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Vesicles containing endocytosed elementary bodies are delivered some two hours after infection into close approximation with the host cell Golgi apparatus. Translocation of the chlamydial entry vacuole requires the active participation of chlamydiae, because if early chlamydial protein synthesis is blocked with the antibiotic chloramphenicol, the vacuole-bound elementary bodies remain distributed throughout the cell cytoplasm [Scidmore et al., 1996a]. Translocation of early C. trachomatis vacuoles to the peri-Golgi region probably involves the host cell cytoskeleton, as aggregated elementary bodies were associated with the micro-tubule organising centre of the cytoskeleton [Clausen et al., 1997], while microtubule inhibitors blocked the aggregation of early inclusions [McBride & Wilde, 1990].

Within the family Chlamydiaceae there are important species differences in this process. In multiply-infected cells, individual C. trachomatis inclusions fuse into a single large inclusion. In many Chlamydophila species, multiple, separate, small inclusions develop. Moreover, although both C. trachomatis and C. pneumoniae form a single large inclusion, the process differs with respect to whether tyrosine phosphorylation of adjacent host cell protein occurs and whether the microtubule motor protein, dynein, is involved [Clausen et al., 1997]. No information is available for the other Chlamydiales, which may well be quite different.

The Golgi apparatus is the intracellular organelle responsible for much of the vesicular / membrane trafficking in the host cell. Clearly the growing chlamydial vacuole must intercept this membrane traffic somehow, in order to enlarge the inclusion membrane. The chlamydial inclusion is not an endosomal inclusion, as shown by the absence of markers of the plasma cell membrane, of fluid phase markers, or markers for either the early (transferrin and its receptor) or late (cation-independent mannose-6-phosphate receptor) endosome or for lysosomes (acid phosphatase, cathepsin D, lysosomal glycoproteins or vacuolar H⁺ ATPase). The interaction of chlamydiae with membrane derived from the Golgi apparatus can be determined by labelling the Golgi with C6-NBD-ceramide, which is endogenously converted into a fluorescent derivative of the membrane lipid, sphingomyelin [Scidmore et al., 1996b]. Endocytosed elementary bodies begin to acquire sphingomyelin from vesicles exocytosed from the Golgi within 2 hours of infection, in an energy-dependent process. Sphingomyelin acquisition and the translocation of C. trachomatis vacuoles to the Golgi region is blocked by inhibitors of chlamydial gene transcription or translation [Scidmore et al 1996b], indicating that chlamydiae actively and rapidly modify the host cell response to their invasion. Moreover the process is specific, in that similar membrane trafficking to another intracellular bacterial pathogen, Coxiella burnetii [Q fever] is not observed [Heinzen et al., 1996]. In intercepting this exocytic pathway, chlamydial vacuoles appear to the host cell as secretory vacuoles, and thus avoid the normal cellular process of fusion of phagocytic vesicles with endosomal vesicles or with lysosomes. Once exposed on the inner surface of the inclusion membrane, the sphingomyelin is readily acquired by chlamydiae within the inclusion and it appears to be essential for their replication [van Ooij et al., 1998]. Chlamydophila pneumoniae also intercepts the exocytic pathway in a similar manner to C. trachomatis [Wolf & Hackstadt, 2001]. Cholesterol, a lipid not normally found in bacteria, has also been identified in purified C. trachomatis elementary bodies and in the inclusion membrane in infected HeLa cells. Chlamydial acquisition of de novo-synthesized cholesterol or of low-density lipoprotein-derived cholesterol was microtubule-dependent and brefeldin A-sensitive, indicating that it was dependent on the Golgi apparatus. Chlamydial protein synthesis was also essential for this cholesterol transport, indicating that this is a pathogen-directed process using a similar mechanism to that for sphingomyelin acquisition [Carabeo et al., 2003]. The properties of the chlamydial inclusion are established by two hours postinfection. Studies using a combination of confocal and electron microscopy show a lack of endocytic or lysosomal markers within the inclusion membrane or lumen, indicating that the nascent chlamydial inclusion is minimally interactive with endosomal compartments early in infection. Even when chlamydial protein synthesis is blocked, vesicles containing chlamydial elementary bodies are very slow to acquire lysosomal characteristics. These results imply a two-stage mechanism for chlamydial avoidance of lysosomal fusion: (i) an initial phase of delayed maturation to lysosomes due to an intrinsic property of elementary bodies and (ii) an active modification of the vesicular interactions of the inclusion requiring chlamydial protein synthesis [Scidmore et al., 2003], including the synthesis of chlamydial inc proteins.

In summary, intrinsic properties of the chlamydial elementary body initially cause the chlamydial entry vacuole to be minimally fusogenic with the host cell endocytic pathway, delaying normal fusion with lysosomes. Subsequently, chlamydial gene products modify the vacuole so that it becomes fusogenic with a subset of sphingomyelin and cholesterol-containing exocytic vesicles from the Golgi apparatus [Hackstadt, 1999; Carabeo et al., 2003]. Glycero-phospholipids acquired by the chlamydial vacuole from the Golgi apparatus, the mitochondria and endoplasmic reticulum, are subsequently modified by the chlamydiae [Wylie et al.,  1997]. The result is that chlamydiae have a phospholipid composition that is much closer to the host cell than to other bacteria [Wylie et al., 1997; Newhall, 1988], including the unusual presence of cholesterol [Carabeo et al., 2003]. For a review see Hackstadt et al., 1997.

Role of early endosomal antigen EEA1

DNA multi-array analysis of genomic transcripts of C. trachomatis D/UW3/Cx [Belland et al., 2003] identified CT147 as an early gene transcript during the developmental cycle [See: developmental regulation]. CT147 encodes a 162 KDalton protein with strong functional homology to the human early endosomal antigen EEA1 involved in endosomal fusion and tethering. In chlamydial infected cells, it is suggested that CT147 tethers chlamydial endosomes but  does not permit  their fusion with lysosomes, perhaps because CT147 lacks the Rab5 GTPase binding domain that in the human protein is involved with regulating endosomal fusion. Although Scidmore et al., 2003 failed to demonstrate the presence of a chlamydial analogue of EEA1 in the chlamydial endosome, Belland et al., 2003 showed by confocal microscopy that CT147 is located in the membrane of the developing chlamydial endosome / inclusion and that it is post-translationally modified. It seems likely that CT147, at least in part, may be the molecular basis of the important observation that the chlamydial envelope is capable of blocking fusion of chlamydial endosomes with lysosomes [Eissenberg et al., 1983; Scidmore et al., 2003].

Effects on cadherins

An interesting effect of C. trachomatis on sheets of cervical epithelial cells in cell culture is their ability to cause epithelial cells to separate from each other without detaching from the substratum. By confocal microscopy, it was found that infected cervical epithelia or Hela cell sheets lost N-cadherin and beta-catenin labeling from the junctional complexes between cells, where it plays a role in intercellular adhesion. This was associated with concomitant appearance of intense beta-catenin labeling associated with the chlamydial inclusion. This might be an important mechanism by which C. trachomatis alters epithelial cell function [Prozialeck et al., 2002]. Furthermore beta catenin released from the junctional complex is free to interact with transcription factors in the nucleus to stimulate the expression of genes associated with apoptosis and cell cycle control [Prozialeck et al., 2002 ; see: apoptosis].

Rab GTPases

Rab GTPases are key regulators of membrane trafficking. Rzomp et al., 2003 therefore examined the intracellular localization of several green fluorescent protein (GFP)-tagged Rab GTPases in chlamydial - infected HeLa cells. GFP-Rab4 and GFP-Rab11, which function in receptor recycling, and GFP-Rab1, which functions in endoplasmic reticulum to Golgi trafficking, are recruited to Chlamydia trachomatis, C. muridarum, and Chlamydophila pneumoniae inclusions. GFP-Rab5, GFP-Rab7, and GFP-Rab9, markers of early and late endosomes, are not. In contrast, GFP-Rab6, which functions in Golgi to endoplasmic reticulum and in endosome to Golgi trafficking, shows species specificity in that it is associated with inclusions of  C. trachomatis inclusions but not C. muridarum or C. pneumoniae. In contrast, the opposite was observed for the Golgi-localized GFP-Rab10. GFP-Rab11's association with the inclusion is not mediated solely through Rab11 association with transferrin-containing recycling endosomes. Finally, GFP-Rab GTPases remain associated with the inclusion even after the microtubule disassembly which disperses recycling endosomes and the Golgi apparatus within the cytoplasm. This suggests there is a specific interaction of Rab GTPases with the inclusion membrane. Consistent with this, GFP-Rab11 colocalizes with C. trachomatis IncG at the inclusion membrane. Thus, chlamydiae recruit key regulators of membrane trafficking to the inclusion, which may regulate both membrane trafficking and the fusogenic properties of the inclusion [Rzomp et al., 2003].

