JOURNAL OF BACTERlOLOGY, Jan. 1970, p. 278-285 Copytight a) 1970 American Society for Microbiology Vol. 101, No. 1 Printed In U.S.A. Electron Microscopic Observations on the Effects of Penicillin on the Morphology of Chlamydia psittaci A. MATSUMOTO AND G. P. MANIRE Department of Bacteriology and Immunology, School o Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 21 August 1969 L ceRs were infected at high multiplicity with meningopneumonitis organisms and incubated in medium containing 200 units per ml of penicillin. At intervals up to 48 hr after infection, cells were removed and thin sections were prepared for electron microscopic studies on the morphology of the developing organism. Penicillin had no effect on the initial reorganization of the infecting elementary body to form the de- velopmental reticulate body (RB), and, up to 12 hr after infection, the treated and untreated cultures were identical. After that time, however, penicillin-treated orga- nisms showed striking differences in that binary fission was prevented, large ab- normal RB forms were seen in great numbers, masses of RB cytoplasmic membranes and envelopes were formed within and outside the RB itself, and large numbers of empty or partially filled small vesicles were pinched off the RB. After 36 hr immature nucleoids were formed within the RB. Throughout all of this period, both the outer cell envelope and the cytoplasmic membrane of these RB were recognized. When infected cells were transferred into penicillin-free medium, the abnormal RB showed recovery to form normal RB both by a budding-like process and by internal frag- mentation or subdivision rather like endosporulation. We have concluded that penicillin inhibits binary fission and prevents the synthesis of certain components essential for the formation of the elementary body envelope. Organisms in the genus Chlamydia undergo a complex growth cycle in which the infectious form [elementary body (EB)] penetrates a sus- ceptible cell and is converted into a large develop- mental form [reticulate body (RB)] which multi- plies by fission, then undergoes maturation to form EB (3). In the case of the meningopneumo- nitis strain of C. psittaci, the conversion of EB to RB occurs within 4 to 6 hr after infection, and RB maturation to form EB begins about 19 hr after infection (8). In a previous publication from this laboratory, observations on the effects of penicillin on this growth cycle were reported (10). Penicillin does not inhibit conversion of EB to RB, but it does prevent any maturation of RB to form EB. The RB forms continue to grow until host cell exhaus- tion, and there is continuous increase in RB forms for at least 40 hr after infection. Both RB enve- lopes and intact RB forms harvested at 40 hr resembled those harvested at 18 hr in chemical composition. Weiss reported in 1950 (13) that penicillin pre- vented binary fission of Chiamydia but caused the growth of large abnormal forms, which could be observed by light microscopy in infected cells. Similar results were reported by Tajima et al. (7), who studied meningopneumonitis (MP) organisms in penicillin-treated yolk sacs by elec- tron microscopy. Abnormally large forms of MP organisms were observed in infected cells, and no evidence of normal maturation was seen. We have extended these observations in a study of the fine structure of the MP organisms during their growth cycle, both in the presence of peni- cillin and after removal of the drug. These obser- vations are described in this paper. MATERIALS AND METHODS Organisms and cells. The MP strain of Chlamydia psittaci was cultured in suspended L cells as pre- viously described (10). Preparation of penicillin-treated cultures. For cultures of L cells infected at high multiplicity, 60 ml of medium containing 2 X 106 L cells per ml were centrifuged at 300 X g for 10 min; the cells were 278 on July 13, 2020 by guest http://jb.asm.org/ Downloaded from
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JOURNAL OF BACTERlOLOGY, Jan. 1970, p. 278-285Copytight a) 1970 American Society for Microbiology
Vol. 101, No. 1Printed In U.S.A.
