JOURNAL OF BACTERIOLOGY, June 1978, p. 1171-1175 0021-9193/78/0134-1171$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 134, No. 3
Printed in U.S.A.
NOTES Association of Long Surface Appendages with AdherenceRelated Functions of the Gram-Positive Species Actinomyces naeslundii R. P. ELLEN,* D. L. WALKER, AND K. H. CHAN Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5G I G6
Received for publication 2 November 1977
Electron microscopy of new isolates of gram-positive Actinomyces naeslundii demonstrated long, fragile appendages. Removal of the appendages impaired attachment to epithelial cells and reaggregation, thus implicating them in attachment-related functions.
Actinomyces naeslundii is a gram-positive species which has been implicated in the etiology of periodontal disease and has been isolated from actinomycotic lesions in humans (11). A report by Girard and Jacius that the one laboratory strain ofA. naeslundii which they studied elaborated "fibril-like" appendages (6) and our observations that sonically dispersed A. naeslundii cells lacked appendages and the ability to reaggregate rapidly (3) prompted the present investigation to determine whether the "fibrils" of gram-positive A. naeslundii cells were morphologically and functionally analogous to the fimbriae or pili of gram-negative bacteria. Ten strains conforming to previous descriptions of A. naeslundii (7, 10, 11) were newly isolated from human dental plaque. Laboratory strain WVU 398A was obtained from M. A. Gerencser, West Virginia University. After two or three laboratory subcultures to assure purity, stock cultures were lyophilized. Cultures for all experimental procedures were grown from the first subculture of lyophilized stock in a chemically defined medium for oral gram-positive rods (S. S. Socransky, C. Smith, and A. D. Manganiello, Int. Assoc. Dent. Res., abstr. no. 120, 1973). For electron microscopy studies, 2-day cultures were grown in an atmosphere of 85% N2, 10% H2, and 5% C02. Most of the cells grew adherent to the glass vessel walls. The adherent deposits were rinsed twice with 0.01 M potassium phosphate-buffered saline at pH 6.0 and resuspended in the same buffer. Samples of 15 ml were taken from each suspension and blended for 60 s at approximately 16,000 rpm (VirTis model 23 homogenizer), a modification of Binton's procedure for removing pili from gram-
negative bacteria (1). Control cells, which had been dispersed by repeated forceful pipetting, and homogenized cells were harvested in a refrigerated centrifuge, washed once, apd resuspended in phosphate-buffered saline. SampIes of these suspensions were negatively stained and examined by transmission electron microscopy (3). In addition, samples of strain C2 were mounted on carbon-coated grids and examined after shadow casting with platinum. Negatively stained control cells ofA. naeslundii 398A and 9 of the 10 fresh isolates possessed surface appendages (Fig. la). The delicate, fairly straight appendages had no discernible pattern of localization. Their length was generally greater than the cross-sectional diameter of the A. naeslundii cell. Platinum-shadowed strain C2 (Fig. 2) demonstrated tangling of separate appendages into bundles. Most cells in the control suspensions demonstrated appendages; in contrast, most (usually all) cells in the homogenized suspensions lacked them (Fig. lb). Strain 398A and four new isolates (designated C2, C10, N8, and N9) were selected for subsequent adherence studies. The abilities of homogenized and control A. naeslundii cells to attach to human buccal mucosa cells were compared using an in vitro method described previously (5). Cells from these same cultures were used to study the reaggregation of dispersed bacteria. Tubes containing phosphate-buffered saline suspensions were diluted to an optical density of 0.8 at 550 nm (Turner model 350 spectrophotometer) and placed in a shaking water bath at 37°C. The degree of interbacterial reaggregation was determined by measuring the decrease in optical density caused by the settling of aggregates to
1171
1172
J. BACTERIOL.
NOTES
\4 * .. ^ !
, S~~~~f s >>c~~~~~~~~~~~~lx
*
. ~
lb¢
't
~
~
~ 5
1
*
~ o;.
