Electron Microscopic Study of Lipopolysaccharide

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JOURNAL OF BACrERIOLOGY, July 1970, p. 238-243 Vol. 103, No. 1 Copyright 0 1970 American Society for Microbiology Printed in U.S.A. Electron Microscopic Study of Lipopolysaccharide from an Avian Strain of Escherichia coli 018 J.

1 JOURNAL OF BACrERIOLOGY, July 1970, p Vol. 103, No. 1 Copyright American Society for Microbiology Printed in U.S.A. Electron Microscopic Study of Lipopolysaccharide from an Avian Strain of Escherichia coli 018 J. LOPESI AND W. E. INNISS Department of Biology, University of Waterloo, Waterloo, Ontario, Canada Received for publication 22 April 1970 The fine structure of lipopolysaccharide (LPS), isolated from an avian strain of Escherichia coli 018, was examined by electron microscopy. In positively stained preparations, ribbonlike structures with frequent branching were observed as previously reported (4). Two densely stained parallel lines were occasionally seen associated with a ribbon. When negative staining was employed, the LPS appeared as a branching ribbon with one central and two lateral zones divided by two relatively dense parallel lines running the complete length of the ribbon. The lateral zones were probably due to the 0-antigenic side chains of the LPS. This interpretation was supported by the fact that the electron microscopic structure of the LPS from two rough strains, E. coli K-12 Gal 23 and Salmonella typhimurium TV1 19 RII, both lacking the 0-specific side chains, did not possess the outer lateral zones. The electron microscopic appearance of lipopolysaccharide (LPS) was originally reported by Schramm et al. (10) to be that of droplets at ph 7 and long ribbons at ph 10. LPS isolated from Escherichia coli B was a ribbon-shaped network (13), whereas LPS from a lysine-requiring mutant of E. coli had the appearance of globules of various dimensions and stacked plates (15). E. coli 0113 LPS, prepared by trichloroacetic acid extraction and sucrose density gradient fractionation, showed various types of sheets in the form of round or oval patches, or strings, or nets. A proportion of the material consisted of more regular spherical particles and slender rods (2). LPS isolated from E. coli K-235 by the phenol-water procedure appeared as a network of ribbon-shaped structures (6). The morphology of LPS from various other microorganisms has also been examined. LPS isolated from cell walls and whole cells of Bordetella pertussis possessed fibers of different lengths among a large number of shorter rodlike structures (7). Bladen and Mergenhagen (3) have shown that negatively stained LPS, isolated from Veillonella by phenolwater extraction and centrifugation at 105,000 X g, appeared as flat or twisted discs. The LPS of Salmonella typhimurium strain 7, when positively stained, appeared primarily as flat, linear ribbons with branching (11), whereas negatively stained LPS from S. typhimurium G-30 consisted of 1 Present address: Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut. tris 238 spherically shaped structures (9). The present report concerns a study of the fine structure of the LPS isolated from an avian strain of E. coli 018 and comparison of its morphology to the LPS isolated from two rough strains of bacteria. MATERIALS AND METHODS Organisms and isolation of LPS. The bacteria used were E. coli K-12 Gal 23, S. typhimurium TV119 RII, and an avian strain of E. coli 018 (12). The E. coli K-12 Gal 23 is a rough strain, defective in uridine diphosphoglucose synthetase (8) and lacking the 0-antigenic side chain in its LPS; it was kindly supplied by A. M. C. Rapin, Harvard Medical School, Boston. S. typhimurium TV119 RII is also a rough strain, lacking the 0-antigenic side chain in its LPS, and was generously supplied by B. A. D. Stocker, Stanford University, California. The cultural conditions used for all three strains were essentially the same (4). LPS was isolated from the two rough strains by extraction from cell acetone powders prepared by suspending the harvested cells in 500 ml of acetone at 0 C. After the acetone was removed by filtration, the cell mass was washed with acetone at 0 C and the solvent was again removed. The cell powder was homogenized in distilled water to give a 1% suspension and then subjected to the phenol-water extraction procedure (14). The LPS was recovered by centrifugation of the dialyzed aqueous phase at 105,000 X g for 3 hr at 4 C, suspended in distilled water, centrifuged, suspended in 100 ml of distilled water, and finally lyophilized. LPS was isolated from the avian strain of E. coli 018 as previously described (4). Electron microscopy. LPS was dissolved in 0.1 M (hydroxymethyl) aminomethane (Tris) buffer (ph

VOL. 103X 1970 LPS FROM E. COLI 239 8.0) and examined by electron microscopy after positive or negative staining.

