PREPARATION AND PROPERTIES OF CELL WALLS OF" THE AGENT OFMENINGOPNEUMONITISI, 2

HOWARD M. JENKINS3 4

Department of Microbiology, University of Chicago, Chicago 37, Illinois

Received for publication April 6, 1960

It is recognized that microorganisms of thepsittacosis group are contained within a rigidenvelope (Hamre, Rake, and Rake, 1947) whichis highly resistant to enzymes (Brown, Itatani,and Moulder, 1952) and mechanical disintegra-tion (Ross and Gogolak, 1957). With the demon-stration that penicillin inhibits the growth ofbacteria by interfering with cell wall synthesis(Park and Strominger, 1957; Hahn and Ciak,1957; Lederberg, 1957), the susceptibility ofmembers of the psittacosis group to penicillin(Hurst, 1953) suggested that their outer en-velopes might be analogous to the cell walls ofbacteria. Since these structures have propertiesnot encountered in other kinds of cells (Cumminsand Harris, 1956; Salton, 1956; Work, 1957;Mitchell, 1959), the association of a reasonablenumber of these unique properties with theouter envelopes of members of the psittacosisgroup would be of decisive weight in establishingtheir evolutionary origin.

This report describes the isolation and chemicalcharacterization of the rigid envelopes of theagent of meningopneumonitis, a typical memberof the psittacosis group. These structures willfrom now on be called cell walls as a matter ofconvenience and without any immediate impli-cation of analogy or lack of analogy with bacterialcell walls. Their serological properties will bereported elsewhere (Jenkin, Ross, and Moulder,unpublished data)

' Supported chiefly by research grant E-1594from the National Institute of Allergy and Infec-tious Diseases, Public Health Service, and in partby grants from the Dr. Wallace C. and Clara A.Abbott Memorial Fund of the University ofChicago and from the Abbott Laboratories, NorthChicago, Illinois.

2 A preliminary account of this work has al-ready been published (Jenkin, 1959).

3 Mr. and Mrs. Frank G. Logan Fellow in Micro-biology, 1959.

4Present address: U. S. Army Chemical Corps,Fort Detrick, Frederick, Maryland.

METHODS

Preparation of purified particles. The Cal 10strain of meningopneumonitis virus was passed2 times at limiting dilution in chick embryo yolksac and then 8 to 10 times in the allantoic cavitybefore being used as an inoculum. Purified intactparticles were separated from infected allantoicfluid by four cycles of high and low speed cen-trifugation at 0 C (Col6n and Moulder, 1958).In each cycle, the particles were sedimented at5,000 X g for 30 min, resuspended in 0.1 Msodium phosphate buffer, pH 7.4, and clarifiedat 800 X g for 10 min. The low speed pelletwas discarded and the particles sedimented athigh speed to begin the next cycle. Immedi-ately after the final centrifugation, the chickembryo LD50 was measured by yolk sac titration(Moulder and Weiss, 1951), the total particlecount was determined by Sharp's (1949) sedi-mentation method as modified by Litwin (1959),and bacteriological sterility was established byinoculation of thioglycolate broth.

Preparation of cell walls. Freshly prepared puri-fied particles were suspended in 0.01 M phosphatepH 7.4, containing 1 per cent sodium deoxycho-late (The Matheson Company, Inc.) and shakenfor 4 hr at 45 C (Schaechter et al., 1957). Thepreparation was cooled to 0 C, centrifuged for20 min at 8,000 X g, and the pellet resuspendedin 0.1 M phosphate, pH 7.4, containing 1 mg perml trypsin (two times recrystallized, in 50 percent MgSO4. Nutritional Biochemicals Corpora-tion). The tryptic digest was shaken 1 hr at 37 C,cooled again to 0 C, and sedimented for 20 minat 8,000 X g. The sediment was then washedthree times in distilled water to yield the finalcell wall preparation which was cultured to testfor bacteriological sterility and counted in thesame manner as intact particles.