P +
FIG. 1. Electronphotomicrographs of negatively stained cells from A. naeslundii C10 control (a) and homogenized (b) suspensions.
VOL. 134, 1978
NOTES
1173
FIG. 2. Electronphotomicrograph of platinum-shadowed A. naeslundii C2. Bundles and branches of individual appendages are evident (arrows).
1174
J. BACTERIOL.
NOTES
the bottom of the suspensions (3). Cells in control suspensions of all five strains attached well to human buccal epithelial cells (Fig. 3). An average of between 30 and 100 bacteria became attached per epithelial cell. In contrast, the ability of "bald" homogenized cells to attach was significantly impaired. Blending suspensions of A. naeslundii cells to remove
1S
appendages had a similar inhibitory effect on their reaggregation (Fig. 4). Decreases in optical density occurred more rapidly in control than in homogenized suspensions during the first 4 h. Even after 20 h, the homogenized cells had not reaggregated to the same extent as the control
cells.
Bacteria colonizing bathed surfaces have evolved diverse surface components which probably determine their selective affinities for dis111 tinct infection sites (4). This investigation has proposed attachment-related functions for the long, slender appendages of newly isolated -J strains of A. naeslundii, a gram-positive species which establishes on oral mucosal surfaces of .. _ infants and remains a numerically significant member of the oral flora into adulthood (2). Attachment to epithelial cells by other gramas 750 positive bacteria (e.g., Streptococcus salivarius, S. pyogenes, S. mitior) is usually mediated by their much shorter "fuzz." 90C0-N C2 0 The selection of a term to describe A. naeslundii appendages is a contentious issue; their chemical nature has not yet been determined. 10 However, we feel that a strictly morphological description justifies the terms fimbria (thread) or pilus (hair) and that their proposed attachment functions, once firmly established, will eventually support either term. Previous reports N9 NS C2 CIO 3W8A of fimbriated or piliated gram-positive bacteria CONTROL A.nuslundl Stra4n TREATMENT I4OMOGEN IZED have not been common, being limited to memFIG. 3. Attachment of control and homogenized A. bers of the genus Corynebacterium (8, 13, 14). naeslundii cells to human buccal mucosa cells in Similar to gram-negative species, some corynebacteria elaborate long, slender, nonflagellar apvitro. HOMOGEN I ZED
CONTROL
.9
.8
z
4~ ~
~
~
~~'
A. naslundll strain
¾%
NB
N9 C2
CIO 39A
..... ...... --------
TIME ( HRS. ) FIG. 4. Interbacterial aggregation of control and homogenized cells ofA. naeslundii.
VOL. 134, 1978
pendages (termed pili by their discoverers) which confer hemagglutinating activity and the ability to attach to mammalian cells in culture (8, 13). Unlike most pili of gram-negative bacteria, but similar to the structures containing K88 antigen in enteropathogenic E. coli (9, 12), appendages on corynebacteria and the strains of A. naeslundii studied herein are occasionally branched or bundled and not necessarily rigid in appearance. It is reasonable to assume that among gram-positive bacteria the possession of long surface appendages is not a characteristic unique to the genera Actinomyces and Corynebacterium. Additional gram-positive genera will probably be found to attach to surfaces which they naturally infect via long appendages once thought to be limited to gram-negative bacteria. This investigation was supported by grant MA-5619 from the Medical Research Council of Canada and by Public Health Service research grant RO1-DE-04464-01 from the National Institute of Dental Research. We thank G. Pudy for his assistance during the electron microscopy studies and H. J. Sandham for his helpful suggestions during the preparation of the manuscript.
LITERATURE CITED 1. Brinton, C. C., Jr. 1965. The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27:1003-1054. 2. Ellen, R. P. 1976. Establishment and distribution of Actinomyces viscosus and Actinomyces naeslundii in the human oral cavity. Infect. Immun. 14:1119-1124. 3. Ellen, R. P., and I. B. Balcerzak-Raczkowski. 1977. Interbacterial aggregation of Actinomyces naeslundii
NOTES 4.
5.
6. 7.
