Protein-carbohydrate-lipid complex isolated from the cell envelopes of Chlamydia psittaci in alkaline buffer and ethylenediaminetetraacetate.

Exposure of isolated cell envelopes from purified infectious elementary (EB) of Chlamydia psittaci to sodium carbonate-bicarbonate buffer at pH 10 plus ethylenediaminetetraacetate (EDTA) results in partial solubilization of the total protein. The released materials represent 20% of the dry weight, 16% of the total protein, 40% of the total carbohydrate, and 9% of the total lipid of the cell envelopes. Sucrose density gradient centrifugation, and Sephadex G-200, Sepharose 4B, or diethylaminoethyl-cellulose column chromatography, reveal a protein-carbohydrate-lipid complex of several hundred thousand molecular weight that contains 50% protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated EB cell envelopes reveals two major protein bands, A and B, with estimated molecular masses of approximately 85,000 and 53,000, respectively, both of which also stain for the presence of carbohydrate and lipid. Gel electrophoresis of the protein-carbohydrate-lipid complex reveals two protein bands, C and D, with estimated molecular weights of approximately 17,000 and 13,000, respectively, which contain lipid and a small amount of carbohydrate; bands A and B are not present in the complex. Gel electrophoresis of the cell envelope residues after extraction of the complex with alkali and EDTA shows a single main band, corresponding to the position of band B, which contains protein, carbohydrate, and lipid; band A is completely missing. B and A is believed to be a component of the complex, which is split into two subunits on alkali solubilization.     

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.     

Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes.

Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L2 were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. Our data suggest that these putative chlamydial adhesins play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.     

Surface projections and internal structure of Chlamydia psittaci.

The outermost surface of the small infectious forms of Chlamydia psittaci contain geometrically arranged spikes distributed over approximately 50% of the bacterial surface. The spikes are located opposite the concave side of an electron-dense crescent-shaped chlamydial core.     

Architecture of the cell envelope of Chlamydia psittaci 6BC.

The cysteine-rich envelope proteins of the elementary body form of chlamydiae are thought to be located in the outer membrane on the basis of their insolubility in the weak anionic detergent N-lauryl sarcosinate (Sarkosyl). We found, however, that the insolubility of the small (EnvA) and the large (EnvB) cysteine-rich proteins of Chlamydia psittaci 6BC in Sarkosyl is dependent on the maintenance of a supramolecular disulfide-cross-linked complex and is unlikely to be a valid indicator of outer membrane location. Consequently, we used other methods to characterize the architecture of the cell envelope of C. psittaci 6BC. We found that disulfide-reduced EnvA, previously shown to be a lipoprotein, segregated into the detergent phase during Triton X-114 partitioning experiments and was recovered from the membrane fraction of elementary bodies lysed by nondetergent means. In contrast, disulfide-reduced EnvB segregated to the aqueous phase in partitioning experiments and was found in the soluble fraction of elementary bodies lysed in the absence of detergents. The hydrophobic affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine labeled the major outer membrane protein and EnvA but did not label EnvB. Treatment of intact elementary bodies of C. psittaci with trypsin had no effect on the cysteine-rich proteins, although the major outer membrane protein was partially degraded. On the basis of these and other observations, we propose that EnvA is anchored to the outer membrane by its lipid moiety, with a hydrophilic peptide portion extending into the periplasm, and that EnvB is located exclusively within the periplasm. We further propose that disulfide-cross-linked polymers of EnvB are the functional equivalent of peptidoglycan, forming a disulfide-cross-linked network with the periplasmic domains of EnvA and other membrane proteins, which accounts for the osmotic stability of elementary bodies.     

Pyrimidine metabolism by intracellular Chlamydia psittaci.

Pyrimidine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined mutations affecting pyrimidine metabolism. C. psittaci AA Mp cannot synthesize pyrimidines de novo, as assessed by its inability to incorporate aspartic acid into nucleic acid pyrimidines. In addition, the parasite cannot take UTP, CTP, or dCTP from the host cell, nor can it salvage exogenously supplied uridine, cytidine, or deoxycytidine. The primary source of pyrimidine nucleotides is via the salvage of uracil by a uracil phosphoribosyltransferase. Uracil phosphoribosyltransferase activity was detected in crude extracts prepared from highly purified C. psittaci AA Mp reticulate bodies. The presence of CTP synthetase and ribonucleotide reductase is implicated from the incorporation of uracil into nucleic acid cytosine and deoxycytidine. Deoxyuridine was used by the parasite only after cleavage to uracil. C. psittaci AA Mp grew poorly in mutant host cell lines auxotrophic for thymidine. Furthermore, the parasite could not synthesize thymidine nucleotides de novo. C. psittaci AA Mp could take TTP directly from the host cell. In addition, the parasite could incorporate exogenous thymidine and thymine into DNA. Thymidine kinase activity and thymidine-cleaving activity were detected in C. psittaci AA Mp reticulate body extract. Thus, thymidine salvage was totally independent of other pyrimidine salvage.     

Purine metabolism by intracellular Chlamydia psittaci.

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell. Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highly purified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract.     

鹦鹉热衣原体B细胞表位的表达及抗原鉴定

利用噬菌体PⅢ蛋白为载体对鹦鹉热衣原体单抗17筛选得到的B细胞抗原表位进行了研究,利用改构后的原核表达载体pQE30,构建含有B细胞表位基因的重组表达质粒,表达的蛋白经ELISA及Western Blot实验证实能够与单抗C17发生特异反应,蛋白免疫动物后得到了抗鹦鹉热衣原体的抗体,验证了所筛选表位的真实性。     

Effect of Penicillin on the Multiplication of Meningopneumonitis Organisms (Chlamydia psittaci)

Although formation of infectious particles of meningopneumonitis organism in L cells was completely inhibited by 1 or more units of penicillin per ml, multiplication of reticulate bodies was observed, by light microscopy, in the presence of 200 units of penicillin per ml in stained smears of infected cells. When reticulate bodies were purified from cultures containing penicillin after 18, 30, and 45 hr of incubation, continuously increasing yields were obtained. When penicillin was added to infected cultures 0 to 15 hr after infection, no increase in infectivity was observed at 40 hr, but when antibiotic was added between 20 and 35 hr, partial synthesis of infectious particles was observed at 40 hr. On the other hand, removal of penicillin from an infected culture before 15 hr after infection did not affect the final yields of infectivity when assayed at 40 hr, but elimination of penicillin after 20 hr resulted in a decrease in infectivity. In suspensions of (32)P-labeled purified reticulate bodies grown in cultures containing penicillin and harvested 18 and 40 hr after infection, the (32)P distributions obtained by acid fractionation were similar to those of reticulate bodies from penicillin-free cultures. Cell membranes of reticulate bodies were also prepared from 40-hr cultures with penicillin. The size and shape of purified membranes, as seen by electron microscopy, and their amino acid compositions were similar to membranes prepared from reticulate bodies grown without penicillin, except that very small structures were observed in membranes from cultures containing penicillin. These results indicated that penicillin does not inhibit reproduction of reticulate bodies and formation of their cell membranes, but does inhibit the formation of elementary body cell envelopes.     

Immunoelectron microscopy of Chlamydia psittaci with monoclonal antibodies.

An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydia psittaci and infected cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.     

Detection of Chlamydia psittaci by Immunofluorescence

A direct fluorescent-antibody (FA) test was developed to detect Chlamydia psittaci in dural impressions from specimen-inoculated mice. Technical procedures for the test were compared. C. psittaci was found in mice after infection as early by the FA technique as it was by cytochemical staining methods usually used. The lymphogranuloma venereum organism was also stained by conjugated antibody to C. psittaci. A distinctive advantage of the described FA test is that organisms are identified immunologically as members of the genus Chlamydia simultaneously with their detection.     

Processing of McCoy cell cultures infected with Chlamydia trachomatis: sequential isolation of chlamydial elementary bodies and lipopolysaccharide

Abstract Triton X-100 (TX100) enhances the liberation of chlamydial elementary bodies (EB) from host cells and dissolves the host cell membrane. In the presence of TX100 only differential centrifugation is needed to isolate reasonably pure EBs. The remaining high-speed supernatant still contains a large part of the chlamydial lipopolysaccharide (LPS), which can be isolated with the standard phenol-chloroform-petroleum ether extraction.     

Adenine nucleotide and lysine transport in Chlamydia psittaci.

Isolated reticulate bodies of Chlamydia psittaci were found to transport ATP and ADP by an ATP-ADP exchange mechanism. ATP uptake activity was not detected in elementary bodies. The apparent Km of transport for both ATP and ADP was approximately 5 microM, and the calculated Vmax for both was about 1 nmol of nucleotide transported per min per mg of protein. ADP competitively inhibited ATP transport with a Ki of 4.5 microM. Other nucleotides tested had no effect on the uptake of ATP. A magnesium-dependent, oligomycin-sensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was associated with reticulate bodies, and most of the transported ATP was hydrolyzed to ADP, which was exchanged for additional, extracellular nucleotide. Some ADP was hydrolyzed to AMP, which exited the cells slowly. Lysine was transported against the electrochemical gradient by reticulate bodies in the presence of ATP. Oligomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited ATP-dependent lysine transport. Lysine exited reticulate bodies when the reticulate bodies were incubated in the presence of ADP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or a reduced concentration of ATP. The results support the concept that chlamydiae are energy parasites which are capable of drawing upon the adenine nucleotides of their hosts, hydrolyzing ATP, and establishing an energized membrane.     

Characterization of lipoprotein EnvA in Chlamydia psittaci 6BC.

The primary sequence of the small cysteine-rich protein (EnvA) of Chlamydia psittaci 6BC has been shown to possess a potential lipid modification/signal peptidase II-processing site, and the mature protein was labeled by a [3H]palmitic acid precursor. We further characterized the mature EnvA, showing that it lacks the N-terminal methionine of the primary peptide, is hydrophobic despite a peptide sequence that is predicted to be hydrophilic, and appears to be lipid modified at an N-terminal cysteine in a manner analogous to that of murein lipoproteins of gram-negative bacteria. We also report the fatty acid content of the small cysteine-rich proteins of C. psittaci and Chlamydia trachomatis L2 as determined by combined gas chromatography-mass spectrometry.     

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.     

Experimental Chlamydia psittaci Infection of Japanese Quail

Japanese quail were used for the infection model of avian chlamydiosis. One-day-old Japanese quail were highly susceptible to lethal infection by a Chlamydia psittaci strain of budgerigar origin upon inoculation via the air sac route with 10^4.1 FFU of the organism, showing an acute and lethal course with chlamydial propagation. In contrast, 7-day-old quail developed resistance to the infection as shown by the lack of lethal effect with the same dose. The resistance of 7-day-old birds was abolished by immunosuppressive treatment with cyclophosphamide. Upon inoculation with a sublethal dose of 10^2.1 FFU, latent infection was established in 1-day-old birds with a minimum number of the organism. The latent infection in the birds was converted to the lethal form by treatment with cyclophosphamide along with chlamydial propagation and suppression of antibody production.     

Arrays of hemispheric surface projections on Chlamydia psittaci and Chlamydia trachomatis observed by scanning electron microscopy.

Scanning microscopy of two strains of Chlamydia psittaci and four strains of Chlamydia trachomatis representative of the wide diversity in origin and behavior of members of the genus revealed patches of regular arrays of hemispheric projections on the surfaces of elementary bodies of all six strains. These distinctive and perhaps unique surface structure are probably present in all populations of chlamydiae.