Ultrastructure of Actinomyces viscosus and Actinomyces naeslundii

Archs oral Bid

Vol

19. pp. 71 to 79 Pergamon Prrs

1974. Printed in Great Britain

ULTRASTRUCTURE OF ACTINOM YCES VZSCOSUS ACTINOMYCES NAESLUNDII A. E. Department

of Microbiology,

GIRARD

University Connecticut

AND

and B. H. JACIUS of Connecticut 06032, U.S.A.

Health

Center,

Farmington.

Summary-Electron micrographs of freeze-etched, thin-sectioned, shadowed and negatively-stained cells were presented. Fibril-like structures were observed on the outer surface of both organisms in all preparations. Thin sections revealed a layered cell wall in both microbes. but the layering was particularly distinct in A. ncreslundii. Freeze-etching of both species demonstrated two layers in the cytoplasmic membrane. The outermost layer of the plasma membrane was shown to have numerous particles, while the second layer had a smooth surface with evenly dispersed dome-like structures. Since plaque organisms have many of the structures described here, they may be members of the genus Actinomyces. The demonstration of fibres on the outer surface of these bacteria provides structural evidence for a potential mechanism of adhesion. These observations are of suecial interest as both organisms induce experimental periodontal disease in hamsters and gnotobiotic rat’s

METHODS

INTRODUCTION Organistns

Actinomyces viscosus and Actinomyces naeslundii have been shown to induce experimental periodontal disease in hamsters and gnotobiotic rats (Jordan and Keyes, 1964; Socransky, Hubersak and Propas, 1970; Jordan, Keyes and Bellack, 1972). Despite the importance of these organisms to the oral biologist, few ultrastructural studies have been made of members of this genus (Duda and Slack, 1972; Overman and Pine, 1963). The study by Duda and Slack (1972) .was primarily designed to yield ultrastructural information of taxonomic value. A few micrographs of thin sections of A. naeslundii were presented; no micrographs of A. uiscosus were published, although the morphological features were described and the width of the cell wall noted. Overman and Pine (1963) reported on the cytoplasmic structures in the Actinomyces. Newman (1972) has placed particular emphasis on the finding that most plaque organisms have “hairy” outer layers. The significance of this observation is that these structures may be just as important in maintaining the integrity of plaque as the inherent tackiness of extracellular polysaccharides (Gibbons and Nygaard, 1970). It has been further suggested that the fibrils observed consist of polysaccharide, and that their form, as well as adhesive properties, enable the organisms to stay in contact with one another and the tooth surface. This report presents data of an ultrastructural study of A. uiscosus and A. naeslundii utilizing freeze-etching, thin-sectioning, shadow-casting and negative-staining techniques. The objective of the investigation was to disclose further structural information of the outer surface (“fuzzy layer”). cell wall and cytoplasmic membrane of these organisms.

The organisms used in this study were obtained from the Virginia Polytechnic Institute (VPI) Anaerobe Lab and are designated Acrir~~~ctis nue.s/undii (VP1 7604) and Acrid,I~~WS ris~o,s~~,s(VPI 7281). Each culture was streaked for purity and the pure cultures were characterized. The results obtained were in agreement with the reactions noted for these species in the VP1 Anaerobe Laboratory Manual (Holderman and Moore, 1972). Culture conditions The organisms were grown in Trypticase Soy Broth with added dextrose in a candle jar at 37‘C for 18 hr or 5 days. Preparation

of cells for electron microscop)

(a) Negative stain und shadowing. The cells were centrifuged, washed once in saline and resuspended in fresh saline. The cell suspension was placed on copper grids (200 mesh) layered with a thin film of carbon-coated Formvar. The material was then dried and shadowed employing carbonplatinum pellets (Ladd Industries. Inc.). Cells prepared in the same manner were negatively stained with 2 per cent phosphotungstic acid (PTA). (b) Preparation for thin-sectioning. An equai volume of 6 per cent glutaraldehyde was added to cells grown as previously described. The cells were allowed to stand in glutaraldehyde for 1 hr. Following glutaraldehyde fixation. the cell suspension was washed with saline, resuspended in 2 per cent osmium tetroxide (pH 7.3) and allowed to stand overnight. The doubly fixed bacteria were embedded in agar, dehydrated through a graded series of ethanol and then embedded in Epon 812. Ultrathin sections were cut with a diamond knife on an ultramicrotome (LKB Ultratome), stained with uranyl acetate followed by lead citrate (Reynolds, 1963) and examined in a Hitachi HU-llE-1 electron microscope at an acceleration voltage of 75 kV. (c) Frwr-etching. The cells were centrifuged and the cell pellet resuspended in the small amount of supernatant fluid 71