8. 9.
10.
11. 12.
13.
14.
1175
and dental plaque streptococci. J. Periodontal Res. 12:11-20. Gibbons, R. J. 1974. Bacterial adherence to mucosal surfaces and its inhibition by secretory antibodies, p. 315-325. In J. Mestecky and A. R. Lawton (ed.), The immunoglobulin A system. Plenum Publishing Corp., New York. Gibbons, R. J., and J. van Houte. 1971. Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. Infect. Immun. 3:567-573. Girard, A. E., and B. H. Jacius. 1974. Ultrastructure of Actinomyces viscosus and Actinomyces naeslundii. Arch. Oral Biol. 19:71-79. Holmberg, K., and C.-E. Nord. 1975. Numerical taxonomy and laboratory identification of Actinomyces and Arachnia and some related bacteria. J. Gen. Microbiol. 91:17-44. Honda, E., and R. Yanagawa. 1975. Attachment of Corynebacterium renale to tissue culture cells by the pili. Am. J. Vet. Res. 36:1663-1666. Jones, G. W. 1975. Adhesive properties of enteropathogenic bacteria, p. 137-142. In D. Schlessinger (ed.), Microbiology-1975. American Society for Microbiology, Washington, D.C. Slack, J. M. 1974. Genus I. Actinomyces Harz 1877, 485, p. 660-667. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. Slack, J. M., and M. A. Gerencser. 1975. Actinomyces ifiamentous bacteria. Biology and pathogenicity. Burgess Pulishing Co., Minneapolis. Stirm, S., F. Orskov, I. 0rskov, and A. Birch-Andersen. 1967. Episome-carried surface antigen K88 of Escherichia coli. III. Morphology. J. Bacteriol. 93:740-748. Yanagawa, R., and E. Honda. 1976. Presence of pili in species of human and animal parasites and pathogens of the genus Corynebacterium. Infect. Immun. 13:1293-1295. Yanagawa, R., and K. Ostuki. 1970. Some properties of pili of Corynebacterium renale. J. Bacteriol. 101:1063-1069.
Vol. 134, No. 3
Printed in U.S.A.
NOTES Association of Long Surface Appendages with AdherenceRelated Functions of the Gram-Positive Species Actinomyces naeslundii R. P. ELLEN,* D. L. WALKER, AND K. H. CHAN Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5G I G6
Received for publication 2 November 1977
Electron microscopy of new isolates of gram-positive Actinomyces naeslundii demonstrated long, fragile appendages. Removal of the appendages impaired attachment to epithelial cells and reaggregation, thus implicating them in attachment-related functions.
Actinomyces naeslundii is a gram-positive species which has been implicated in the etiology of periodontal disease and has been isolated from actinomycotic lesions in humans (11). A report by Girard and Jacius that the one laboratory strain ofA. naeslundii which they studied elaborated "fibril-like" appendages (6) and our observations that sonically dispersed A. naeslundii cells lacked appendages and the ability to reaggregate rapidly (3) prompted the present investigation to determine whether the "fibrils" of gram-positive A. naeslundii cells were morphologically and functionally analogous to the fimbriae or pili of gram-negative bacteria. Ten strains conforming to previous descriptions of A. naeslundii (7, 10, 11) were newly isolated from human dental plaque. Laboratory strain WVU 398A was obtained from M. A. Gerencser, West Virginia University. After two or three laboratory subcultures to assure purity, stock cultures were lyophilized. Cultures for all experimental procedures were grown from the first subculture of lyophilized stock in a chemically defined medium for oral gram-positive rods (S. S. Socransky, C. Smith, and A. D. Manganiello, Int. Assoc. Dent. Res., abstr. no. 120, 1973). For electron microscopy studies, 2-day cultures were grown in an atmosphere of 85% N2, 10% H2, and 5% C02. Most of the cells grew adherent to the glass vessel walls. The adherent deposits were rinsed twice with 0.01 M potassium phosphate-buffered saline at pH 6.0 and resuspended in the same buffer. Samples of 15 ml were taken from each suspension and blended for 60 s at approximately 16,000 rpm (VirTis model 23 homogenizer), a modification of Binton's procedure for removing pili from gram-
