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Chlamydial entry into host cells.

Typically, chlamydiae are observed attached to the host cell near the base of microvilli (see: attachment), from which site they are actively endocytosed by the host cell in tight endocytic vesicles. The actual process of chlamydial entry is relatively efficient, but not understood. Electron microscopic studies suggest two possible mechanisms for chlamydial entry. One mechanism advocated by the author involves a sequential, zipper-like [Finlay & Cossart, 1997], microfilament-dependent process of phagocytosis requiring direct circumferential contact between bacterial adhesins and host cell receptors [see: Fig Entry 1 below] [Ward & Murray, 1984]. The second, involves uptake into clathrin -coated vesicles by receptor mediated endocytosis [Hodinka et al., 1988], a process normally used for the uptake of physiologically essential, large molecules into the host cell. However, chlamydial elementary bodies are much larger than the normal clathrin-coated vesicles involved in receptor mediated endocytosis [Fig Entry 2 below] , even though they appear capable of exploiting it. One study of C. caviae and C. trachomatis entering McCoy cells found that a small proportion only of either organism occurred in coated vesicles;  clathrin-coated cytoplasmic membrane generally pinched off into small vesicles ahead of the chlamydiae [Reynolds & Pearce, 1990]. It seems probable that chlamydiae, dependent on the strain, the host cell and other circumstances, may be able to enter cells by either route, with the mode of inoculation (static or centrifuge assisted) one of the factors [Prain & Pearce, 1989]. This is supported by the fact that, in polarized human endometrial cells, chlamydiae tended to enter via clathrin-coated vesicles if the cells were grown on collagen and in non-coated vesicles when the cells were on plastic. Again, culture conditions and route of inoculation appear to be important [Wyrick et al., 1989].

More recent evidence increasingly favours the importance of the microfilament network, along with the kinesin and dynein microtubule motor proteins, in the internalisation and further development of Chlamydophila psittaci. Cytochalasin D disruption of actin filaments, and blockage of the motor proteins through the introduction of monoclonal antibodies into the host cells at various times indicated that, although Chlamydophila psittaci can make use of both microfilament-dependent and independent entry pathways in both cell types,  internalisation and development mainly involved  microfilaments (in L929 fibroblasts) or microtubules (in Buffalo green monkey kidney epithelial cells). In both cell types mutual participation of the actin and tubulin networks was necessary  for optimal growth [Escalante-Ochoa et al., 2000].

Another study investigated the role of membrane lipids in chlamydial entry. It was found that C. trachomatis serovar K enters HEp-2 and HeLa 229 epithelial cells and J-774A.1 mouse macrophage/monocyte cells via caveolin-containing sphingolipid and cholesterol -enriched raft microdomains in the host cell plasma membranes . The evidence for this was that filipin and nystatin, drugs that specifically disrupt raft function via cholesterol chelation ,  impaired the entry of C. trachomatis. Moreover, the chlamydia-containing endocytic vesicles in which replication takes place specifically reacted with antisera against caveolin. These are the vesicles which intercept the exocytic pathway from the Golgi apparatus that recycles sphingolipids and cholesterol to the plasma membrane [see: membrane trafficking]. The late-stage inclusions retain high levels of caveolin. It was suggest that the atypical raft-mediated entry process might have important consequences for the host-pathogen interaction well after entry has occurred, enabling the chlamydial vesicle to avoid acidification and fusion with lysosomes, to traffic to the Golgi region, and to intercept sphingolipid-containing vesicles from the Golgi [Norkin et al., 2001].

Clathrin-independent endocytosis of the type described above occurs through cholesterol-rich lipid microdomains, which are relatively detergent insoluble. It has been shown that C. trachomatis serovar L2 and C. caviae bind to detergent-resistant lipid microdomains (DRMs) of HeLa cells. Even after internalisation, chlamydiae remain associated with cholesterol-rich, detergent resistant domains. Furthermore, extraction of plasma membrane cholesterol inhibits infection of HeLa cells by C. trachomatis L2. Although many of the membrane proteins associated with these detergent resistant domain are anchored to membrane glycosylphosphatidylinositol, a role of these proteins in the entry process has yet to be identified.  It was suggested that the binding of chlamydiae to cholesterol-rich domains might lead to coalescence of the chlamydial cells, perhaps triggering internalisation Jutras et al., 2003.  