Electron Microscopic Observations on the Effects ofPenicillin on the Morphology
of Chlamydia psittaciA. MATSUMOTO AND G. P. MANIRE
Department ofBacteriology and Immunology, School o Medicine, University ofNorth Carolina, Chapel Hill, North Carolina 27514
Received for publication 21 August 1969
L ceRs were infected at high multiplicity with meningopneumonitis organisms andincubated in medium containing 200 units per ml of penicillin. At intervals up to 48hr after infection, cells were removed and thin sections were prepared for electronmicroscopic studies on the morphology of the developing organism. Penicillin had noeffect on the initial reorganization of the infecting elementary body to form the de-velopmental reticulate body (RB), and, up to 12 hr after infection, the treated anduntreated cultures were identical. After that time, however, penicillin-treated orga-nisms showed striking differences in that binary fission was prevented, large ab-normal RB forms were seen in great numbers, masses ofRB cytoplasmic membranesand envelopes were formed within and outside the RB itself, and large numbers ofempty or partially filled small vesicles were pinched off the RB. After 36 hr immaturenucleoids were formed within the RB. Throughout all of this period, both the outercell envelope and the cytoplasmic membrane of these RB were recognized. Wheninfected cells were transferred into penicillin-free medium, the abnormal RB showedrecovery to form normal RB both by a budding-like process and by internal frag-mentation or subdivision rather like endosporulation. We have concluded thatpenicillin inhibits binary fission and prevents the synthesis of certain componentsessential for the formation of the elementary body envelope.
Organisms in the genus Chlamydia undergo acomplex growth cycle in which the infectiousform [elementary body (EB)] penetrates a sus-ceptible cell and is converted into a large develop-mental form [reticulate body (RB)] which multi-plies by fission, then undergoes maturation toform EB (3). In the case of the meningopneumo-nitis strain of C. psittaci, the conversion of EB toRB occurs within 4 to 6 hr after infection, andRB maturation to form EB begins about 19 hrafter infection (8).
In a previous publication from this laboratory,observations on the effects of penicillin on thisgrowth cycle were reported (10). Penicillin doesnot inhibit conversion of EB to RB, but it doesprevent any maturation of RB to form EB. TheRB forms continue to grow until host cell exhaus-tion, and there is continuous increase in RB formsfor at least 40 hr after infection. Both RB enve-lopes and intact RB forms harvested at 40 hrresembled those harvested at 18 hr in chemicalcomposition.Weiss reported in 1950 (13) that penicillin pre-
vented binary fission of Chiamydia but caused thegrowth of large abnormal forms, which could beobserved by light microscopy in infected cells.Similar results were reported by Tajima et al.(7), who studied meningopneumonitis (MP)organisms in penicillin-treated yolk sacs by elec-tron microscopy. Abnormally large forms of MPorganisms were observed in infected cells, and noevidence of normal maturation was seen.We have extended these observations in a study
of the fine structure of the MP organisms duringtheir growth cycle, both in the presence of peni-cillin and after removal of the drug. These obser-vations are described in this paper.
MATERIALS AND METHODSOrganisms and cells. The MP strain of Chlamydia
psittaci was cultured in suspended L cells as pre-viously described (10).
Preparation of penicillin-treated cultures. Forcultures of L cells infected at high multiplicity, 60 mlof medium containing 2 X 106 L cells per ml werecentrifuged at 300 X g for 10 min; the cells were
VOL. 101, 1970 MORPHOLOGICAL EFFECTS OF PENICILLIN ON C. PSITTACI
washed with fresh medium, and resuspended in 5 mlof medium containing 2 X 108 particles per ml ofpartially purified MP organisms. After adsorptionfor 60 min at 37 C with continuous agitation, thecells were washed three times in phosphate bufferedsaline (PBS), washed once with fresh medium, thensuspended in 120 ml of fresh medium to make two 60-ml cultures. To one bottle, penicillin G sodium wasadded to a final concentration of 200 units per ml.Both treated and control cultures were incubated at37 C on a rotary shaker. Beginning at 4 hr after cellinfection, 3 ml of suspension of each culture wasremoved at 3- to 4-hr intervals up to 48 hr [multi-plicity of infection (MOI) = 10].
For cells infected at low multiplicity, the inoculumwas diluted 1 to 100, and the procedure was carriedout as above (MOI = 0.1).