negative bacteria (1). Control cells, which had been dispersed by repeated forceful pipetting, and homogenized cells were harvested in a refrigerated centrifuge, washed once, apd resuspended in phosphate-buffered saline. SampIes of these suspensions were negatively stained and examined by transmission electron microscopy (3). In addition, samples of strain C2 were mounted on carbon-coated grids and examined after shadow casting with platinum. Negatively stained control cells ofA. naeslundii 398A and 9 of the 10 fresh isolates possessed surface appendages (Fig. la). The delicate, fairly straight appendages had no discernible pattern of localization. Their length was generally greater than the cross-sectional diameter of the A. naeslundii cell. Platinum-shadowed strain C2 (Fig. 2) demonstrated tangling of separate appendages into bundles. Most cells in the control suspensions demonstrated appendages; in contrast, most (usually all) cells in the homogenized suspensions lacked them (Fig. lb). Strain 398A and four new isolates (designated C2, C10, N8, and N9) were selected for subsequent adherence studies. The abilities of homogenized and control A. naeslundii cells to attach to human buccal mucosa cells were compared using an in vitro method described previously (5). Cells from these same cultures were used to study the reaggregation of dispersed bacteria. Tubes containing phosphate-buffered saline suspensions were diluted to an optical density of 0.8 at 550 nm (Turner model 350 spectrophotometer) and placed in a shaking water bath at 37°C. The degree of interbacterial reaggregation was determined by measuring the decrease in optical density caused by the settling of aggregates to
1171
1172
J. BACTERIOL.
NOTES
\4 * .. ^ !
, S~~~~f s >>c~~~~~~~~~~~~lx
*
. ~
lb¢
't
~
~
~ 5
1
*
~ o;.
P +
FIG. 1. Electronphotomicrographs of negatively stained cells from A. naeslundii C10 control (a) and homogenized (b) suspensions.
VOL. 134, 1978
NOTES
1173
FIG. 2. Electronphotomicrograph of platinum-shadowed A. naeslundii C2. Bundles and branches of individual appendages are evident (arrows).
1174
J. BACTERIOL.
NOTES
the bottom of the suspensions (3). Cells in control suspensions of all five strains attached well to human buccal epithelial cells (Fig. 3). An average of between 30 and 100 bacteria became attached per epithelial cell. In contrast, the ability of "bald" homogenized cells to attach was significantly impaired. Blending suspensions of A. naeslundii cells to remove
1S
appendages had a similar inhibitory effect on their reaggregation (Fig. 4). Decreases in optical density occurred more rapidly in control than in homogenized suspensions during the first 4 h. Even after 20 h, the homogenized cells had not reaggregated to the same extent as the control
cells.
Bacteria colonizing bathed surfaces have evolved diverse surface components which probably determine their selective affinities for dis111 tinct infection sites (4). This investigation has proposed attachment-related functions for the long, slender appendages of newly isolated -J strains of A. naeslundii, a gram-positive species which establishes on oral mucosal surfaces of .. _ infants and remains a numerically significant member of the oral flora into adulthood (2). Attachment to epithelial cells by other gramas 750 positive bacteria (e.g., Streptococcus salivarius, S. pyogenes, S. mitior) is usually mediated by their much shorter "fuzz." 90C0-N C2 0 The selection of a term to describe A. naeslundii appendages is a contentious issue; their chemical nature has not yet been determined. 10 However, we feel that a strictly morphological description justifies the terms fimbria (thread) or pilus (hair) and that their proposed attachment functions, once firmly established, will eventually support either term. Previous reports N9 NS C2 CIO 3W8A of fimbriated or piliated gram-positive bacteria CONTROL A.nuslundl Stra4n TREATMENT I4OMOGEN IZED have not been common, being limited to memFIG. 3. Attachment of control and homogenized A. bers of the genus Corynebacterium (8, 13, 14). naeslundii cells to human buccal mucosa cells in Similar to gram-negative species, some corynebacteria elaborate long, slender, nonflagellar apvitro. HOMOGEN I ZED
CONTROL
.9
.8
z
4~ ~
~
~
~~'
A. naslundll strain
¾%
NB
N9 C2
CIO 39A
..... ...... --------
TIME ( HRS. ) FIG. 4. Interbacterial aggregation of control and homogenized cells ofA. naeslundii.