2 VOL. 103X 1970 LPS FROM E. COLI ) and examined by electron microscopy after positive or negative staining. Positive staining was carried out by placing a drop of LPS on a carbon-coated copper grid which had previously been treated with bovine serum albumin to facilitate even spreading. After 3 min the excess was removed by filter paper and the grid was air-dried, reacted with 2% uranyl acetate for 8 min, gently washed with distilled water, and air-dried. For negative staining, an equal volume (0.5 ml) of LPS preparation and 2% sodium tungstate were mixed, and a drop of the mixture was placed on a carbon-coated copper grid and quickly removed by filter paper. The grid was then air-dried for 30 min before use. To determine the effect of a surface tension depressant on the structure of the LPS, 1 ml of sodium lauryl sulfate (1 mg/ml) and 1 ml of avian E. coli 018 LPS (2 mg/ml) were mixed and incubated at 37 C for 20 min. The incubated mixture was then positively stained and examined. All the electron micrographs were taken with a Philips EM 300 (Philips Electronics Industries Ltd., Toronto, Ontario) operating at an acceleration voltage of 60 kv. RESULTS In positively stained preparations, the fine structure of avian E. coli 018 LPS appeared as a network of flat ribbonlike structures branching freely as previously shown (4). Infrequently, the ribbon showed a region marked by two dense parallel outer lines as indicated previously (4) and as also reported for LPS from Salmonella typhimurium (11). Figure 1 shows the appearance of the avian E. coli LPS after negative staining. Although the LPS presented the same ribbonlike profile as seen with positively stained material, much more of its structural details were observed. An unstained core region ran along the entire length and was enclosed longitudinally between two parallel lines. Another unstained zone with a slightly irregular margin was on either side of the parallel lines, increasing the observed width from approximately 16.7 nm (average ribbon width in positively stained preparations) to 31.4 nm. At the branching regions, the inner unstained core bifurcated without the outer parallel lines crossing each other. When negatively stained LPS from the two rough strains (E. coli K-12 Gal 23 and S. typhimurium TV119 RII) were examined, ribbonlike structures, some of which were rather coiled, were again evident (Fig. 2 and Fig. 3). However, the outer unstained region, observed with the avian E. coli 018 LPS, was completely lacking in the LPS of these rough strains. Figure 4 shows positively stained avian E. coli 018 LPS after treatment with sodium lauryl sulfate. The LPS ribbons appeared to be broken down into smaller lengths. The parallel outer FIG. 1. LPS (negatively stained) from avian E. coli 018. The outer region (a) is considered to be 0 antigenic side chains. Tlhe inner region (b) which shows clear bifurcation (c) is considered to be the lipid portion of the LPS. The marker represents 0.1 j.m.

Marker represents 0.1,um. 0 FIG. 3. LPS (negatively stained) from S.

3 240 LOPES AND INNISS J. BACTERIOL. FIG. 2. LPS (negatively stained) from E. coli K-12 Gal 23 consisting ofribbons lacking the outer region (arrows). Marker represents 0.1,um. 0 FIG. 3. LPS (negatively stained) from S. typhimurium TV]I 9 RI! consisting ofribbons lacking the outer region (arrows). Marker represents 0.1 im.

4 VOL. 103, 1970 LPS FROM E. COLI 241 ; 'i,i:^ 8;-'. '* w!-, i.-.- e 01.~~~~~~~~~1 Ji -Iw * FIG. 4. LPS (positively stained) from avian E. coli 018 after treatment with I mg of sodium lauryl sulfate per ml. Double-line structures are apparent at various locations on the ribbonis (arrows). Marker represents 0.1 jam. lines, infrequently seen in the untreated LPS, were more frequently present and appeared more prominently. DISCUSSION The electron microscopic examination of positively stained avian E. coli 018 LPS showed ribbonlike structures with branching (4). Occasionally a rather dense parallel line structure was observed to be associated with a ribbon (4) similar to that reported for LPS from S. typhimurium (11). To explain such a structure, a trilaminar model was proposed by Shands et al. (11). According to the model, the LPS has a tendency to orientate itself on its widest surface and twists appear when the LPS stands on its thin edge, thus causing the appearance of the double-line structure. When negatively stained LPS from avian E. coli 018 was examined, ribbons with nearly twice the width as observed with positive staining were seen. The structural details can be interpreted by a comparison with the two models of LPS, one as proposed by Shands et al. (11) and the other as proposed by Rothfield and Horne (9). According to the first model, the double lines observed with negative staining would be due to a continuous orientation of the LPS on its thin edge, which seems unlikely. One mode of orientation of the LPS ribbon in the positive staining and a different mode of orientation in the negative staining does not account for the uniform staining pattern of the ribbon (including the branching points) observed in the positively stained LPS (4) and the continuous bifurcation of the inner unstained region seen in the negatively stained LPS (Fig. 1). If, in the negative staining (Fig. 1), the LPS were to be on its thin edge and thus represent longitudinal cross section of the ribbon, then in the positive staining representing the wider surface, the branches would be densely stained at the branching points due to overlapping and vice versa. On the other hand, Rothfield and Horne (9) have attributed unstained regions, observed in electron micrographs of negatively stained phosphatidyl ethanolamine, to the hydrophobic moiety in the organized lipid structure as originally suggested for lecithin by Bangham and Horne (1) and Lucy and Glauert (5). They proposed a model of LPS in which a basic unit in the structure consists of a hydrophobic lipid moiety and a polysaccharide part. Such units combine by opposing their hydrophobic regions in the center, leaving their polysaccharide parts external (Fig. 5). Thus, the central hydrophobic region