A. E. Girard

72

and B. H. Jacius

remaining after decanting. This cell suspension was frozen in Freon-22, stored in liquid N,, and transferred into the Balzers apparatus (Moor et al., 1961) for subsequent freezefracturing and -etching. as described by Moor and Miihlethaler (1963). The replicas were freed of cell residues by treatment with 50 per cent sulphuric acid and 5.25 per cent sodium hqpochlorite solutions. The clcancd replicas wcrc picked up on Formvar-coated grids and examined in the electron microscope. HESL’LTS

The negatively-stained cells of both A. mws/undii (Fig. 1) and A. ciscosu.s are shown to have an exterior fibrillar layer: the fibrils appear to extend a considerable distance from the cell surface and have a very small diameter (4-8 nm). This outer layer’of A. naes/undii is also clearly demonstrated in Fig. 2.

Fibril-like structures can bc seen (Figs. 3 and 4) in the I- and 5-day cultures of A. ~i,scosus; this feature is not as prominent in the cultures of A. nueslundii, but short hair-like structures are seen at the outer surface in l- and 5-day cultures (Figs. 5 and 7). ActinomJres naeslundii is shown to have a thicker cell wall than A. ~~i.scosz~.s. as reported previously by Duda and Slack (1972). It should be noted that A. wes/~u~dii has a double layered cell wall; this is clearly demonstrated in the region of septation (Fig. 5). In addition there seems to be a cleft (Fig. 6) which forms on the outer layer of the cell wall. The outer layer apparently can be broken more easily than the inner; such a break is illustrated inFig. 5 and it is clear that the two cells share the outer layer. Both layers appear to have a “fuzzy” surface. The cytoplasm of the l-day cultures of both A. oiscosus and A. naes/undii is densely packed with ribosomes; no mesosomes are seen in A. naeslundii, while these structures are clearly demonstrated in A. aiscosus. The older cells of both organisms have generally poorly defined ribosomes and most cells are in various phases of degeneration. Freeze-etched

cells

The outer layer of A. l;iscosus is seen in the freezeetch preparation (Fig. 8) as fibrils. The outer wall has fractured but the thickness may be misleading because of the cute&c shell produced in the etching process as noted by De Voe, Costerton and MacLeod (1971) and others. Just beneath the outer wall is a rough layer composed of numerous particles (Figs. 8-12). These 78 nm particles. like those reported in the cell membranc of ~\&~i(~/~it~ c,o/i (Bayer and Remscn. 1970: Nanninga. I9 70) and P.scudouww.s ueluyinosa (Weiss and Fraser, 1973) appear to be embedded in a smooth surface. Hemispherical or dome-like structures appear in areas devoid of particles; this feature was also noted in P. crertryifzosa (Weiss and Fraser, 1973), and Bncil/u.s stc,trrothcrrllo/,hillr\(Slcytr. 1970). but not E. w/i (Bayer

and Remsen, 1970). The diameter of the freeze-etched domes is 15-18 nm. The surface of the plasma membrane facing the cytoplasm (Fig. 12) appears identical to the surface adjacent to the particle layer and thus differs from other reports concerning this membrane (Holt and Edward. 1972; Branton, 1966; Branton. 1969). The cytoplasm appears as a very rough region. A non-etched preparation of A. ~~i.scosusis shown in Fig. 10. The micrograph is presented as a control to support the observations noted on the thickness of the outer wall. Since etching was omitted, the eutectic shell was eliminated. Clearly the thickness of the outer wall does not differ significantly from the dimensions noted in the etched samples. The particle-studded arcas ;IS well as the smooth regions in the mcmbranc are demonstrable without etching but arc not as distinctly seen. The freeze-etch preparations of ,4. rltrc,s/~nrlii are shown in Figs. I1 and 12; generally A. ~~eslmdii has all the features noted above for A. ci.scost~s.