Preparation of thin sections for electron microscopy.The 3-ml suspensions removed at intervals weresedimented at 300 X g for 0.5 min in a clinicaltable top centrifuge. Before fixation, cells were ex-amined in stained smears. The packed cells were fixedin 1% Os04-Veronal acetate buffer at pH 7.3 for 90min in an ice bath, dehydrated in acetone, and em-bedded in Vestopal W. Thin sections were cut on aReichert ultramicrotome and stained with uranylacetate (12) and lead hydroxide (6). All sections wereexamined in the Akashi TRS-50EI microscope withthe 50 JAm objective aperture.
RESULTS
Observations on the sequential development oforganisms in the presence of penicillin. In speci-mens prepared from 20-hr cultures, no differencescould be seen in the inclusions in cells infected athigh and low multiplicities, except for the relativenumber of inclusions per cell. The results aredescribed, consequently, without regard to MOI.
Observations on the control series in each casewere essentially the same as previously describedby others (2, 3). A typical inclusion of untreatedRB at 24 hr after infection is shown in Fig. 1Awhere several organisms undergoing binary fis-sion are clearly seen. These RB forms are all 0.5to 1 Am in diameter and have relatively homoge-neous internal structure. In Fig. 1B, a similarinclusion at 48 hr is seen in an untreated cell. Inthis inclusion, there are typical RB forms under-going binary fission, completely mature EB forms,and at least two intermediate forms in whichmaturation is beginning. The diameter of thesetransition forms is similar to that of RB.
In Fig. IC through 3A, the growth of MP orga-nisms in penicillin medium is shown at 12 to 48hr after infection. In Fig. IC, each RB is seen in aseparate inclusion, indicating that no fission hastaken place. The inserted figure shows the inclu-sion membrane, envelope, and cytoplasmic mem-brane of this organism. These RB organisms were
not morphologically different from RB seen at12 hr in untreated cultures.
After 18 hr, the RB organisms show definiteabnormal growth (Fig. 1D). The RB is enlarged,and some empty or partially filled vesicles areenclosed in the inclusion membrane.
In Fig. 1E, two fused inclusions are seen, eachcontaining one enlarged RB and numerous smallRB-like structures and empty vesicles. The upperRB shows definite internal membranes. This sec-tion is from a culture harvested 24 hr after infec-tion. In Fig. 2A, internal membrane formation isobvious. This was never seen before 20 hr butbecame progressively more prominent with time.The cytoplasm of this RB appears to be distrib-uted homogeneously, with fine membranes inthe center as indicated by the arrow. Numerousempty and partly filled vesicles are also con-tained within this inclusion. The origin of thesestructures is not certain, but there is evidence inolder cultures that they are pinched-off portionsof the RB envelope.
After 20 hr, some quite normal appearing RBare seen in inclusions. In Fig. 2B, two or three ofthese are seen in the inclusion with one large RBat 28 hr after infection. The arrows indicate aflat membrane sheet surrounding the RB. Thisstructure was not observed before 28 hr. Figures2C and 2D show two inclusions at 30 hr afterinfection. Within the large RB, some areas areseen that appear to be encircled with membrane.In Fig. 2C, a small RB is seen which appears to bewithin the large RB envelope but to have its ownlimiting cytoplasmic membrane. The areas indi-cated by solid arows in Fig. 2D show connec-tions between large and small RB forms suggest-ing a buddinglike process. The broken arrowpoints to a very narrow connection between asmall RB and an empty membrane in what verylikely indicates the origin of these small emptyvesicles.Many large RB forms are seen in Fig. 2E,
which was prepared 36 hr after infection. Of par-ticular interest is the very large amount of enve-lope production under the influence of penicillin.These appear to be in layers arising from the RBenvelopes. These are not tubular projections butthe same flat sheets seen at 28 hr in Fig. 2B.Whether they contain both cell wall and cyto-plasmic membrane is not known.RB forms grown in penicillin medium for
44 hr are shown in Fig. 2F. In these RB, therewas a tendency for single or multiple "nucleoid"formation, such as one sees in the intermediateform in untreated cultures. No evidence of fur-ther maturation was seen. These nucleoid forma-tions were seen in many RB organisms after 44 hr
FIG. 1. C. psittaci in L cells. Bar, I pm. (A) Inclusion seen in untreated cell at 24 hr after infection. Sev-eral organisms show binary fission. X 16,000. (B) Part of an inclusion in untreated cell 48 hr after infection.Many mature elementary bodies, reticulate bodies, and intermediate forms with dispersed cores are seen. X 16,000.(C) Reticulate bodies seen at 12 hr of incubation in penicillin. Each organism is located within an inclusion mem-brane. X 16,000. The insert at higher magnification shows the outer reticulate body envelope (solid arrow) andcytoplasmic membrane (broken arrow). X 32,000. (D) Reticulate body at 18 hr of incubation in penicillin. Largereticulate body, small vesicles (solid arrows), and empty vesicles (broken arrows) are seen. X 20,000. (E) Fusion oftwo inclusions seen at 24 hr. Internal membrane is seen in the upper RB. X 16,000.
