (STD), into lactylpeptidesand disaccharides.

Rather, this trace amount of NaOH-resistant monomer wasmost likely a nonreducing 1,6-anhydromuramyl PG deriva-tive. Thus, pertussis PG was non-O-acetylated and pos-sessed a small number of anhydromuramyl ends.Muramidase sensitivity ofPG and PG-protein. Extensive 0

acetylation characteristically renders PG resistant to eggwhite lysozyme relative to its non-O-acetylated counterpart(3, 10, 12, 28); then based on the above data, pertussis PGmight be expected to be lysozyme sensitive. Indeed, purifiedpertussis PG from either phase III (Fig. 3) or phase I (datanot shown) was degraded completely by egg white lysozymeas well as by muramidase SG, consistent with the structuralevidence that it lacks O-acetyl substituents (Fig. 2). PG-

TABLE 2. Peptide cross-linking of B. pertussis PG as determinedfrom distribution of muramidase SG digestion fragments

Distribution of fragments (% of total)a Peptide cross-B. pertussis lnig()Monomer Dimer Trimer Tetramer linking (%)b

Phase I 17.1 47.6 25.7 9.6 48.1Phase III 19.6 44.0 27.5 8.9 47.0

a Means of two independent determinations on different lots of PG fromphase I cells and three independent determinations on different lots of PGfrom phase III organisms.

b Percent cross-linking = (percent dimer x 0.50) + (percent trimer X0.667) + (percent tetramer x 0.75).

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az

IC

a

IL

a

44

I-.

00-

75-

50-

25-

*00

75-

50-

25-

100

75-

50-

25

2 4 6Hr

FIG. 3. Degradation of [3H]DAP-labeled PG-protein and purifiedPG (each obtained from phase III organisms) by proteinase K, eggwhite lysozyme, and muramidase SG; each enzyme was present ata concentration of 5 ,ug/ml.

protein complexes, on the other hand, were relatively resist-ant to egg white lysozyme, and approximately 25% of the[3H]DAP-labeled PG in such complexes remained insolubleeven at completion of the reaction. Thus, protein affordedthe PG at least some protection from enzymatic hydrolysisby lysozyme.

Interestingly, some (typically 10 to 20%) of the 3H inPG-protein complexes was degraded by proteinase K (Fig.3). Because purified 3H-PG was not degraded by proteinaseK (Fig. 3), there was no reason to believe that the proteinasewas contaminated with PG hydrolase; because all of the 3Hin PG-protein was present in DAP exclusively (Fig. 1), therewas no reason to believe that the [3H]DAP had been metab-olized significantly and incorporated into proteinase-sensitive protein. A plausible interpretation of these data isthat a small fraction of the PG in PG-protein was relativelylow molecular weight and was insoluble solely by virtue ofits complexing with a trichloroacetic acid-insoluble protein.

Structural analysis of purified disaccharide peptide mono-mers by fast atom bombardment-mass spectrometry andtandem mass spectrometry. Amino acid analysis revealedthat the muramidase-derived monomer fraction from phaseIII bacteria was free of detectable non-PG contaminants andcontained glucosamine, muramic acid, alanine, glutamicacid, and DAP in molar ratios similar to those of the intactPG from which it was derived (Table 1). Reversed-phaseHPLC further revealed that this muramidase-derived mono-mer fraction was a surprisingly homogeneous set of PGfragments. In fact, greater than 96% of the PG detected waspresent in only two peaks (Fig. 4), which, when isolatedand subjected to fast atom bombardment-mass spectrome-try, both yielded the same parent protonated molecularion, (M+H)+940 (Table 3). Thus, the two peaks resolvedby HPLC were anomers of a single compound with a mass

of 939 daltons. The most probable structure of such a

compound (Table 3) is N-acetylglucosaminyl-N-acetylmur-amyl-alanyl--y-glutamyl-diaminopimelyl-alanine (NAG-NAM-Ala-Glu-DAP-Ala). Indeed, unambiguous structuraldetermination of (M+H)+940 by tandem mass spectrometryverified this sequence. The mass spectrum of (M+H)+940

