Electron Microscopic Observations on the Fine Structure of Cell Walls of Chlamydia psittaci

L-cell cultures were infected with elementary bodies (EB) of meningopneumonitis organisms. Cell walls were prepared from reticulate bodies (RB), which are the intracellular developmental forms into which EB are converted, and from EB at appropriate times after infection. When fragmented EB cell walls were shadowcast with platinum palladium alloy, about one-half of the fragments were seen to be composed of hexagonally arrayed structures on the inner side of the cell wall. When EB cell walls were negatively stained with phosphotungstic acid, they all showed this fine structural array. These macromolecular units were estimated to be about 18 nm in diameter. RB cell walls, harvested at various times after infection, were similarly stained; about 20% of RB walls at 15 hr after infection showed traces of these regular structures, but only 2% of them had the structures at 24 hr. When RB cell walls prepared from penicillin-containing culture were examined, they were observed to be similar to RB without penicillin. When EB cell walls were treated with formamide at 160 C, and then centrifuged in a 10 to 40% potassium tartrate density gradient, hexagonal particles about 20 nm in diameter were obtained as a middle band in the gradient column. These particles were not obtained from RB cell walls harvested from cultures with or without penicillin. It is concluded that the particles are macromolecular subunits located on the inner side of the EB cell walls, that the subunits probably provide the structural rigidity found in the EB, and that their synthesis is inhibited by penicillin.     

Fine Structures of Cell Envelopes of Chlamydia Organisms as Revealed by Freeze-Etching and Negative Staining Techniques

The cell walls of Chlamydia psittaci (meningopneumonitis strain) were examined by the freeze-etching and negative staining techniques. It was observed that the cleaved convex surface of the developmental, reticulate body was covered with numerous non-etchable particles 9 to 10 nm in diameter, these particles being rarely seen on the concave surface. Similarly, the convex surface of the mature, elementary body (EB) was covered with many particles but the concavity lacked these particles. After etching, the smooth concave surface of the EB appeared to have a hexagonally arrayed subunit structure, on which the button structure (B structure) was observed. Each B structure had a diameter of 27 nm and several B structures were grouped together in a hexagonal arrangement with a center-to-center spacing of 45 nm. In a limited area of the negatively stained EB cell wall, hexagonally arrayed rosette structures were present, with a center-to-center spacing similar to the B structures seen in the freeze-etched preparation. Each rosette, about 19 to 20 nm in diameter, appeared to be composed of a radial arrangement of nine subunits. The freeze-fractured cell wall-cytoplasmic membrane complexes indicated that the outer surface of the cytoplasmic membrane which appeared as the convex surface was covered with the fine particles, and thus it was likely that frozen EB was cleaved at the gap between the cell wall and ctyoplasmic membrane. On the cleaved inclusion, several groups of fine particles were observed. In each group, the particles were arranged hexagonally with the spacing ranging from 20 to 50 nm.     

Separation of the Polypeptides of Chlamydia and Its Cell Walls by Polyacrylamide Gel Electrophoresis

The polypeptide composition of Chlamydia was examined by acrylamide gel electrophoresis. When the polypeptide patterns of purified infectious elementary bodies (EB) of C. psittaci meningopneumonitis strain, 6BC strain, and C. trachomatis T'ang strain were compared, no significant differences were observed. The polypeptide patterns of whole EB and reticulate bodies (RB) appeared to overlap, but differences were found. In EB cell walls, nine main and several minor bands of polypeptides were observed in gels containing sodium lauryl sulfate, and the eighth main band from the top of the gel stained positive with periodic acid-Schiff reagent. On the other hand, the polypeptides in bands 3, 6, and 8 in EB cell walls were missing or minor in RB cell walls, and the ninth band was clearly stained by PAS. Band 8 was also stained slightly. Purified subunits, which occur as a lattice structure on the inside layer of EB cell walls but are largely missing in RB cell walls, contained bands 4, 6, and 8, and band 8 was PAS positive. These results indicate that significant polypeptide synthesis or reorganization in the cell walls occurs during the growth cycle.     

Electron microscopic observations of surface projections on Chlamydia psittaci reticulate bodies.

Electron microscopic observations were carried out to confirm the presence of surface projections on Chlamydia psittaci reticulate bodies (RBs). The morphology of the projections on RBs was identical with that on elementary bodies (EBs); one end of each projection was connected with the cytoplasmic membrane, but the other end of the projection protruded beyond the cell wall through a fine hole or rosette in the cell wall. The results demonstrated that the rosettes seen in RB cell walls were morphological markers indicating the presence of the surface projections. A statistical anaylsis of the number of projections on EBs and the number of rosettes in RB cell walls prepared at 10, 15, and 20 h after infection demonstrated that all RBs had the projections and that the number of projections was maximal by 10 h after infection and then decreased gradually to approximately the same number of projections on EBs.     