The demonstration of hairy outer surfaces on both A. ~isco.su.s and A. rxwslur~dii by shadowing, negativestaining, thin-sectioning and freeze-etching provides ultrastructural evidence that these organisms have a potential mechanism which may enable them to stay in contact with one another and with the tooth surface. The chemical nature of this layer is still unknown, but further studies are planned to determine at least the general composition (i.e. protein, carbohydrate or lipid). The finding that A. norslundii and A. ~i.scosus have a double-la!crcd cell wall ma! bc of taxonomic value. A similarly structured cell wall has been reported in higher organisms such as Penicillium chrysogenum (McCoy. Girard and Kornfeld. 1971). as well as in Btrc~illus/XJ/~JU!YLI (Nermut and Murray. 1967) and Ustwiu (Ghosh and Murray, 1967). The cleft that was seen in thin sections of A. naeslundii may also be of taxonomic value. The formation of this structure is apparently a result of the release of single cells from a chain, leaving the cleft behind with the mother cell. The cells examined here were either l- or 5-day cultures. It is clear that the 5-day cultures are composed largely of dead or dying cells. We feel, therefore, that the morphological description of these organisms must be made on young cultures (18-24 hr) or at least on actively growing cells. This may cast some doubt on the morphological features described for these organisms by Duda and Slack (1972). but we do concede

that

the cultural

conditions

L+ct-e dillerent

and

thus they may have had an actively growing culture after 4 days. Particles similar to those reported here are present on the surfucc of -I~~/Io/~~/~/~~,\~~J(~ /rfit//~/l~ ii and ZIJ.c,o/~/tr\~rw (/cI//~,\c’/‘I~c.~/~~I (Maniloll‘ and Morowitl. 1972). Since mycoplasmas lack a cell wxll and therefore contain on11 ;I c! toplasmic mcmbranc as their outer struc-

Ultrastructure

of A. viscosus and A. naeslundii

ture, these particles probably represent structures within this membrane. It has been suggested that the particles noted on the mycoplasma membrane may represent proteins within the membrane since they are not removed by lipid solvents (Das and Maniloff, 1971, Biophys. Sot. Abstr., p. 291a). The hemispheres reported in this paper which form part of the smooth layer beneath the particle layer are not seen in A. laidlawii or M. gallisepticum, but are noted in P. aeruginosa (Weiss and Fraser, 1973), and B. stearothermophilus (Sleytr, 1970). Weiss and Fraser (1973) suggested that the particle studded layer represents the outer layer of the cytoplasmic membrane and that the smooth layer containing the larger mounds is the second layer of this membrane. The reports noted above support the interpretation that freeze-etching of both Actinomyces species demonstrated two layers in the cytoplasmic membrane. Many organisms occurring in plaque exhibit the particle layer with a smooth, dome-covered surface beneath (Newman, 1972). These organisms also have hairy outer layers. There is a strong suggestion that the organisms described as plaque microbes may be in fact members of the genus Actinomyces. The hairy outer layers may also be present in many other oral bacteria; further data are required to be conclusive. At a time when much effort is being directed towards understanding dental plaque, investigators are now examining the complex ultrastructure of this bacterial community. To interpret the structures observed in such a mixed population, a knowledge of the morphology of the expected groups of plaque microorganisms such as the Actinomyces is necessary.

REFERENCES

Bayer M. E. and Remsen C. C. 1970. Structure of Escherichia coli after freeze-etching. J. Bact. 101, 304-313. Branton D. 1966. Fracture faces of frozen membranes. Proc. ~totn. Acad. Sci. C’.S..4. 55, 1048-1056. Branton D. 1969. Membrane structure -1,111.RL,I.. PI, Ph,~.\io/. 20,209-238. De Voe I. W., Costerton J. W. and MacLeod R. A. 1971. Demonstration by freeze-etching of a single cleavage plane in the cell wall of a Gram-negative bacterium. J. Bacr. 106,659-67 I. Duda J. J. and Slack J. M. 1972. Ultrastructural studies on the genus Acfinornycrs. J. qen. Microhiol. 71, 63-68.

