Chapter 20 : The Chlamydial Developmental Cycle

Line drawing of a generalized chlamydial developmental cycle. Infection begins when an infectious but metabolically inactive EB comes in contact with a host cell (A) and is endocytosed (B). The phago-cytic vacuole (the inclusion) migrates toward the Golgi apparatus, and the EB differentiates into the noninfectious but metabolically active RB (C). RB division ensues, and the inclusion increases in size (D). Reticulate bodies then begin to reorganize back into EBs, and the inclusion grows until it occupies the entire cytoplasm of the infected cell (E). The inclusion lyses, the host cell lyses, and EBs are freed to infect another cell. While there are differences in this cycle among the different chlamydial strains and species, the general process is similar. N, nucleus.

Electron micrographs of the sequential changes from attachment of EBs to host cells through the division of RBs. These micrographs show C. psittaci Cal 10 infecting L929 cells. (A and B) 30 min p.i. Electrostatic and receptor-mediated adhesions between the EB and the cell can be observed. (C) 60 min p.i. Note the nucleoid structure and the apparent fusion of a tiny vesicle to the phagocyte. (D) 100 min p.i. Two developmental forms can be observed: a typical EB very soon after phagocytosis (top) and another located in the deeper cytoplasm. Note the larger size and apparent reorganization of the nucleoid. The images in panels C and D also represent the sequential change from the EB to intermediate body—a structure that is found both at the beginning and the end of the cycle. (E and F) 6 h p.i. The conversion from EBs to RBs is complete. At this point inclusions will be found in the region of the Golgi apparatus. Note the larger size of the RB in panel F. This may represent growth prior to the initial division process. (G) 8 h p.i. Note the RB constriction prior to binary fission and the fine comb-like structure on the right side of the RB. (H and I) 10 h p.i. Terminal stages of first division. Mitochondria can be seen in the vicinity of the inclusion, but not associated with the inclusion membrane. Magnification of all micrographs, X 60,000; bar in panel I = 1 μm.

Electron micrographs of the later stages of inclusion development in both C. psittaci and C. pneumoniae. (A to C) C. psittaci Cal 10-infected L929 cells cultured at 37°C. (A) 18 h p.i.; (B) 24 h p.i.; (C) 34 h p.i. In each panel, note the relative numbers of RBs and EBs and the association between the inclusion and host cell mitochondria. (D to F) C. pneumoniae TW-183 in HEp-2 cells, cultured at 37°C. (D) 18 h p.i.; (E) 24 h p.i.; (F) 34 h p.i. Note the apparent lack of mitochondria! association, the more spherical nature of the inclusion, and the slight compression of the host nuclei. The inclusions in panels A, D, and E contain exclusively RBs, while the remaining panels show the asynchronous nature of the later inclusions. Bars = 1 μm.

Effects of detergents and reducing agents on the integrity of EB and RB developmental forms. In both panels the arrows point to the 60-kDa EnvB (Omp2) and the 12-kDa EnvA (Omp3) proteins, and the arrowhead points to the 40-kDa MOMP band. (A) Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) profiles of insoluble residues of C. psittaci EBs and RBs after extraction with detergents plus or minus 2-mercaptoethanol (2-ME). Purified developmental forms were treated with the extracting agent and subjected to high-speed centrifugation. The material in the pellets was then prepared for standard PAGE. Lanes: 1, EB extracted with phosphate-buffered saline (PBS); 2, EBs extracted with Sarkosyl; 3, EBs extracted with SDS; 4, EBs extracted with SDS plus 2-ME; 5, RBs extracted with PBS; 6, RBs extracted with Sarkosyl; 7, RBs extracted with SDS; 8, RBs extracted with SDS plus 2-ME. Note the absence of detectable EnvA and EnvB in the RB preparations but the abundance of MOMP in both EBs and FU3s. (B) A similar experiment with EBs cultured in the presence of either [35S]cysteine or [35S]methionine. Purified EBs were then extracted with either PBS (lanes 1 and 2) or SDS (lanes 3 and 4). Insoluble material was collected by centrifugation and prepared for standard PAGE. The resulting gel was dried and exposed to film. The samples in lanes 1 and 3 represent EB labeled with [35S]cysteine. The samples in lanes 2 and 4 represent EB labeled with [35S]methionine. Note the distinction between detectable SDS-insoluble EB proteins under the two labeling conditions. (Data reproduced from Hatch et al., 1984 , with permission of the authors and ASM.)

Morphologies of highly purified C. psittaci EBs fixed differently prior to thin sectioning. (A) EBs were doubly fixed with glutaraldehyde and OsO4 and embedded in Epon. Thin sections were doubly stained with uranylacetate and lead citrate solutions. Note the lack of visible surface projections. (B) EBs were fixed with glutaraldehyde and treated with tannic acid. The sections were examined without subsequent staining with uranylacetate and lead citrate. While the internal structures are not visible, the regular nature of the surface projections is clearly demonstrated. (C) Purified EBs were fixed with glutaraldehyde and treated with ruthenium red. Thin sections were then stained with uranyl acetate and lead citrate. Note that the surface projections are located only on the EB surface opposite the nucleoid structure. Magnifications of all micrographs, X 90,000. Bar (panel C) = 0.1 μm.

Freeze-etch micrographs of the external surfaces of chlamydial inclusions. (A) C. psittaci Cal 10 inclusion 16 h p.i. Note the surface projections enlarged in the inset (X76,000). (B) Similar micrograph of Cal 10 at 18 h p.i. Magnification, X 30,000. Bars = 1 μm (full-scale images) and 0.1 μm (inset).

Aberrant RB forms produced during culture of C. trachomatis L2/434/Bu in the presence of penicillin G (128 μg/ml). (A) Thin section of penicillin-treated, infected cells fixed 24 h p.i. Note the extremely large and vacuolated single RB within a spacious inclusion and the extra folds of membrane adjacent to the RB. (B) A higher magnification of the region of contact between the RB and the inclusion membrane (indicated by an arrow in panel A). Note the connections between the inclusion membrane and RB at the point of contact. (C) Freeze-etch preparation of a similar inclusion. Note the surface projections extending through the surface of the inclusion (arrowhead), as seen in Fig. 6 with normal inclusions. The effect of penicillin on C. trachomatis is very similar to that seen with other chlamydiae.