Fig 1. Entry of Chlamydia trachomatis LGV elementary bodies (E) into HeLa cells. Note the host cell microvilli enveloping the elementary bodies. The bar represents 0.25 microns. 39 Kb. Figures provided by Michael Ward.	Fig 2. A Chlamydia trachomatis elementary body (EB) in the process of entry into a HeLa cell. Tannic acid stained to enhance visualisation of chlathrin. Note the clearly demarcated outer envelope of the EB and the surrounding membrane (m) of the vacuole. Also note how small the clathrin coated pit (ccp) is compared to the chlamydial endosome. The bar represents 0.1 microns. 75Kb.

Cyclic nucleotides and calcium

The host cell signalling processes that trigger chlamydial entry are only partially understood. Early studies by this author suggested there might be a role for cyclic nucleotides in C. trachomatis LGV biovar entry into HeLa cells as cGMP and its potentiators tended to increase infectivity whereas cAMP analogues suppressed it [Ward & Salari, 1982]. Calcium ionophores similarly increased infectivity and there was some [weak] evidence for an influx of calcium ions [Murray & Ward, 1984]. However a more recent study found that chlamydiae did not induce a flux of calcium across the cell membrane, the level of calcium remaining constant during endocytosis and early intracellular development [Majeed & Kihlstrom, 1991]. A physiological concentration of intracellular cytosol-free calcium was required for the formation of chlamydial aggregates and inclusions. The calcium-binding fusogenic proteins, annexins , were selectively translocated to the proximity of the intracellular C. trachomatis [Majeed & Kihlstrom, 1991] as were three main intracellular calcium storage proteins [Majeed et al., 1999]. It was suggested that a local accumulation of calcium in the vicinity of chlamydial aggregates might trigger the association of certain proteins, such as the annexins, with chlamydial containing endosomes thereby regulating vacuole-membrane interactions downstream of entry [Majeed et al., 1999]. This requires further study.

In infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp), C. caviae (GPIC), and C. trachomatis serovars E, F and K. Caveolin-2 also associates with C. trachomatis serovars A, B and C, independently of caveolin-1. Furthermore, caveolin-2 was associated with these chlamydiae at the inclusion membranes. In caveolin-1 deficient FRT cells, caveolin-2 is not normally transported out of the Golgi in the absence of caveolin-1. Nevertheless, provided that the chlamydiae are viable, caveolin-2 in FRT cells colocalizes with chlamydial inclusions. This implies that chlamydial gene expression is necessary for the acquisition of caveolin-2 from the host cell Golgi apparatus [Webley et al., 2004]. 

Removal of cholesterol from the plasma membrane with methyl-beta-cyclodextrin inhibits uptake of C. trachomatis [Jutras et al., 2003]. However C. trachomatis EBs are reported not to be associated with detergent resistant lipid rafts nor do they localise with caveolin 1 [Gabel et al., 2004]. The role of lipid raft domains is therefore uncertain. The lipid raft-dependent pathway of entry might partially equate to the clathrin-independent chlamydial entry pathway previously described  by this reviewer [Ward & Murray, 1984] and others [Hodinka et al., 1988; Reynolds & Pearce, 1990].  However as Stuart and colleagues have demonstrated, lipid raft dependent entry does not necessarily involve caveolae. Caveolae and caveolins are structurally and functionally quite distinct from clathrin and from clathrin-coated pits [see: Webley et al., 2004, prepublication responses].  