E

C:

0

0

0

C

0

.0

8

-k

0.00 7.00*1 1

14.00 21.00 28.00

Minutes

FIG. 4. Reversed-phase HPLC on a C18 column of muramidase-derived PG monomers obtained from B. pertussis. A binary solventsystem consisting of (i) H20 (containing 0.05% trifluoroacetic acid)and (ii) CH3CN (containing 0.035% trifluoroacetic acid) was em-ployed with a linear gradient of 3 to 30% (ii) in 30 min at a flow rateof 1 ml/min.

was practically identical to that of the same compoundisolated from N. gonorrhoeae (17). In addition to(M+H)+940, minor amounts of only three other PG mono-mers, including (M+H)+922, NAG-(1,6-anhydro)NAM-Ala-Glu-DAP-Ala, were detected based on fast atom bombard-ment-mass spectrometry of peaks isolated by HPLC (Table3). Consistent with Fig. 2, no 0-acetylated monomers werefound.

It is not surprising that NAG-NAM-Ala-Glu-DAP-Ala wasthe principal compound detected among pertussis PG mono-mers, since this represents one of the more common subunitstructures in gram-negative PG. However, given the exten-sive complexity of and diversity in analogous monomerpreparations obtained from other gram-negative bacteria sofar tested, e.g., E. coli (9, 20) and N. gonorrhoeae (5), B.pertussis PG subunits are remarkable for their apparenthomogeneity.The objective of this study was to provide structural

details on PG derived from B. pertussis. We have found thatpertussis PG (i) exists in complexes containing about 40%protein, (ii) has a gross composition reminiscent of classical

TABLE 3. Structural determination of disaccharide peptidemonomers purified from pertussis PGa

Protonated % Of

molecular Structure total

ion observed

869 NAG-NAM-Ala-Glu-DAP 1.5922 NAG-(1,6-anhydro)NAM-Ala-Glu-DAP-Ala 1.4940 NAG-NAM-Ala-Glu-DAP-Ala 96.11011 NAG-NAM-Ala-Glu-DAP-Ala-Ala 1.0

a Muramidase SG-derived PG monomers were purified by reversed-phaseHPLC and subjected to fast atom bombardment-mass spectrometry.

* PG

O PG-PROTEIN PROTEINASE K

LYSOZYME(MURAMIDASE)

SG MURAMIDASE

- *me f Y

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STRUCTURE OF PERTUSSIS PG 4227

chemotype I PG (8), (iii) is more extensively cross-linkedthan PG of many other gram-negative bacteria, (iv) is notsubstituted by O-acetyl groups, consistent with its suscepti-bility to egg white lysozyme, (v) possesses at least someanhydromuramyl residues at the ends of glycan chains, (vi)consists almost exclusively of monomer subunits with thestructure NAG-NAM-Ala-Glu-DAP-Ala, and (vii) appearsstructurally similar in both phase I and phase III organisms.The structural background provided by these data shouldenhance efforts to define the basis for the pertussis PG-mediated toxicity to ciliated epithelium (11), a propertywhich might conceivably be common to other pathogens thatmust colonize mucosal surfaces (18).

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantsAI-14826 and AI-20110 from the National Institute of Allergy andInfectious Diseases. Mass spectra were obtained at the NationalInstitute of Health Division of Research Resources Mass Spectrom-etry Facility at the Massachusetts Institute of Technology (grantRR00317, K. Biemann, Principal Investigator).We are grateful to Sandra Wilson for excellent assistance in

preparation of this manuscript and to W. S. Wegener for sharing hisunpublished data that [3H]DAP is incorporated efficiently intoBordetella macromolecules and for providing critical review of themanuscript.

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