Electron microscopy. The electron microscopewas used to count particles and to follow thecourse of purification of intact particles and cellwalls. Both types of particles were suspended in

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TABLE 1Relatiotns between dry weight, total nitrogen, particle count, and LD50

Dry Weight per Total Nitrogen per Total Nitrogen per Particles per ChickParticle Particle Chick Embryo LDso Embryo LD5o

X 1-14 g

No. of preparations ...... 11 7 8 8Intact particles.......... 1.87 + 0.13a 0.14 i 0.01 10.0 i 1.0 66 i litbCell walls ............... 0.85 ± 0.07 0.08 i 0.02

a Mean + standard error.b Similar values have been reported by Crocker (1954) ancl Smith and Manire (1959).

dlistilled water or the volatile salt ammoniumacetate. They were (leposited on formvar-coatedcopper gridIs directly or by sedimenting or spray-ing. The samples were (Iried in air and examinedin an RCA electron microscope, with and withoutchromium shadowing at an angle of 16°.

Analytical methods. Dry weights were deter-mined by drying to constant weight at 60 C invacuo.

Total nitrogen wAas (letermined accotding toGroman (1951); a-amino nitrogen according toSobel, Hirschman, an(d lBesman, (1945) andSpeck (1949).

Phosphorus partitions were performed bySchneider's (1945) method as modified by Zahler(1953). Bartlett's (1959) conditions were usedfor phosphate determination.

Two-dimensional chromatography of aminoacids was carried out according to Cummins andHarris (1956). Samples were hydrolyzed in 6 NHCl for 16 hr at 100 C. The solvents werephenol:water (80:20) over 1 per cent NH40Hand NaCN followed by 2, 6-lutidine:ethanol:water :diethylamine (55:25:18:2).

Total nucleic acid was measured in terms of the260 m,u absorbancy of hot perchloric acid extracts(Ogur and Rosen, 1950). Deoxyribonucleic acidwas determined byN the method of Webb andLevy (1955); ribonucleic acid by that of Webb(1956).Reducing sugars were measured according to

Park and Johnson (1949) before and afterhydrolysis in 2 N HCl for 2 hr at 100 C. Hex-osamines were released only after 8 hr in 6 NHCl at 100 C. They were determined by themethod of Dische (1955).

Qualitative lipid analyses were made by thechromatographic procedure of Hack (1953).

RESULTS

It is difficult to apply the concepts of purityand homogeneity to populations of members ofthe psittacosis group which are made up ofparticles of widely different size and internalorganization (Litwin, 1959). However, the intactparticle preparations w-ere almost completelyfree of extraneous particles when viewed in theelectron microscope and were of relatively con-stant composition as judged by the reproduci-bility of the dry weight per particle, total nitrogenper particle, total nitrogen per chick embryoLD50, and particles per LD50 (table 1).

Disruption of particles by mechanical forces.TMechanical means of (lisruption are generallypreferred for preparing bacterial cell walls be-cause they minimize extraction of cell wall com-ponents during the course of purification. How-ever, the meningopneumonitis particles weredisrupted only partiallv or not at all in the Mickleapparatus and the French pressure cell or byalternate freezing and thawing. Sonic disintegra-tion was not satisfactory because the particlesresisted 9 ke for 3 hr an(l then fragmented intosmall pieces (Ross and Gogolak, 1957).

Effect of deoxycholate on intact particles. Sincemechanical methods for particle disruption wereunsuccessful, the method of Schaechter et a].(1957) for preparing cell walls of rickettsiae wasnext tried. As observed by these workers forRickettsiae mooseri, maximal loss of opticaldensity occurred at 45 C in the presence of 1 percent sodium deoxycholate (figures 1 and 2).Almost all nucleic acid, as measured by the 260m,u absorbency of perchloric acid extracts, wasalso lost under these conditions, and infectivitywas rapidly destroyed. However, electron micro-graphs of deoxycholate-treated particles revealedonly slight loss of electron-dense internal mate-

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1MENINGOPNEUMoNITIS CELL WALL

u.21 I0 20 40 240

MINUTES

Figure 1. Effect of different concentrations ofdeoxycholate on the agent of meningopneu-monitis. Various concentrations of deoxycholatewere added to intact particles in 0.01 M phosphatebuffer, pH 7.4. The mixtures were incubated at45 C, samples removed at different times, and theoptical density measured at 460 msA. Each pointrepresents the mean of two independent experi-ments.

rial (figure 3C). These results are difficult tocompare with those of Crocker (1956) whotreated the agent of meningopneumonitis withdeoxycholate under a different set of conditionsand studied the effect of this reagent in differentways.