73

Ghosh B. K. and Murray R. G. E. 1967. Fine structure of Listeria monocytogenes in relation to protoplast formation, J. Bact. 93.411-426. Gibbons R. J. and Nygdard M. 1970. Interbacterial aggregation of plaque bacteria. Archs orai Bioi. 15, 1397-1400. Holdcmon 1. V. and Moore W. E. C. 1972. -I~~rro~>hcLtrhorutory Manuul. The VP1 Anaerobe Laboratory, Blacksburg, Virginia. Holt S. C. and Edward M. R. 1972. Fine structure of the thermophilic blue-green alga Synechococcus liuidus Copeland. A study of frozen-fractured-etched cells. Can. J. Microbial. 18, 175-181. Jordan H. V. and Keyes P. H. 1964. Aerobic, Gram-positive tilamentous bacteria as aetiologic agents of experimental periodontal disease in hamsters. Archs orul Biol. 9, 401414. Jordan H. V.. Keyes P. H. and Bellack S. 1972. Periodontal lesions in hamsters and gnotobiotic rats infected with actinomyces of human origin. J. periodont. Rex 7, 21b28. Maniloff J. and Morowitz H. J. 1972. Cell biologv of the mycoplasmas. Butt. Rec. 36, 263-290. McCoy E. C.. Girard A. E. and Kornfeld J. M. 1971. Fine structure of resting and germinating Pcvlicilliunl c~hr~~soyer~u~~~ conidiospores. Protoplusitlu 73, 443-456. Moor H. and Miihlethaler K. 1963. Fine structure in frozenetched yeast cells. J. Cell Biol. 17, 609-628. Moor H.. Miihlethaler K., Waldner H. and Frey-Wlssling A. 196 I. A new frcejing ultratoms. J. hioph Y.S.hiochvru. Cl&~/. 10, I-13. Nanninga N. 1970. Ultrastructure of the cell envelope of Escherichia co/i B after freeze-etching. J. Bact. 101, 297303. Ncrmut M. V. and Murray R. G. E. 1967. Ultrastructure of the cell wall of Bacillus poIy/11yxu. J. Bact. 93, 1949-1965. Newman H. N. 1972. Freeze-etching and dental research. J. periodont. Res. 7, 91-101. Overman F. H. and Pine L. 1963. Electron microscopy of cytoplasmic structures in facultative and anaerobic Actinomyces. J. Bact. 86, 656-665. Reynolds E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. Slcltr 1:. 9. 1970. Fracture fnces in intact cells and protoplasts of Bocillr,\ ,~r~~~~r~orltr~~~~rrt,l,liil~~~. A stud! b! coll\entional freeze-etching and freeze-etching of corresponding fracture moieties. Protoplasrna 71, 295-312. Socransky S. S., Hubersak C. and Propas D. 1970. Induction of periodontal destruction in gnotobiotic rats by a human oral strain of Actinomycrs naeslundii. Archs oral Biol. 15, 993-995. Weiss R. L. and Fraser D. 1973. Surface structure of intact cells and spheroplasts of Pseudomonas aeruqinosa. J. Bact. 113,963 968.

RBsumtSUne Ctude de microscopic Clectronique est rCaliste h partir de bactt-ries prkparies par cryodt?capage, coupes fines. ombrage et coloration nkgative. Des structures fibrillaires sont visibles ti la surface externe des micro-organismes dans toutes les prCparations. Des coupes fines montrent une paroi cellulaire h plusieurs couches dans les 2 types de microbes, mais une paroi complexe est surtout visible chez A. nar.s/urniii. Le cryodecapage permet de mettre en (_vidence 2 couches dans la membrane cqtoplasmique des dcux espkces. La couche externe dc cette membrane est form&e de nombrcuses particules. alors que la scconde couche a une surface lisse avec des structures “en d8mc” reguli&rement disposkes. Comme les bactCries de la plaque posst+dent souvent de telles structures. ils pourraient appartenir au groupe des Acfinom),ces. La prCsence de fibres sur la surface externe de ces bactCries semble apporter une preuve structurale d’un m&an&me possible d’adhision. Ces observations sont int&essantes, car les dcux organismes provoquent des parodontolyses experimentales chez le hamster ct le rat ynotohiotc.