VOL. 134, 1978
pendages (termed pili by their discoverers) which confer hemagglutinating activity and the ability to attach to mammalian cells in culture (8, 13). Unlike most pili of gram-negative bacteria, but similar to the structures containing K88 antigen in enteropathogenic E. coli (9, 12), appendages on corynebacteria and the strains of A. naeslundii studied herein are occasionally branched or bundled and not necessarily rigid in appearance. It is reasonable to assume that among gram-positive bacteria the possession of long surface appendages is not a characteristic unique to the genera Actinomyces and Corynebacterium. Additional gram-positive genera will probably be found to attach to surfaces which they naturally infect via long appendages once thought to be limited to gram-negative bacteria. This investigation was supported by grant MA-5619 from the Medical Research Council of Canada and by Public Health Service research grant RO1-DE-04464-01 from the National Institute of Dental Research. We thank G. Pudy for his assistance during the electron microscopy studies and H. J. Sandham for his helpful suggestions during the preparation of the manuscript.
LITERATURE CITED 1. Brinton, C. C., Jr. 1965. The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27:1003-1054. 2. Ellen, R. P. 1976. Establishment and distribution of Actinomyces viscosus and Actinomyces naeslundii in the human oral cavity. Infect. Immun. 14:1119-1124. 3. Ellen, R. P., and I. B. Balcerzak-Raczkowski. 1977. Interbacterial aggregation of Actinomyces naeslundii
NOTES 4.
5.
6. 7.
8. 9.
10.
11. 12.
13.
14.
1175
and dental plaque streptococci. J. Periodontal Res. 12:11-20. Gibbons, R. J. 1974. Bacterial adherence to mucosal surfaces and its inhibition by secretory antibodies, p. 315-325. In J. Mestecky and A. R. Lawton (ed.), The immunoglobulin A system. Plenum Publishing Corp., New York. Gibbons, R. J., and J. van Houte. 1971. Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. Infect. Immun. 3:567-573. Girard, A. E., and B. H. Jacius. 1974. Ultrastructure of Actinomyces viscosus and Actinomyces naeslundii. Arch. Oral Biol. 19:71-79. Holmberg, K., and C.-E. Nord. 1975. Numerical taxonomy and laboratory identification of Actinomyces and Arachnia and some related bacteria. J. Gen. Microbiol. 91:17-44. Honda, E., and R. Yanagawa. 1975. Attachment of Corynebacterium renale to tissue culture cells by the pili. Am. J. Vet. Res. 36:1663-1666. Jones, G. W. 1975. Adhesive properties of enteropathogenic bacteria, p. 137-142. In D. Schlessinger (ed.), Microbiology-1975. American Society for Microbiology, Washington, D.C. Slack, J. M. 1974. Genus I. Actinomyces Harz 1877, 485, p. 660-667. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. Slack, J. M., and M. A. Gerencser. 1975. Actinomyces ifiamentous bacteria. Biology and pathogenicity. Burgess Pulishing Co., Minneapolis. Stirm, S., F. Orskov, I. 0rskov, and A. Birch-Andersen. 1967. Episome-carried surface antigen K88 of Escherichia coli. III. Morphology. J. Bacteriol. 93:740-748. Yanagawa, R., and E. Honda. 1976. Presence of pili in species of human and animal parasites and pathogens of the genus Corynebacterium. Infect. Immun. 13:1293-1295. Yanagawa, R., and K. Ostuki. 1970. Some properties of pili of Corynebacterium renale. J. Bacteriol. 101:1063-1069.