Effect of trypsin on deoxycholate-treated particles.Ribonuclease and deoxyribonuclease had no effecton deoxycholate-treated particles. However,trypsin destroyed the electron-dense internalmaterial of these particles to yield the structuresshown in figure 3D which are called cell wallsbecause (1) they are rigid enough to determinethe size and shape of the intact particles fromwhich they were derived, (2) they are almostcompletely free of electron-dense internal mate-rial, and (3) they contain only traces of nucleicacid.

In marked contrast, untreated intact particleswere unaffected by trypsin (Brown et al., 1952)(figure 3B). This sharp change from trypsinresistance to trypsin sensitivity suggests a chemi-cal alteration of the cell wall by deoxycholate.

MIorphology of cell walls. Purified preparations

07

>z * - ..jI

Id

Q

05

-J @ 0

0.4-5

0.310 30 60 240

MINUTES

Figure 2. Effect of temperature on the agent ofmeningopneumonitis in the presence of 1 per centdeoxycholate. Conditions were as given in figure 1.

of intact particles contained two morphologicaltypes, a dense-centered particle 250 to 300 m,uin diameter and a larger, flatter particle of 350to 1000 m,u diameter. This particle showed scat-tered areas of electron-dense material instead ofa single central mass (figures 3A and 4A). Thesetwo kinds of particles appear to be present inmature populations of all members of the psitta-cosis group (see Litwin (1959) for discussion).The cell walls were greatly altered in appearance,although it was readily apparent which walls werederived from dense-centered particles and whichcame from flat ones (figures 3D and 4B). In form-ing cell walls, the dense-centered particles losttheir central mass, and the residual structureresembled a collapsed and empty envelope. Theflat particles became even flatter, lost theirelectron-dense granules, and acquired a roughersurface texture. These visible surface differencesare paralleled by differences in surface charge ofthe two kinds of intact particles as revealed bystarch electrophoresis and cellulose-anion chro-matography (Jenkin and Moulder, unpublisheddata).

Relation of cell wall mass to that of the intactparticles. About 30 per cent of the dry weight ofintact particles was recovered in the form of cellwalls, roughly the same recovery generally re-ported for the cell walls of bacteria. However,total particle counts showed a significant loss of

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Cr 4

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S

D

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.; .. ..

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Figure 3. Electron micrographs showing the effects of trypsin and deoxycholate in the preparation ofmeningopneumonitis cell walls. Unshadowed air-dried preparations (X13,500). A. untreated intactparticles; B. trypsin-treated particles; C. deoxycholate-treated particles; D. deoxycholate-trypsin-treated particles-the final cell wall preparation

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1960]~~MENJNGOPNEUMONITJS CELLWALL64

44~~~~~~~~~~~~~~~......

D

E

lp

Figure 4. Electron micrographs comparing deoxycholate-trypsin-treated meningopneumonitis agent,Escherichia coli and Staphylococcus aureus. Chromium shadowed, air-dried preparations (X 12,500).A: meningopneumonitis-intact particles; B: meningopneumonitis-deoxycholate-trypsin-treated par-tidles (cell walls); C: E. coli-intact cells; D: E. coli-deoxycholate-trypsin-treated cells; E: S. aureus-intact cells; F: S. aureus-deoxycholate-trypsin-treated cells.

4

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TABLE 2

Chemical composition of intact particles and cell walls

Dry WeightConstituent No. of

PreparationsIntact particles Cell walls

ca-Amino acid nitrogen.......................Total nitrogen...............................Proteinb ...................................

Hexosamine ..................................Reducing sugar (hydrolyzed).................Muramic acid................................Carbohydratec ...............................

Ribonucleic acid.............................Deoxyribonucleic acid........................Total nucleic acid............................

Phospholipidd ... ......................

a Mean ± standard error.

33

342

333

3

5.30 + 0.15a6.50 i 0.40

33.1

0.362 ± 0.041.05 + 0.05

Trace1.67

2.50 ± 0.083.40 ± 0.065.62 i 0.30

7.5

5.207.20

32.5

4 0.31+ 0.30

0.44 i 0.051.14 i 0.02

Trace1.58

TraceTrace

0.40 i 0.10

1.5

I Calculated by multiplying the a-amino acid nitrogen by a factor of 6.5.c Calculated from the sum of the hexosamine and reducing sugar; hydrolytic conditions for reducing

sugar were insufficient to release hexosamine.d Calculated from the alcohol-ether soluble phosphorus fraction by multiplying by a factor of 25.

particles during cell wall preparation. When therecovered cell wall weight was corrected for thisloss, the yield was increased to 45 per cent of thedry weight of intact particles (table 1). The cor-rected recovery of total nitrogen was also about45 per cent.