(Fig. 1 on page 281)
FIG. 2. C. psittaci in L cells. (A) Large reticulate body containing internal membrane (solid arrow) and smallempty or partly filled vesicles after 20 hr of incubation. X 20,000. (B) Inclusion seen at 28 hr. Large and smallreticulate bodies and empty vesicles are seen. Flat membrane structures have appeared (solid arrow). X 14,000.(C) Inclusion seen at 30 hr. The arrow indicates a small reticulate body produced within cell envelope of largebody. X 20,000. (D) Inclusions at 30 hr. The production ofsmall RB from large abnormal form is indicated bysolid arrow. The broken arrow indicates empty vesicle being formedfrom small RB. X 15,000. (E) Inclusion at 36hr. Large irregularly shaped RB with sheets offlat layers of envelope emergingfrom RB envelope. X 16,000. (F)Inclusion at 40 hr. Some RB contain several nucleoids. Arrows indicate structures which resemble intermediateforms between RB and EB. X 14,000.
(Fig. 2 on page 282)
FIG. 3. C. psittaci in L cells. (A) Inclusion at 48 hr containing two large RB forms. One is subdivided into smallareas containing dense nucleoids indicating some maturation. Large amounts of internal membrane are evident.X 16,000. (B) Inclusion at 28 hr after infection and 8 hr after removal of penicillin. Normal RB forms are seen,and it appears that the large RB is undergoing budding. X 14,000. (C) Inclusion at 44 hr after infection and 24hr after removal ofpenicillin. Normal RB and the budding process are evident (arrows). X 16,000. (D) Inclusion at44 hr after infection and 24 hr after removal ofpenicillin. Most RB forms appear normal with old RB envelopefragments still evident. X 15,000. (E) Inclusion at 36 hr after infection and 16 hr after removal of penicillin.Part ofold RB envelope can be seen around normal RB. X 15,000.
but not at all before 36 hr. Very large numbers ofempty or partially filled small vesicles were seenin each inclusion. In Fig. 3A, two large RB areseen in which nucleoid condensation and internalmembrane formation are evident. In none of thesedid complete maturation occur.Recovery of MP from penicillin treatment. Two
L cell cultures (60 ml per flask) were infected athigh MOI and treated with penicillin (200 units/ml) as before. After 20 hr of incubation, the cellswere washed 3 times in PBS, washed twice infresh medium, and suspended in fresh medium. Toone was added penicillin (200 units/ml) and theother was left untreated. Both were incubated onthe rotary shaker. Beginning at 20 hr after infec-tion, samples were removed at 4-hr intervals andsections were prepared for electron microscopy.