A. E. Girard

and B. H. Jacius

ZusammenfassungEs wcrden elcktroncnmikroskopische Bildcr van Bakterienzellen dargestellt. die durch Gerfrier~itzung. Diinnschnitt. Bedampfung und Negativfirbung erhalten wurden. FibrillenPhnliche Strukturen burden an dcr iiui3ercn Obcrtliichc beidcr Mikroorganismen bei allen Prsparationsarten bcobachteL Diinnschnitte IieDcn bei beiden Mikroorganismen eine geschichtete Zellwand erkennen, wobei die Schichtung bei A. ~r,slunrlii besonders deutlich hervortrat. Die GefrierHtzung lien bei beiden Spezies 2 Schichten in der rytoplasmatischen Membran hervortreten. Die Buf3erste Schicht der Plasmamembran cnthielt zahlrciche Partikel. wiihrcnd die zweitc Schicht eine glatte Oberfllche mit gleichm%&g vcrteiltcn kuppclnrtigcn Strukturcn besal3. Da Plaquemikroorpanismcn viele der hier beschiebenen Strukturen besitrcn. konntcn lie Mitglicdcr dcr .-l~ri,io,,rl.~~,\-Art scin. Der Nachwcis van Fascrn an der lul3cren Obetliichc diesel- Baktel-icn untcrstii(rt die slrukturellr Vorstcllung eines potentiellen AdhHsionsmechanismus. Diesc Beobachtungcn sind van bcsondcrcm Interesse. da beidc Mikroorganismen im Experiment bci Hamstern und gnotohiotischcn Rattcn Parodontopathien induzieren.

75

76

A. E. Girard

and B. H. Jack

Ultrastructure

of

A. ciscosus

and

A. ~a~s~i~~~~ij

Pumi

1

A.O.B. f.p. 76

A. E. Girard

and

B. M. Jacks

PLATE 4

Ultrastructure

of A.

uiscosus

and A.

77

rxwslundii

Fig. 4. Thin section of A. ~i.scosus (Sday). Aggregation of the cytoplasm, fibrils (F), and the branching and filamentous morphology typical of this genus are evident. x 58.400 Fig. 5. Thin section

of A. naeslundii

(24 hr). The fuzzy (F) outer x 71.600

(W-l) and inner (W-2) layers

are seen.

Fig. 6. Thin section of A. naeslundii (24 hr). The cell wall layers (W-l, W-2) and cleft (C) \I hich may represent a portion of the outer cell wall remaining after fragmentation are shown. x 67.800 Fig. 7. Thin section of A. wesiuntlii (S-day). Aggregation of the cl toplasm is seen as uell as formation of intraqtoplasmic vesicles (V). The cleft (C)ah described in Fig. 6 is present. x 65.600

A. E. Girard

78

and B. H. Jaciua

PI Fig. 8. Freeze-etched shown. The direction

III

3

preparation of A. ri.sco.tu.s. Fibrils (F), the particle layer (P). and dome layer (D) are of the shadow is indicated by (As) on this and the following micrographs of freezeetched samples. x 56,000

Fig. 9. Frccrc-ctchcd preparation of il. hcows. Man> features noted in Fig. 8 are present in this micrograph: in addition a fracture may be seen in the area of the septum (Sp). x 71,000 F1.g. IO. Frccrc-fractured but non-etched preparation of .-I. IY.~UNIS.EssentialI) the same structures noted in Figs. 8 and 9 arc demonstrated such as: cell wall (C‘W); particle layer (P); dome layer (D); and cytoplasm (Cy). The eutectic effect is avoided by omitting the etching process. x 73.700

Ultrastructure

of A. viscosus and A. narslundii

PLATE 3 A.O.B.

f.p. 78

A. E. Girard

and

E. H. Jacius

PLATE 4

Ultrastructure

of il. t~i~co.w and A. ruresluntlii

79

PLATER Fig. 11. Freeze-etched preparation of A. nueslunciii. Structures similar to those seen in A. ~~i.w~su.s are present. including fibril layer (F). particle layer (P). dome layer (D) and the cytoplasm (Cy.) x 63.000 Fig. 12. Freeze-etched preparation of A. ~~slur~dii. The surface of the cytoplasmic cytoplasm is seen (I) as a concave (-_) smooth layer containing the “domes”. is seen as a convex (-) surface. x 63.000

membrane The outer

facing the of the cell