The Structure of Pili (Fimbriae) of Moraxella bovis C. F. Simpson, F. H. White and T. S. Sandhu*

ABSTRACT

INTRODUCTION

Cells from rough and smooth colonies of Moraxella bovis were examined by electron microscopy utilizing both shadowing and thin sectioning techniques. Pili were found on the surfaces of cells from rough but not smooth colonies. Pili had a peritrichous distribution and appeared as delicate (6.5-8.5 nm in diameter), elongated unbranched filaments. When bacteria were sectioned pili did not contain central pores and appeared to originate from opacities on the surface of the cell wall.

Pili (fimbriae) are long, thin appendages present on the surfaces of several species of gram negative and some gram positive bacteria (2, 15). Recently, pili have been observed on a fungus, Ustilago violacea (8). In most studies the morphology of pili was ascertained from the electron microscopic examination of negative stained and shadowed organisms. Studies of pili by thin sectioning techniques have not been extensive (5, 8, 14). In a recent electron microscopic study in this laboratory it was determined that cells from rough colonies of Moraxella bovis, the probable etiological agent of infectious keratoconjunctivitis of cattle, were piliated, while cells from smooth colonies were nonpiliated (11). Since pili were plentiful on the surfaces of cells from rough colonies it was decided to determine the ultrastructural morphology of these appendages and their relation to the cells using shadowing and ultrathin sectioning techniques. The findings were compared with those of similar studies made of the surfaces of nonpiliated cells from smooth colonies of M. bovis. The results of this investigation are the subject of the present paper.

RESUMEk Les auteurs se sont servi de la microscopie electronique, en utilisant la technique d'ombrage et les coupes ultra-fines, pour examiner des bacteries Moraxella bovis provenant de cultures rugueuses et lisses. Seuls les microorganismes des cultures rugueuses s'avererent peritriches. Leurs cils consistaient en des filaments simples, longs, delicats et d'un diametre variant de 6.5 'a 8.5 nm. La section de bacteries peritriches revela que leurs cils etaient depourvus de pores centrales et qu'ils semblaient originer de petits points opaques situ6s sur la face externe de la membrane cellulaire.

MATERIALS AND METHODS *Department of Veterinary Science, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32611. Submitted May 7, 1975.

Volume 40

-

January, 1976

Three isolates of M. bovis grown on tryptose agar base with 5% sheep blood were

I

studied. Since both smooth and rough colonies grew on the same agar plate, pure rough or smooth cultures were obtained by selection and transfer to separate bloodI agar plates. Colonies were classified as. rough or smooth on the basis of colonial morphology and texture (11). For electron microscopy, rough or smoothl colonies, following incubation at 37°C fori 24 hours, were removed from blood agar plates and faixed in 1 % 2S04 (3) for 30 minutes. Cells were examined with a Philips EM200 electron microscope by two methods. A suspension of fixed cells after being dropped on grids was shadowed with chromium at an angle of 200. In addition, a pellet of fixed cells was dehydrated in a series of five concentrations of ethanol ranging from 10 to 100% at ten minute intervals and two changes of propylene oxide cut thinly after embedment in Araldite, and sections on grids were stained with uranyl acetate and lead citrate (10) prior to being examined with the electron microscope.

Fig. 2. A cell from smooth colonies does not have pili X20,000.

on its surface. Chromium shadowed.

RESULTS Rough colonies were flat, umbonate, f irm. dry, adhesive and autoagglutinated when suspended in distilled water. Smooth colonies lacked adhesive and autoagglutinating

properties.

The surfaces of shadowed cells from rouLlgh colonies of the three isolates had a lperitrichous distribution of delicate, elongated pili on their surfaces and these structures were often intertwined a short distance from the cell surface. Unattached pili were often seen near cells, and this was considered evidence of fragility. Pili were more or less straight or mildly curved and of tiniform diameter throughout their length. Their average diameter was 6.58.5 nm and therefore they cast an imperceptible shadow. Each pilus was an elonFig. 1. Cells from a rough colony showing that pill (P) have a peritrichous distribution. Some segments of pili (P,) have fracturedl from the cell. Chromium shadowed. X20.000.

2

gated, unbranched, single stranded structure ( Fig. 1). Shadowed cells from smootlh

colonies were

free of pili (Fig. 2).

Can. J. comp. Med.

It was difficult to obtain sections of pili much beyond their places of origin because of their small diameter and fragile nature but their peritrichous distribution was readily apparent (Fig. 3). High magnification micrographs (Fig. 4) of longitudinally sectioned pili indicated that they originated from small opacities of the outer cell wall, did not have a central pore, were beaded in appearance and averaged 6.5-8.5 nm in diameter. There was no evidence that they extended beneath the cell wall. Observations of tangential sections through the cell wall corroborated that pili originated from opacities of the outer cell wall. Neither pili nor opacities from which they seem to originate were seen on the surfaces of cells from smooth colonies sectioned in the longitudinal or tangential planes.

4 i-

P.

iL. .. !.