In cells harvested at 20 and 24 hr, there was nodifference between the treated and untreated cul-tures, and no evidence of recovery of the largeRB was seen. At 28 hr after infection, or 8 hr afterdrug removal, definite evidence of recovery andactive binary fission were seen. Figure 3B wasprepared from cells at 28 hr, and the arrows indi-cate the plane of fission, although large RB arestill present. It would appear that the first normalRB was perhaps formed by budding, and theothers by binary fission.
Figure 3C shows inclusions at 42 hr afterinfection and 22 hr after drug removal. Thisshows a large RB with connections to RB formswith more normal morphology. Numerousnormal RB forms and some complete EB formsare seen in this inclusion.In Fig. 3D and 3E, evidence of internal repro-
duction within a large RB is seen. In both ofthese it appears that a large RB gave rise tonumerous normal RB forms by a process akinto endosporulation, and that parts of the originalenvelope can still be seen around these groups.These have been seen in numerous preparationsof cells 16 to 28 hr after drug removal.
DISCUSSIONIn the normal development of C. psittaci in L
cells, the EB is enclosed within an inclusionmembrane from the time of penetration into thecell, and this membrane is derived from the hostcell membrane. Within 6 hr the EB is convertedinto the reproductive form referred to as RB,and within 12 hr after infection the RB formsbegin to undergo binary fission (3). This processcontinues as long as the cell can support growth,with most host cells showing lysis in 40 to 48 hr.Even at that time numerous RB forms can beseen in unlysed cells. The process of maturationof RB to form EB begins at about 18 to 20 hr
after infection, when some RB undergo a con-densation to form a central dense structurereferred to as a nucleoid.
In previous reports, we have shown that theenvelopes of EB and RB are significantly differ-ent, in that EB envelopes are rigid and resistantto physical treatment and contain all commonamino acids and significant amounts of phospho-lipids (5). RB envelopes, however, are fragile,thin, and flexible, and contain no cystine ormethionine and little phospholipid (9). Pre-liminary observations by electron microscopy ofuntreated and detergent-extracted envelopes ofboth RB and EB forms have shown that the cellwall or envelope is continuous throughout thegrowth cycle, thus indicating that the changeswhich occur in the envelope during the conver-sion of EB to RB and RB to EB are not due tothe removal or synthesis of the envelope, but aredue to changes in the nature of the membrane.Tamura and Manire recently studied the effect
of penicillin on the chemical composition oforganisms grown in penicillin, in which RB wereharvested at different times during a 40- to 48-hrgrowth period (10). No infectious EB were pro-duced when penicillin was in the medium, butthe RB showed continuous growth throughoutthe incubation period as measured by dry weight.The envelopes of penicillin-treated organismsharvested at 18 or 40 hr were similar in contentand structure to those harvested from normal18-hr RB forms.In the studies reported in this paper, we have
confirmed the observations of Weiss (13), Walz(11), and Tajima et al. (7), all of whom reportedthat penicillin causes the production of large ab-normal forms of the organism. In our studies onthe fine structure of the penicillin-treated orga-nisms, typical binary fission was almost never seen,but the RB forms became progressively largerand more abnormal in morphology. Occasionally,two or more large forms were seen within oneinclusion, but careful observation indicated thatthese arose by fusion of the inclusion membraneand not by fission. Each of the older inclusionscontained many small vesicles, some of whichwere empty and some appeared to be normal RBforms.These were formed by a budding process,and this was occasionally seen when the new"bud" appeared to have its own cytoplasmicmembrane while still enclosed in the RB envelope.A striking feature of the development of these
organisms in penicillin is the great amount ofmembrane formation associated with theseabnormal RB forms. Large flat sheets of mem-branes which we presume to be RB envelopeswere present 28 hr after infection and increased
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up to 48 hr. By 40 hr after infection, the processof irternal material condensation was seen in thelarge RB forms, as if the maturation process wasbeing initiated. In no case, however, were typicalEB seen in these inclusions.However, the viability of these abnormal RB
forms was demonstrable when penicillin wasremoved from the L cell medium 20 hr after in-fection. These RB forms could still divide to formnormal RB which could undergo maturation toform EB. This process of division appeared tooccur by two different means. In the most com-mon, newRB forms were formed by a buddinglikeprocess, with the new RB forms undergoingbinary fission. In the other, the large RB formsappeared to be subdivided internally into numer-ous normal RB forms which were surrounded byfragments of the old RB envelope.On the basis of these studies and previous
reports, we have concluded that penicillin doesnot prevent the growth of Chlamydia, but growthis limited to the ability of the host cell to supportthis intracellular organism. Penicillin does notinhibit the formation of the envelope of theorganism during this growth period. The drugdoes inhibit binary fission, and it does preventthe formation of EB envelope and of infectiousorganisms. Galasso and Manire (1) studiedChlamydia in a persistently infected culture ofHeLa cells and found that such cultures could notbe freed of their infection by penicillin. After aslong as 100 days of treatment with penicillin, theChlamydia reappeared as infectious EB within ashort time after drug removal.