Type III Secretion, Contact-dependent Model for the Intracellular Development of Chlamydia

The medically significant genus Chlamydia is a class of obligate intracellular bacterial pathogens that replicate within vacuoles in host eukaryotic cells termed inclusions. Chlamydia's developmental cycle involves two forms; an infectious extracellular form, known as an elementary body (EB), and a non-infectious form, known as the reticulate body (RB), that replicates inside the vacuoles of the host cells. The RB surface is covered in projections that are in intimate contact with the inclusion membrane. Late in the developmental cycle, these reticulate bodies differentiate into the elementary body form. In this paper, we present a hypothesis for the modulation of these developmental events involving the contact-dependent type III secretion (TTS) system. TTS surface projections mediate intimate contact between the RB and the inclusion membrane. Below a certain number of projections, detachment of the RB provides a signal for late differentiation of RB into EB. We use data and develop a mathematical model investigating this hypothesis. If the hypothesis proves to be accurate, then we have shown that increasing the number of inclusions per host cell will increase the number of infectious progeny EB until some optimal number of inclusions. For more inclusions than this optimum, the infectious yield is reduced because of spatial restrictions. We also predict that a reduction in the number of projections on the surface of the RB (and as early as possible during development) will significantly reduce the burst size of infectious EB particles. Many of the results predicted by the model can be tested experimentally and may lead to the identification of potential targets for drug design.     

Hemagglutinin in Cell Walls of Chlamydia psittaci

Intact purified elementary bodies (EB) of Chlamydia psittaci agglutinate chicken erythrocytes in low titer, whereas homogenates of EB and of EB cell walls agglutinate at much higher titers depending on the extent of disruption by shaking and sonication. The hemagglutinin is contained in the cell envelope and can be purified with cell wall fractions. Treatment of cell wall with sodium dodecyl sulfate completely inactivated the hemagglutinin. Purified hemagglutinin was found to have an identical polypeptide composition to EB cell walls. Preparations of purified reticulate forms, the reproductive intracellular form of the organism, were almost totally devoid of hemagglutinin.     

Unique ultrastructure in the elementary body of Chlamydia sp. strain TWAR.

The ultrastructure of two prototype strains (TW-183 and AR-39) of Chlamydia sp. strain TWAR was described. The TWAR elementary body (EB) demonstrated a unique morphology and structure distinct from those of other chlamydial organisms. It was pleomorphic but typically pear shaped. The average size was 0.38 micron, with a long axis of 0.44 micron, a short axis of 0.31 micron, and a ratio of the long to the short axes of 1.42. The cytoplasmic mass was round, with an average diameter of 0.24 micron. There was a large periplasmic space. Small, round electron-dense bodies (0.05 micron in diameter), which were attached to the cytoplasm by a stringlike structure, were seen in the periplasmic space. These features are in contrast to those of other chlamydiae, which are typically round with a narrow or barely discernible periplasmic space. The TWAR reticulate body (RB) was morphologically and structurally similar to those of other Chlamydia species, having an average diameter of 0.51 micron and being circular in shape. The ultrastructural observations of the intracellular growth of TWAR in HeLa cells revealed that TWAR underwent the same developmental cycle as do other chlamydiae, i.e., transformation of EB to RB, multiplication by binary fission, and maturation by transformation of RB to EB via the intermediate-form stage.     

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis survival within amoebal cysts

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.     

The persistence of Chlamydia trachomatis elementary body cell walls in human polymorphonuclear leucocytes and induction of a chemiluminescent response

Human polymorphonuclear leucocytes (HPMN) were incubated with [35S]methionine-labelled Chlamydia trachomatis (serovar L2/434/Bu) elementary bodies (EB) and EB cell walls. No net loss in the TCA-precipitable radioactivity was observed over 24 h in the HPMN that had taken up EB cell walls. SDS-polyacrylamide gel electrophoresis of the labelled C. trachomatis EB and EB cell wall proteins extracted from the HPMN at 2 and 24 h after infection demonstrated the persistence of most of the chlamydial cell wall polypeptides. Analysis of extracts of the HPMN that had taken up either EB or EB cell walls on Urografin density gradients at 2 and 24 h after infection, and electron microscopic observations on fractions representing the peaks, demonstrated the persistence of the EB cell walls in the HPMN. Electron microscopic observations of HPMN that had taken up EB or EB cell walls demonstrated EB cell walls in the HPMN phagosomes at 24 h after infection. The HPMN exposed to EB and EB cell walls of C. trachomatis gave chemiluminescent (CL) responses with peaks respectively 12 and 7 times greater than the peak value of the control. The significance of the persistence of the EB cell wall polypeptides and cell walls in the HPMN and activation of the HPMN to produce oxygen radicals (i.e. a CL response), and its possible relation to rheumatic diseases, is discussed.     

Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.     

Ultrastructure and partial characterization of a regular array in the cell wall of Lactobacillus brevis.

The cell wall of Lactobacillus brevis was revealed by electron microscopy to have an outer layer composed of a regular array. The morphological unit of the regular array appeared to consist of four spherical subunits, each about 2 nm in diameter, which were arranged in a tetragonal pattern about 4.5 by 7.0 nm in dimension. The regular array was composed of the tetragonal units in rows in two directions at an angle of about 75 degrees to each other. The average spacing between the rows was about 10 nm in one direction and about 7 nm in the other. The tetragonally arranged subunits were removed from the cell wall by treatment with guanidine hydrochloride, urea, or sodium dodecyl sulfate (SDS) but not by the action of ethylenediaminetetraacetate, nonionic detergents, or proteolytic enzymes except pepsin. The regular subunits were shown to be composed of a protein with a molecular weight of about 51,000 by SDS-polyacrylamide gel electrophoresis.     

THE CELL WALL OF PYROCYSTIS SPP. (DINOCOCCALES)1,2

The cell of Pyrocystis spp. is covered by an outer layer of material resistant to strong acids and bases. Internal to this layer much of the cell wall is composed of cellulose fibrils. The presence of cellulose fibrils was established by staining raw and ultra-violet–peroxide-cleaned cell walls and by combining X-ray diffraction spectroscopy with electron microscope observation. Carbon replicas of freeze-etched preparations and thin sections of P. lunula walls show outer layers, inside them ca. 24 layers of crossed parallel cellulose fibrils (4–5 nm thick, ca. 12 nm wide), then a region of smaller (ca. 6–12 nm diameter) fibrils in a disperse texture, and then the plasma membrane. Cellulose fibrils in the parallel texture are constructed of 3–5 elementary fibrils ca. 3 nm in diameter. Walls of P. fusiformis and P. pseudonctiluca also have cellulose fibrils in a crossed parallel texture similar to those of P. lunula. The Gymnodinium-type swarmer from lunate P. lunula appears to have a cell wall ultrastructure typical of other “naked” dinoflagellates.     

Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy

Summary Glucuronoxylans (GXs), the main hemicellulosic component of hardwoods, are localized exclusively in the secondary wall of Japanese beech and gradually increase during the course of fiber differentiation. To reveal where GXs deposit within secondary wall and how they affect cell wall ultrastructure, immuno-scanning electron microscopy using anti-GXs antiserum was applied in this study. In fibers forming the outer layer of the secondary wall (S₁), cellulose fibrils were small in diameter and deposited sparsely on the inner surface of the cell wall. Fine fibrils with approximately 5 nm width aggregated and formed thick fibrils with 12 nm width. Some of these thick fibrils further aggregated to form bundles which labelled positively for GXs. In fibers forming the middle layer of the secondary wall (S₂), fibrils were thicker than those found in S₁ forming fibers and were densely deposited. The S₂ layer labelled intensely for GXs with no preferential distribution recognized. Compared with newly formed secondary walls, previously formed secondary walls were composed of thick and highly packed microfibrils. Labels against GXs were much more prevalent on mature secondary walls than on newly deposited secondary walls. This result implies that the deposition of GXs into the cell wall may occur continuously after cellulose microfibril deposition and may be responsible for the increase in diameter of the microfibrils.Abbreviations GXs glucuronoxylans - PBS phosphate-buffered saline - RFDE rapid-freeze and deep-etching technique - FE-SEM field emission scanning electron microscope - TEM transmission electron microscope     

Regular Array in the Cell Wall of Lactobacillus fermenti as Revealed by Freeze-Etching and Negative Staining1

Freeze-etching of Lactobacillus fermenti F-4 (NCTC 7230) revealed that the outer layer of the cell wall was composed of a regular array in which parallel lines ran obliquely to the longitudinal axis of the cell with an average distance between the centers of about 9.6 nm and were intersected by thinner lines with an average periodicity of approximately 6.2 nm at an angle of about 75°. Occasionally the direction of the striation was discontinuously shifted near one end of the cell. Beneath the regular array the middle cell wall layer packed with granules and the smooth inner cell wall layer were discernible and the mesosomes were also visible in the cytoplasm. When the ultrastructure of isolated outer cell wall fragments was examined by negative staining, the regular array appeared to be composed of subunits, about 3.6 nm in diameter, which were arranged in a tetragonal pattern. The tetragonal array consisted of the subunits in rows in two directions at an angle of about 75° to each other. The average spacing between the rows was about 9.3 nm in one direction and 5.5 nm in the other direction.     

Electron Microscopic Observations on the Effects of Penicillin on the Morphology of Chlamydia psittaci

L cells 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 developmental reticulate body (RB), and, up to 12 hr after infection, the treated and untreated cultures were identical. After that time, however, penicillin-treated organisms showed striking differences in that binary fission was prevented, large abnormal 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 fragmentation 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.     

Bacteriophage T4D receptors and the Escherichia coli cell wall structure: role of spherical particles and protein b of the cell wall in bacteriophage infection.

The nature of the interaction of bacteriophage T4D and the outer cell wall of its host, Escherichia coli B, has been investigated. Bacteria with altered or modified cell walls have been obtained by two different growth procedures: (i) growth in high osmolarity medium or (ii) growth in broth in the presence of divalent heavy metal ions. When these altered host cells were washed and subsequently added to regular growth medium, they interacted with added phage particles, but successful infection did not occur. Most of the phage particles released from these treated cells were observed to have full heads and an altered tail structure. The altered phage tails had contracted sheaths and unusual pieces of the bacterial cell wall attached to the distal portion of the exposed phage tail tube. Phage released from bacteria grown in the high osmolarity medium had attached cell wall pieces of two major types, these pieces being either 40 or 21 nm in diameter. The smaller-type cell wall pieces (21 nm) were formed by three spheres each measuring 7 nm in diameter. Phage particles released from cells previously exposed to the divalent metal ions had only one 7-nm cell wall sphere attached to the distal end of the tail tube. It was found that these 7-nm spheres (i) are normal components of the cell wall and are morphologically similar to endotoxin, (ii) are held in place on the cell wall by a component of the cell wall called protein b, and (iii) are most likely the site of penetration of the phage tail tube through which the phage DNA enters the host cell.     

Origin and Structure of the Group-Specific, Complement-Fixing Antigen of Rickettsia rickettsii

Rickettsia rickettsii was treated with ether and examined by negative-contrast electron microscopy. Group-specific complement-fixing antigen was seen to be originating from the cell wall. The antigen was composed predominately of round particles 10 to 60 nm in diameter. Intact R. rickettsii and antigen from ether-treated organisms were purified by density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. The whole rickettsial cell was composed of a minimum of 30 proteins which ranged in molecular weight from about 23,000 to 155,000. The "soluble" antigen contained nine proteins ranging in molecular weight from about 28,000 to 150,000.     

Mode of Host Cell Penetration by Bacteriophage φX174

Bacteriophage phiX174 is an icosahedral phage which attaches to host cells without the aid of a complex tail assembly. When phiX174 was mixed with cell walls isolated from the bacterial host, the virions attached to the wall fragments and the phage deoxyribonucleic acid (DNA) was released. Attachment was prevented if the cell walls were treated with chloroform. Release of phage DNA, but not viral attachment, was prevented if the cell walls were incubated with lysozyme or if the virions were inactivated with formaldehyde. Treatment of the cell walls with lysozyme released structures which were of uniform size (6.5 by 25 nm). These structures attached phiX174 at the tip of one of its 12 vertices, but the viral DNA was not released. The virions attached to these structures were oriented with their fivefold axis of symmetry normal to the long axis of the structure. No virions were attached to these structures by more than one vertex. Freeze-etch preparations of phiX174 adsorbed to intact bacteria showed that the virions were submerged to one half their diameter into the host cell wall, and the fivefold axis of symmetry was normal to the cell surface. A second cell could not be attached to the outwardly facing vertex of the adsorbed phage and thus the phage could not cross-link two cells. When the virions were labeled with (3)H-leucine, purified, and adsorbed to Escherichia coli cells, about 15% of the radioactivity was recovered as low-molecular-weight material from spheroplasts formed by lysozyme-ethylenediaminetetraacetic acid. Other experiments revealed that about 7% of the total parental virus protein label could be recovered in newly formed progeny virus.     

Structural differentiation of the Bacillus subtilis 168 cell wall.

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.