Chemical properties of intact particles and cellwalls. Table 2 compares analytical results on in-tact particles and cell walls. The two kinds ofparticle preparation gave almost identical resultsfor total nitrogen and a-amino nitrogen. Thetotal protein calculated from the a-amino nitro-gen was lower than comparable values for bac-teria.The amino acid composition of cell walls is of

particular interest because gram-positive bac-terial cell walls contain as few as 3 amino acids,most frequently alanine, lysine (or diamino-pimelic acid), and glutamic acid, whereas gram-negative walls yield most of the amino acids com-monly found in proteins (Salton, 1956; Work,1957). Twelve to 14 amino acids were positivelyidentified in hydrolyzates of intact particles andonly 9 to 10 in cell walls. Aspartic acid, alanine,glutamic acid, glycine, and methionine/valinewere present in highest concentrations in both

preparations. Isoleucine/leucine was found inhigher concentration in intact particles than incell walls. Small amounts of threonine, serine,and phenylalanine appeared in both intactparticles and cell walls, whereas traces of tyrosineand proline were found only in intact particles.A small amount of lysine, but no diaminopimelicacid, was detected in both intact particles andcell walls.Hexosamines and reducing sugars were pres-

ent in low and approximately equal concentra-tions in both preparations. A sensitive test formuramic acid (Strominger, J. L., personal com-munication) revealed a trace of this amino sugarin both intact particles and cell walls. This is ofinterest since muramic acid has been found onlyin bacterial cell walls.

Table 3 shows the results of a phosphorus par-tition according to Schneider (1945). The acid-soluble phosphorus fraction was markedlyvariable, despite repeated washing of the particlesin distilled water to remove inorganic phosphate.This probably reflects the lability of the acid-soluble fraction demonstrated by Zahler and1\oulder (1953) in experiments with radioactivephosphate. If the acid-soluble fraction is disre-

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garded, then relatively constant phosphorusvalues were obtained for the other three fractions.The total nucleic acid phosphorus content ofintact particles was 0.37 per cent, equivalent to4 per cent nucleic acid. This is a little lower thanthe total nucleic acid value of 5.8 per cent givenin table 2, probably because ribonucleic acid was

lost during the extra washing in distilled water(Rappaport et al., 1960). The cell walls containedonly 0.04 per cent nucleic acid phosphorus. Thus,this determination, measurement of 260 m,u ab-sorbancy and estimation of ribonucleic acid anddeoxyribonucleic acid by specific color tests, allindicated the almost complete absence ofnucleic acid from the cell wall preparations.The lecithin equivalent of the phospholipid

phosphorus was 7.5 per cent for intact particlesand 1.5 per cent for cell walls (table 3). The lossof phospholipid during cell wall preparation prob-ably occurred during the deoxycholate treatmentand was associated with the loss of group-specificcomplement-fixing antigen because the phospho-lipid containing group antigen (Ross and Gogo-lak, 1957) was quantitatively recovered in thedeoxycholate extract (Jenkin, Ross and Moulder,unpublished data). Present data do not allow a

definite decision as to whether the phospholipidand the group antigen were extracted from theendoplasm of the particles or from their cellwalls, but the change in sensitivity to trypsinafter deoxycholate treatment suggests that thephospholipid came from the cell walls.

Filter paper chromatography (Hack, 1953)showed the absence of detectable amounts ofcholesterol and the presence of other nonphos-phorus lipids such as neutral fat in both cellwalls and intact particles. Attempts to estimatetotal lipid by direct extraction with lipid solventsgave erratic results, probably because of the verysmall amounts of material available for study.However, there is no doubt that both intactparticles and cell walls contain large amounts oflipid. Only about 50 per cent of the dry weight of

either type of preparation is accounted for by theanalyses reported in table 2; most of the re-

mainder is probably lipid of as yet unknowncomposition.