It would appear to us that penicillin affectsthose components of the cell envelope present inEB but missing in RB. We are currently workingon one such component. This is the subunitreported by Manire (4) to be seen in hexagonalarray on the inside face of envelopes of Chla-mydia. More recent studies (A. Matsumoto andG. P. Manire, Bacteriol. Proc. 1969, p. 109)have shown these to be present in all EB forms,but to be absent in normal RB forms and those
harvested from penicillin-treated cultures. Fur-ther studies on the nature of these structures arenow underway.
ACKNOWLEDGMENTS
This investigation was supported by Public Health Servicegrant AI-00868 from the National Institute of Allergy and In-
fectious Diseases.
LITERATURE CITED
1. Galasso, G. J., and G. P. Manire. 1961. Effect of antiserum
and antibiotics on persistent infection of HeLa cells withmeningopneumonitis virus. J. !mmunology 86:382-385.
2. Higashi, N. 1964. The mode of reproduction of psittacosis-lymphogranuloma-trachoma group viruses. Int. Rev. Exp.Pathol. 3:35-64.
3. Higashi, N. 1965. Electron microscopic studies on the modeof reproduction of trachoma virus and psittacosis virus incell cultures. Exp. Mol. Pathol. 4:24-39.
4. Manire, G. P. 1966. S!ucture of purified cell walls of denseforms of meningopneumonitis organisms. J. Bacteriol.91:409-413.
5. Manire, G. P., and A. Tamura. 1967. Preparation and chemicalcomposition of the cell walls of mature infectious denseforms of meningopneumonitis organisms. J. Bacteriol.94:1178-1183.
6. Millonig, G. 1961. A modified procedure for lead staining ofthin sections. J. Biophys. Biochem. Cytol. 11:736-739.
7. Tajima, M., T. Samejima, and Y. Nomura. 1959. Morphologyof meningopneumonitis virus exposed to penicillin as
observed with the electron microscope. J. Bacteriol. 77:23-34.
8. Tamura, A., and N. Higashi. 1963. Purification and chemicalcomposition ofmeningopneumonitis virus. Virology 20:596-604.
9. Tamura, A., and G. P. Manire. 1967. Preparation and chemicalcomposition of the cell membranes of developmentalreticulate forms of meningopneumonitis organisms. JBacteriol. 94:1184-1188.
10. Tamura, A., and G. P. Manire. 1968. Effect of penicillin on
the multiplication of meningopneumonitis organisms(Chlamydia psittaci). J. Bacteriol. 96:875-880.
11. Walz, A. 1963. Morphologische Studien an einem Psit-tacosestamm und seine Veranderungen unter dem Einflussvon Penicillin. Zentralbl. Bakteriol. Parasitenk. Infektion-skr. hyg. Abt. I Orig. 188:29-61, 174-189.
12. Watson, M. C. 1958. Staining of tissue sections for electronmicroscopy with heavy metals. J. Biophys. Biochem. Cytol.4:475-478.
13. Weiss, E. 1950. The effect of antibiotics on agents of thepsittacosis-lymphogranuloma group. J. Infec. Dis. 87:249-263.