For an update see: Chlamydial entry

IncA and inclusion membrane fusion

For an account of the role of IncA in facilitating fusion of C. trachomatis vacuoles to form the characteristic single inclusion, see the next article.

[MEW] November 2003

NEXT: Chlamydial inclusion proteins.

Index: Biology index.

References

Belland, R. J., Zhong, G., Crane, D. D., Hogan, D., Sturdevant, D., Sharma, J., Beatty, W. L. & Caldwell, H. D. (2003). Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis. Proceedings of the National Academy of Sciences of the U S A. 100, 8478 - 8483.

Carabeo, R. A., Mead, D. J. & Hackstadt, T. (2003). Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion. Proceedings of the National Academy of Sciences U S A. 100, 6771 - 6776.

Clausen, J. D., Christiansen, G., Holst, H. U. & Birkelund, S. (1997). C. trachomatis uses the host cell microtubule network during early events of infection. Molecular Microbiology 25, 441 - 449.

Fields, K. A., Fischer, E. & Hackstadt, T. (2002). Inhibition of fusion of Chlamydia trachomatis inclusions at 32 degrees C correlates with restricted export of IncA. Infection and Immunity 70, 3816 - 3823.

Hackstadt, T. (1999). Cell biology. In: Chlamydia: Intracellular Biology, Pathogenesis, and Immunity, pp. 101-138. Edited by R. S. Stephens. Washington, D.C.: ASM Press. ISBN 1-55581-155-8.

Hackstadt, T., Fischer, E. R., Scidmore, M. A., Rockey, D. D. & Heinzen, R. A. (1997). Origins and functions of the chlamydial inclusion. Trends in Microbiology 5, 288 293. [Review].

Hackstadt, T., Rockey, D. D., Heinzen, R. A. & Scidmore, M. A. (1996). Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane. EMBO Journal 15, 964 - 977.

Hackstadt, T., Scidmore, M. A. & Rockey, D. D. (1995). Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proceedings of the National Academy of Sciences USA. 92, 4877 - 4881. Full article

Heinzen, R. A., Scidmore, M. A., Rockey, D. D. & Hackstadt, T. (1996). Differential interaction between endocytic and exocytic pathways distinguish parasitiphorous vacuoles of Coxiella burnetii and C. trachomatis. Infection and Immunity 64, 796 - 809. Full article 

McBride, T. & Wilde III, E (1990). Intracellular translocation of C. trachomatis. Pages 36-39. In: Chlamydial infections. Proceedings of the eighth international symposium on human chlamydial infection. (Bowie W. R. et al., eds.). Cambridge University Press, Cambridge, UK.

Newhall, W. J. (1988). Macromolecular and antigenic composition of chlamydiae. Pages 47-70. In: Microbiology of Chlamydia, pp. 47-70. (Barron, A. L. ed.). Boca Raton, Florida, CRC Press.

Prozialeck, W. C., Fay, M. J., Lamar, P. C., Pearson, C. A., Sigar, I. & Ramsey K. H. (2002). Chlamydia trachomatis disrupts N-cadherin-dependent cell-cell junctions and sequesters beta-catenin in human cervical epithelial cells. Infection and Immunity 70, 2605 - 2613.  Full article

Rzomp, K. A., Scholtes, L. D., Briggs, B. J., Whittaker, G. R. & Scidmore, M. A. (2003). Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner. Infection and Immunity 71, 5855 - 5870.

Scidmore, M. A., Fischer, E. R. & Hackstadt, T. (1996b). Sphingolipids and glycoproteins are differentially trafficked to the C. trachomatis inclusion. Journal of Cell Biology 134, 363 - 374.

Scidmore, M. A., Fischer, E. R. & Hackstadt, T. (2003). Restricted fusion of Chlamydia trachomatis vesicles with endocytic compartments during the initial stages of infection. Infection and Immunity 71, 973 - 984. Full article

Wolf, K. & Hackstadt, T. (2001). Sphingomyelin trafficking in Chlamydia pneumoniae-infected cells. Cellular Microbiology 3, 145 152.

Wylie, J. L., Hatch, G. M. & McClarty, G. (1997). Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis. Journal of Bacteriology 179, 7233 - 7242. Full article 

NEXT: Chlamydial inclusion proteins.