:::

Fig. 4. Longitudinal section of part of a cell showing that pili (P) are solid, but appear beaded because of the angle of sectioning. They apparently originate fronm opacities (arrows) of the cell wall. X74,000.

croorganisms. Pili of Pseudornonas aeruginosa were not found to originate in the cell wall (14) and were not seen to penetrate into the cell (13). Pili of Proteus mirabilis are said to arise from the cytoplasm or cytoplasmic membrane (6) and pili were DISCUSSION found to originate below the cell wall in the fungus, Ustilago violacea (8). The reThe present electron microscopic study sults of the present work with M. bovis utilizing shadowing and thin sectioning indicate that pili originate from opacities techniques has characterized pili and their of the outer surface of the cell wall and relationship to the surfaces of cells from do not penetrate into the cell. The beaded rough colonies of M. bovis. The delicate, appearance of the filaments in thin sections rod-like nature and observed diameter of was presumed due to the impossibility of 6.5-8.5 nm agree with the description of cutting the delicate structures in a completely flat plane. In this work, OS04 fixathe pili of M. bovis (1, 7). It is generally agreed that it is difficult tion was utilized because preliminary stuto section pili (4, 13). This problem has re- dies indicated that this chemical produced sulted in a paucity of references pertaining better fixation of pilial morphology than to the morphology and origin of pili in did formalin or glutaraldehyde followed by sectioned material. In the limited number OSO4. Interestingly, it has been reported of studies that have utilized this technique that fixation of the pili of U. violacea with considerable variation in the place of origin glutaraldehyde caused degeneration maniof pili has been observed with various mi- fested by tight coiling of the filaments (8). The knowledge (1, 7, 11) that upon repeated transfer rough colonies (piliated cells) of M. bovis and other bacteria (13) disassociate to smooth colonies (nonpiliated cells) is of practical importance because the presence of pili is apparently associated with the ability of some bacteria to produce disease (7, 9, 11, 12, 14).

ACKNOWLEDGMENTS Fig. 3. Cross section of a cell from a rough colony showing peritrichous distribution of pili (P) on the surface of the cell. X47,570.

Volume 40 - January, 1976

The technical assistance of J. W. Carlisle Agricultural Experiment Stations Journal Series No. 5710. is acknowledged. Florida

3

REFERENCES 1. BOVRE, K. and L. 0. FROHOLM. Variation of colony morphology reflecting fimbriation in Moraxella bovis and two reference strains of M. nonliquefaciens. Acta path. microbiol. scand. Sect. B 80: 629-640. 1972. 2. BRINTON, C. C. The structure, function, synthesis and genetic control of bacterial pili and a moleculal model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27: 10031054. 1965. 3. CAULFIELD, J. B. Effect of varying the vehicle for OS04 in tissue fixation. J. biophys. biochem. Cytol. 3: 827-830. 1957. 4. DEVOE, I. W. and J. E. GILCHRIST. Ultrastructure of pili and annular structures on the cell wall surface of Neisseria meningitidis. Infection & Immunity 10: 872-876. 1974. 5. FUERST, J. A. and A. C. HAYWARD. Surface appendages similar to fimbriae (pili) on Pseudomonas species. J. gen. Microbiol. 58: 227-237. 1969. 6. HOENIGER, J. F. M. Development of flagella by Proteus mirabilis. J. gen. Microbiol. 40: 29-42. 1965. 7. PEDERSON, K. B., L. 0. FROHOLM and K. BOVRE. Fimbriation and colony type of Moraxella bovis in relation to conjunctival colonization and

4

development of keratoconjunctivitis in cattle. Acta path. microbiol. scand. Sec. B 80: 911-918. 1972. 8. POON, H. and A. W. DAY. Fimbriae in the fungus Ustilago violacea. Nature, Lond. 250: 648-649. 1974. 9. PUNSALANG, A. P. and W. D. SAWYER. Role of pili in the virulence of Neisseria gonorrhoeae. Infection & Immunity 8: 255-263. 1973. 10. REYNOLDS, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17: 208-212. 1963. 11. SANDHU, T. S., F. H. WHITE and C. F. SIMPSON. Association of pili with rough colony type of Moraxella bovis. Am. J. vet. Res. 35: 437-439. 1974. 12. SILVERBLATT, F. J. Host-parasite interaction in the rat renal pelvis. A possible role for pili in the pathogenesis of pyelonephritis. J. exp. Med. 140: 1696-1711. 1974. 13. WEISS, R. L. The structure and occurrence of pili (fimbriae) on Pseudomonas aeruginosa. J. gen. Microbiol. 67: 135-143. 1971. 14. WEISS, R. L. and H. D. RAJ. The structure and isolation of pili (fimbriae) of Pseudomonas aeruginosa. Aust. J. exp. Biol. med. Sei. 50: 559-566. 1972. 15. WISTREICH, C. A. and R. F. BAKER. The presence of fimbriae (pili) in three species of Neisseria. J. gen. Microbiol. 65: 167-173. 1971.

Can. J.

comp. Med.

The structlre of pili (fimbriae) of Moraxella bovis.

Cells from rough and smooth colonies of Moraxella bovis were examined by electron microscopy utilizing both shadowing and thin sectioning techniques. ...
916KB Sizes 0 Downloads 0 Views