5 242 LOPES AND INNISS J. BACTERIOL. POLYSACCHARIDE CHAIN LIPID HYDROPHOBIC LIPID REGION ASSEMBLY OF LPS UNITS TO FORM RIBBON SHAPED PROPOSED flu fyy)ryyvvwewt LL- - [H11lcIH(a I 4 OBSERVED 7 OUTER UNSTAINED REGION / CENTRAL UNSTAINED REGION CORRELATION OF OBSERVED AFTER PROPOSED MODEL WITH NEGATIVE STAINING DISC SHEET SPHERE cross section PROPOSED ASSEMBLY OF LPS UNITS TO FORM VARIOUS S PREVIOUSLY OBSERVED (1,2,7) FiG. 5. Schematic representations of the LPS structure.

6 VOL. 103, 1970 LPS FROM E. COLI 243 might not be penetrated by sodium phosphotungstate used in the negative staining and would be demarcated by a line of stain on either side, resulting in a parallel double-line appearance. The polysaccharide side chains would lie together on either side of the center. The space between them might be too small for complete penetration of the sodium phosphotungstate giving rise to a lightly stained region on either side of the central zone. Such a structure would be quite stable because the polar groups (i.e., phosphates and residual amino acids) on the surfaces would shield the internal hydrophobic residues from the surrounding aqueous environment. A region of 0-specific side chains on either side of the LPS ribbon has also been suggested by the use of ferritin-labeled antibodies (11). The two outer regions observed by negative staining of avian E. coli 01 g LPS appear to consist of these 0-specific side chains. This conclusion is supported by the absence of similar outer regions on the structure of the negatively stained LPS from the two rough strains which lack 0-antigenic side chains. The treatment with sodium lauryl sulfate appeared to result in the rearrangement of the groups with high affinity for uranyl acetate, which is used for positive staining of the LPS. The reactive groups uniformly present on the surface of the ribbon appeared to arrange in two parallel lines on either side of the ribbon (Fig. 4). This might be a step before a complete disintegration of the LPS structure by the detergent as reported by McIntire et al. (6). ACKNOWLEDGMENTS This investigation was supported by National Research Council of Canada grant A We thank K. Zachariah for his help with the electron microscopy. LITERATURE CITED 1. Bangham, A. D., and R. W. Horne Negative staining of phospholipids and their structural modification by surface-active agents as observed in the electron microscope J. Mol. Biol. 8: Beer, H., A. I. Braude, and C. C. Brinton, Jr A study of particle sizes, shapes, and toxicities present in a Boivintype endotoxic preparation. Ann. N.Y. Acad. Sci. 133: Bladen, H. A., and S. E. Mergenhagen Ultrastructure of Veilloniella and morphological correlation of an outer membrane with particles associated with endotoxic activity. J. Bacteriol. 88: Lopes, J., and W. E. Inniss Electron microscopy of effect of polymyxin on Escherichia coli lipopolysaccharide. J. Bacteriol. 100: Lucy, J. A., and A. M. Glauert Structure and assembly of macromolecular lipid complexes composed of globular micelles. J. Mol. Biol. 8: Mclntire, F. C., H. W. Sievert, G. H. Barlow, R. A. Finley, and A. Y. Lee Chemical, physical, and biological properties of a lipopolysaccharide from Escherichia coli K-235. Biochemistry 6: Milner, K. C., R. L. Anacker, K. Fukushi, W. T. Haskins M. Landy, B. Malmgren, and E. Ribi Symposium on relationship of structure of microorganisms to their immunological properties. II. Structure and biological properties of surfatce anitigens fromn gram-negative bacteria. Bacteriol. Rev. 27: Rapin, A. M. C., and H. Mayer Complex polysaccharides in different strains of Escherichia coli K-12. Ann. N.Y. Acad. Sci. 133: Rothfield, L., and R. W. Horne Reassociation of purified lipopolysaccharide and phospholipid of the bacterial cell envelope: Electron microscopic and monolayer studies. J. Bacteriol. 93: Schramm, G., 0. Westphal, and 0. Luderitz Jber bakterielle Reizstaffe. Ill. Mitt.: Physikalisch-chemisches Verhalten eines hochereinigten Colipyrogens. Z. Naturforsch. 76: Shands, J. W., Jr., J. A. Graham, and K. Nath The morphologic structure of isolated lipopolysaccharide. J. Mol. Biol. 25: Truscott, R. B., and W. E. Inniss Studies on a lesioninducing factor of avian strains of Escherichia coli. Can. J. Microbiol. 13: Weidel, W., H. Frank, and H. N. Martin The rigid layer of the cell wall of Escherichia coli, strain B. J. Gen. Microbiol. 22: Westphal, O., and K. Jann Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure, p Itt R. L. Whistler (ed.), Methods in carbohydrate chemistry, vol. 5. Academic Press Inc., New York. 15. Work, E., K. W. Knox, and M. Vesk The chemistry and electron microscopy of an extracellular lipopolysaccharide from Escherichia coli. Ann. N.Y. Acad. Sci. 133: