The effect of purification on the ultrastructure and infectivity of egg-attenuated Chlamydia psittaci (6BC).

The effect of purification on the ultrastructure and infectivity of egg-attenuated Chlamydia psittaci (6BC)l

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Texas A&M University on 11/14/14 For personal use only.

J . W. COSTERTON Departtnent of B i o l o g y , U n i i ~ e r s i t yof Grlgnr-y, Ccrlgnry, A l b o r n

AND

LINDA POFFENROTH, J . C. WILT,A N D NONNAKORDOVA Depnrtt?letlt of M e d i c a l M i c r o b i o l o g y , U t ~ i i - r r s i f of y M a n i t o b a , Winnipeg, M a n i t o b a

Accepted May 12. 1975

J . C. WILT. and N. KORDOVA.1975. The effect of COSTERTON.J. W., L. POFFEVROTH, purification on the ultrastructure and infectivity of egg-attenuated C / ~ / r r t n y d i npsirtnci (6BC). Can. J. Microbiol. 21: 1448-1463. A procedure is described for the pusification of mixed populations of the three different morphological forms of Cklatnydicr psirrcrci (6BC) from infected yolk sac membranes. Elementary bodies and small intermediate bodies are not perceptibly damaged during purification which involves homogenization of the host cells. differential centl.ifilgation. sedimentation through 20% sucrose. and treatment with trypsin. The observation that elementary bodies undergo plasmolysis in 2% sucrose is interpreted as indicating that the cytoplasmic memhmne of these particles is intact at that stage in the purification. Reticulate bodies and large intermediate bodies are damaged. to a degree. by the homogenization step. This damage takes the form of discontinuities of the outer envelope memhl-ane. and results in the loss of the regular coccohacillary shape of these particles and in an increase in their size. Treatment with a combination of RNase and DNase was found to cilcise profound damage to all three morphological forms of the chlamydiae. COSTERTON.J . W., L. POFFENROTH.J. C. WILT et N. KORDOVA. 1975. T h e effect of and infectivity ofegg-attenuated ClrInt71ydicrpsittcrci (6BC). purification on the ~~ltrastructure Can. J . Micsobiol. 21: 1448-1463. Une methode est decsite pour la purification d e populations mixtesde trois formes rnorphologiques differentes de Chlertngciict ~ ~ s i t r c r(6BC) ci obtenues des membranes infecteesdu jaune d'ceuf. Les particules elementaires et les petites particules intermedi;rires ne sont pas endommagees de faqon perceptihle a u cours d e la purification q ~ cornpol-te ~ i une homogen6isation des cellules h6tes. une centrifugation differentielle. une sedimentation travers une solution de sucrose i 20%. et un traitenlent h la trypsine. L'observation d'une plasmolyse chez les pal-ticules 6ltment:lires dansla solution de sucrose h20% est une indication que la membranecytoplasmiq~~e de ces pal-ticules est intacte ;I ce stade de la purification. Les pal-ticules reticuldes e t les grasses particules intei-mediaires sont quelque peu endommagees a u cours d e I'homogenCisation. Ce dommage consisle en des discontinuites dans I'enveloppe membranaire externe et conduit B une r e ces pasticules ninsi qu'h un accroissement d e leur perte de la forme coccobacillaire I t g ~ ~ l i ede dimension. Un traitement par une combinaison de RNase e t de DNase cause des dommages importants aux trois formes morphologiques de Chlamydia. [Traduit par le journal]

Introduction Chlamydia1 agents exhibit a form of pleomorphism which is unique among the gram-negative bacteria with which they are presently classified (10) and their several life forms differ in ultrastructure, stability, and infectivity (14). This morphological heterogeneity leaves the investigator with two alternatives in a purification procedure: to attempt to separate each morphologically distinct form (4, 5, 7-9, 12), or to obtain enriched preparations of the mixed 'Received December 24, 1974.

population of chlamydia1 particles (1 1, 15). Both methods have been used in recent years to obtain chlamydia from tissue-culture cells (5, 7-9, 14-16). However, only a limited number of chlamydia1 strains can be maintained in cultured cells (14). Our previous work with a n egg-maintained 6BC strain was directed to the first alternative (12). The objective of this present study was to examine the effects of currently used purification procedures (5, 7-9, 14-16) o n the ultrastructure and infectivity of an enriched mixed population of chlamydiae from the chick embryo yolk sac. The aim was to obtain as

COSTERTON ET AL.: CHLAMYDIA PSITTACI 6BC

Infected yolk sacs in TSB I

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Pellet Centrifi~ged;It 27 000 x g fol 120 min

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FIG. 1. The circled numbers indicate samples of material examined by infectivity titrat~onw ~ t hadditional immunofluorescence, and by electron microscopy. Except for the enzyme treatments manipulation of the material was carried out at 4 "C.

complete as possible a mixed population of intact purified particles for further analysis. obTherefore we have examined the tained after each separate step during purification. Materials and Methods Clrlan~ycl~a p ~ i l r a c 6BC, i lot 5 , was obtained from the American Type Culture Collection (ATCC) as a lyophilized preparation of infected chick embryo yolk sacs. Before receipt it was passaged originally in mice, then 15 times in the yolk sac of chick embryo (CE); its mean lethal dose (LD,,) in 1- to 2-day mice after intracerebral inoculation was 105.5/0.03ml.

Thestock material for purification consisted of infected yolk sac membranes from the second Passage of the ATCC strain of 6BC in chick embryos, obtained as follows: 5-day-old en~bryonated eggs were inoc~ilated with seed suspensions of 6BC diluted to give a 25z mortality rate between postinoculation days 5 and 6. On the 6th day, the infected yolk sac membranes were harvested from the surviving eggs, tested for stelility, and stored overnight at 4OC. Man~pulationsof the yolk sac material were-then carried through at 4 O C , without significant interruption: freezing and thawing of the material during purification was avoided. A diagrammatic representation of the purification scheme followed is given in Fig. 1. Samplesof the material for electron microscopy and for determination of the


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infectivity were removed at selected stages of the purification process, as indicated by the circled numbers in this figure. Initially, a 20% suspension of the infected yolk sacs in trypticase soy broth (TSB; Difco) was prepared in closed flasks containing glass beads. Homogenization was done by manual shaking of the yolk sacs and beads for 5 min after which the homogenate was centrifuged at 100 x g for I0 min. The supernatant from this "low-speed" centrifugation was ccntrifuged a t 2700 x g for 2 h and the pellet from this "high-speed" centrifugation was suspended in TSB and subjected to two more cycles o f low- and high-speed centrifugation. The final pellet after high-speed centrifugation was resuspended in TSB and spun through 20% sucrose in 0.033 M tris(hydroxymethy1)aminomethane (Tris), pH 7.4, at 180 000 x g for 2 h, yielding a pellet and a wide band of floating material. The pellet was washed in TSB and centrifuged at 180 000 x g to remove sucrose. The wide floating band was diluted in TSB, sedimented at 150 000 x x, and sampled, but was not processed further. The washed pellet of 6BC from the sucrose centrifugate was treated with 0.14, trypsin (Nutritional Biochemicals, Cleveland, Ohio) for 20 min at 37 "C, then subjected to dityerential centrifugation as above. The final p~~rification step was treatment of the pelleted niaterial with 0.1 mg/nil ribonuclease (RNase) (Worthington Biochemical,

Freehold, New Jersey) and 0.1 mg/ml deoxyribonuclease (DNase) (Worthington Biochemical, Freehold, New Jersey) in 0.1 M acetate buffer (with 0.02 M MgSO,) (2). After digestion, the 6BC suspension was differentially centrifuged and examined. Each of the numbered samples in Fig. 1 was fixed in glutaraldehyde and embedded for electron microscopy as previously described (12). Sections were prepared with an LKB Ultratome 111 using glass knives, collected o n uncoated 400-mesh copper grids, and stained for 30 min with fresh aqueous 1% uranyl acetate, then by lead citrate for 15 min (13). After carbon-backing the grids were examined in a n AEI 801 electron microscope. The infectivity of samples 1, 3, 4, 5, 7, 8, 9, and 10 was assayed in monolayers of L cells. Chlamydia1 inclusions present in the cells after infection were determined by staining with May-Grunwald-Giemsa, and by indirect immunofluorescent staining using antiserum against C. psillaci 6BC as previously described (6).

Results As a control, homogenized yolk sac from non-infected eggs wss examined by electron microscopy. It did not contain any bacterial cells or viral particles which could be confused with chlamydiae (Fig. 2). The homogenate of

F I G .2. Electron micrograph of a section of a honiogenate of ~~ninfected yolk sac membranes to show the ent micrographs indicates absence of bacterial cells and viral particles. The bar in this and s ~ l b s e q ~ ~electron 0.1 I.lm. FIG.3. Electron micrograph of a section of the homogenate of yolk sac membranes infected with C . psillnci (6BC) showing intermediate (1) and elementary (E) bodies. FIGS.4-7. Electron micrographs of sections of the hon~ogenateof infected yolk sac membranes showing o ~ ~ s4), an intermediate body (I) whose spherical reticulate bodies whose outer membrane is d i s c o n t i n ~ ~ (Fig. enveloping menibranes are not closely apposed (Fig. 5), and elementary bodies (Figs. 6 and 7). The inner nienibrane of one of the elementary bodies was of the type I1 variety (Fig. 7, arrow). FIGS.8-10. Electron micrographs of sections of the first low-speed pellet of the homogenate of yolk sac membranes infected with C. psi//rrci(6BC) showing a reticulate body (R), several intermediate bodies (I), and an elementary body (Fig. 10). FIGS. 1 1-17. Electron micrographs of sections of thesecond and third high-speed pellet from the differential centrifugation of the homogenate of yolk sac rnenibranes infected with C. psil~rrci(6BC). These micrographs show reticulate bodies whose outer membrane is distended or lost (Fig. I I), an intermediate body whose enveloping membranes are only slightly separated (Fig. 12), and less-condensed (Fig. 13 and F i n Fig. 17)and highly condensed (Figs. 14-16 and D in Fig. 17) elementary bodies whose enveloping membranes are very closely apposed. FIGS.18-23. Electron micrographs of sections of chlamydial particles after sedimentation through 20% sucrose showing a retic~~late body whose outer membrane is discontinuous (Fig. 18), a well-prcscrvcd intermediate body (Fig. 19), and elementary bodies whose inner membrane is retracted from contact with the outer membrane by plasrnolysis (Figs. 20-23), Note the linear and particulate electron-dense material (arrows) in the space produced by plasrnolysis between the inner and outer membranes. FIGS.24-27. Electron micrographs of the chlamydial particles after sedimentation through 20% sucrose and washing with TSB to remove the sucrose. The reticulate body (Fig. 24) has an appearance unchanged from that in sucrose but the elementary bodies (Figs. 25-27) have deplasmolyzed so that their enveloping membranes are again closely apposed. FIGS.28-40. Electron micrographs of the semipurified chlamydial particles after treatment with trypsin. Note that both enveloping membranes are intact in some reticulate bodies (Figs. 30-33) while the outer membrane is discontinuous in others (Figs. 28 and 29). The intermediate bodies (Figs. 33-36) are intact but there is some disturbance of the contour of their enveloping membranes in most cases. T h e elementary bodies in this preparation are intact and their membranes are very closely apposed (Figs. 37-40). FIGS.41-44. Electron micrographs of semipurified chlamydia1 particles treated with a combination of RNase and DNase. Note the absence of reticulate and intermediate bodies and the presence of very few elementary bodies (E) and damaged particles (Figs. 42 and 43). Small bodies enclosed by a single membrane (arrows) are seen in this preparation, and their extreme electron density suggests that they may be derived from damaged elementary bodies.




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infected yolk sacs (Fig. 1, sample contained a large population of reticulate, intermediate. and elementary bodies (Figs. 3-7). The fragi!e reticulate bodies evidenced a loosely organized cytoplasm surrounded by two double-track membranes, and the inner membrane was intact, while the outer membrane was often incomplete (Fig. 4). This pleomorphism and looseness of organization differ from that seen by direct examination of infected cells (3) and are clear evidence of damage. Both membranes were intact in the intermediate bodies (I in Figs. 3, 5) which showed their characteristic electron-dense condensation of deoxyribonucleic acid (DNA) in a cytoplasm of variable density (compare I in Fig. 5 with I in Fig. 3). The small elementary bodies showed extreme condensation of cytoplasmic and nuclear material ( E in Fig. 3 and particles in Figs. 6, 7) and were enclosed by two intact and very closely apposed membranes Very small numbers (

Cloning and sequence analysis of the major outer membrane protein gene of Chlamydia psittaci 6BC.

Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain.

Immunofluorescence of peritoneal phagocytes after infection of mice with L-cell-attenuated Chlamydia psittaci 6BC.

Australian human and parrot Chlamydia psittaci strains cluster within the highly virulent 6BC clade of this important zoonotic pathogen.

Changes in the ultrastructure of Chlamydia psittaci produced by treatment of the host cell with DEAE-dextran and cycloheximide.

Effect of alkali on the structure of cell envelopes of Chlamydia psittaci elementary bodies.

Ultrastructural cytochemical evidence for the activation of lysosomes in the cytocidal effect of Chlamydia psittaci.

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

Growth of Chlamydia psittaci in macrophages.

Parasite-specified phagocytosis of Chlamydia psittaci and Chlamydia trachomatis by L and HeLa cells.

Protective effect of polyclonal and monoclonal antibodies against abortion in mice infected by Chlamydia psittaci.

Insidious endocarditis caused by Chlamydia psittaci.

Studies on the ultrastructure, virus-like particles and infectivity of Leishmania hertigi.

Use of HeLa cell guanine nucleotides by Chlamydia psittaci.

Prevalence of Chlamydia psittaci and Other Chlamydia Species in Wild Birds in Poland.

The effect of age on mitochondrial ultrastructure.

Immunoelectron microscopy of Chlamydia psittaci with monoclonal antibodies.

Interaction of Chlamydia psittaci reticulate bodies with mouse peritoneal macrophages.

Isolation of Chlamydia psittaci from pleural effusion in a dog.

Factors influencing the infectivity of Chlamydia pneumoniae elementary bodies on HL cells.

Risk factors associated with Chlamydia psittaci infection in psittacine birds.

Evaluation of a commercial solid-phase enzyme immunoassay for the detection of ovine Chlamydia psittaci.

Hepatitis in a family infected by Chlamydia psittaci.

Multiple tandem promoters of the major outer membrane protein gene (omp1) of Chlamydia psittaci.