Effect of deoxycholate on meningopneumonitisvirus and bacteria. With the finding that cell wallscan be obtained from rickettsiae (Schaechter etal., 1957) and from the psittacosis group by treat-ment with deoxvcholate, it became of interest to

TABLE 3Phosphorus partition in intact particles

and cell walls

Dry WeightFraction

Intact particles Cell walls

Acid-soluble P....... 1.44 i0.25a 0.08 4:0.02Alcohol-ether solubleP ................. 0. 30 ±4 0.02 0.06 4± 0.02

Nucleic acid P. 0.37 i 0.03 0.04 i 0.01Phosphoprotein P O. 12 i 0.02 0.04 i 0.00Total P (sum of frac-tions). 2.23 i 0.12 0.22 i 0.01

Total P (direct de-termination). 2.05 0.07 0.20 ± 0.00

a Mean i standard error of 3 independentpreparations.

see what effect this reagent would have on typicalbacteria. Therefore, gram-positive Staphylococ-cus aureus (Oxford strain) and gram-negativeEscherichia coli (strain B) were inoculated intoveal infusion broth, incubated 18 hr at 37 C,washed three times with 0.1 M phosphate buffer,pH 7.4, and treated with deoxycholate and tryp-sin as in the preparation of meningopneumonitiscell walls. Extremely thin and fragile cell wallswere obtained from E. coli (figures 4C and 4D),but S. aureus was not visibly affected by thetreatment (figures 4E and 4F).

DISCUSSION

The isolation of cell walls from the agent ofmeningopneumonitis has provided a new ap-proach to the study of the structure, chemistry,and serology of the psittacosis group. Theseresults and those reported elsewhere (Jenkinet al., unpublished data) on the serology of intactparticles and cell walls should be repeated as soonas possible on cell wall preparations obtained bypurely physical means, so that the question ofchemical alteration of the cell wall during prepa-ration will not arise. Such a method for obtainingcell walls would also yield the endoplasmic con-tents of the particles in a form suitable forchemical examination. A search for an effectivemethod for physically disrupting meningopneu-monitis particles is now in progress.The discovery of appreciable quantities of

diaminopimelic acid, muramic acid, or teichoieacid (Armstrong et al., 1958) in mneningopneu-

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monitis cell walls would have been definite evi-dence of a relation to bacteria. The trace ofmuramic acid found in intact particles and cellwalls is most interesting, but further study isrequired to assess its significance. In the absenceof such clear-cut findings, the relationship be-comes more difficult to discern. It is at once ob-vious that the meningopneumonitis cell walls arefar too complex in chemical composition to becompared with the cell walls of gram-positivebacteria. They more closely resemble the cellwalls of rickettsiae and gram-negative bacteria.However, they contain less carbohydrate andmore lipid than the gram-negative walls so faranalyzed, and their chemical composition is alsoclearly different from the cell walls of R. mooseri(Schaeehter et al., 1957).These results, while not conclusive in them-

selves, add to the growing weight of morpho-logical (Litwin, 1959), enzymological (Col6n,1960), and genetic (Greenland, 1959) evidencethat the members of the psittacosis group re-semble the bacteria inl most of their fundamentalproperties.

ACKNOWLEDGMENTS

I wish to thank Professor J. '. Mfoulder for hisadvice and suggestions throughout the course ofthis investigation. I am also indebted to Dr. J. L.Strominger for his advice and authentic samplesof muramic acid, to Dr. J. A. Hayashi and Dr.J. A. Cifonelli for samples of Park's nucleotides,to Dr. L. E. Rhuland for the diaminopimelic acidanalysis, and to M'I. Rappaport for portions of thenucleic acid analyses. The assistance of J. Mather,0. Keller, and P. Redden in the electron micros-copy is gratefully acknowledged. I also wish tothank the Walter G. Zoller Memorial DentalClinic for the use of their electron microscope inportions of this work.

SUMMARY

Treatment of purified preparations of theagent of meningopneumonitis with deoxycholateand trypsin yielded structures which are calledcell walls because they retained the size and shapeof the intact particles, contained only traces ofnucleic acid, and were almost completely free ofelectron-dense internal material. The morpho-logical and chemical properties of the cell wallswere compared with those of the intact particles.Morphologically, the cell wsalls appeared as col-

lapsed envelopes devoid of the electron-densemasses characteristic of the intact particles.Chemically, they were similar to intact particles,but differed from them in having almost nonucleic acid, a different amino acid composition,and less phospholipid. In some chemical proper-ties and in reaction to deoxycholate, meningo-pneumonitis cell walls resembled those of rick-ettsiae and gram-negative bacteria.

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