Chlamydial-induced host cell phosphorylation and the recruitment of actin

Using scanning and transmission electron microscopy, Coombes & Mahony (2002) demonstrated that attachment of C. pneumoniae to host cells induced the appearance of microvilli at the host cell surface, presumably as a result of actin recruitment and the formation of microfilaments / tubules. Invasion occurred 30-120 min after cell contact, with subsequent loss of membrane microvilli. This process involves phosphorylation of cytoskeleton-related proteins and their appear to be at least two mechanisms. C. pneumoniae entry into Hep2 cells caused a rapid increase in MEK-dependent phosphorylation and activation of ERK1/2, followed by PI 3-kinase-dependent phosphorylation and activation of Akt. Tyrosine phosphorylation of focal adhesion kinase (FAK) preceded its appearance in a complex with the p85 subunit of PI 3-kinase during chlamydial invasion. Isoform-specific tyrosine phosphorylation of the docking protein Shc also occurred at the time of attachment and entry. Chlamydial entry (but not attachment) could be abrogated with specific inhibitors of MEK, PI 3-kinase and of actin polymerization. This shows the importance of these signalling pathways and an intact actin cytoskeleton for C. pneumoniae invasion. The results suggest that activation of cell signalling pathways is an essential strategy for C. pneumoniae invasion of epithelial cells [Coombes& Mahony, 2002].

Entry of C. trachomatis elementary bodies also induces the induction of microvilli and is accompanied by tyrosine-dependent phosphorylation of host cell proteins [Birkelund et al., 1994]. Up to 4 hours post infection, there is only occasional co-localization of the tyrosine phosphorylated proteins with endocytosed elementary bodies. At later times phosphorylated proteins were observed in close approximation to the developing inclusion [Fawaz et al., 1997]. Attachment alone is thought to be sufficient to induce this phosphorylation, as it occurs when microfilament-dependent phagocytosis is blocked [Birkelund et al., 1994]. Some of the phosphorylated protein, at least, is associated with the host cell cytoskeleton protein, cortactin  [Fawaz et al., 1997], which is known to be tyrosine-phosphorylated during uptake of another bacterial pathogen, Shigella flexneri [Dehio et al., 1995]. These findings imply that chlamydial attachment may be sufficient to directly activate the microfilament system.

Another target of tyrosine-phosphorylation is the protein ezrin an 80 KDalton microfilament bundling protein, present in the microvillus core, and a member of the ezrin-radixin-moesin family of proteins which serve as a physical link between host cell receptors and the actin cytoskeleton. Confocal microscopy studies showed colocalization of ezrin and actin at the tips and crypts of microvilli, the site of chlamydial attachment and entry, respectively. Host cells with a dominant negative phenotype for ezrin, or cells treated with ezrin-specific small interfering RNA were significantly less susceptible to infection by human chlamydial strains. It was postulated that the C. trachomatis-specific tyrosine phosphorylation of ezrin might relate to an undefined species-specific mechanism of pathogen entry that involves chlamydial specific ligand(s) and host cell coreceptor usage [Swanson et al., 2007]. Chlamydial attachment and / or entry is also reported to require cellular protein disulphide isomerase [Conant & Stephens, 2007]. A candidate chlamydial ligand is TARP, the chlamydial translocated actin recruiting protein [Jewett et al., 2006], which acts independently of the Arp2/3 complex of 7 host cell binding proteins which may subsequently be required for internalisation through Rac activation [Carabeo et al., 2007; see below].

Carabeo et al., 2004 report that members of the Rho GTPase family are involved in the localized recruitment of actin to sites of chlamydial entry as clostridial toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells. Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA.  Rac but not Cdc42 was activated by chlamydial attachment. Rac was the sole member of the Rho GTPase family recruited to the site of chlamydial entry. Chlamydial induced activation of Rac GTPase, which is required for the localization of WAVE2 at the sites of chlamydial entry. C. trachomatis infection promotes the interaction of Rac with WAVE2 and Abi-1, but not with IRSp53. siRNA depletion of WAVE2 and Abi-1 abrogates chlamydia-induced actin recruitment, significantly reducing chlamydial uptake by the depleted cells. Chlamydia invasion also required the Arp2/3 complex as demonstrated by its localization to sites of chlamydial attachment. Thus, C. trachomatis activates Rac and promotes its interaction with WAVE2 and Abi-1 to activate the Arp2/3 complex [Carabeo et al., 2007].

Hybiske and Stephens 2007 used RNA interference to disrupt proteins with established roles in clathrin-mediated endocytosis (clathrin heavy chain, dynamin-2, heat shock 70-kDa protein 8, Arp2, cortactin, and calmodulin), caveolin-mediated endocytosis (caveolin-1, dynamin-2, Arp2, NSF, and annexin II), phagocytosis (RhoA, dynamin-2, Rac1, and Arp2), and macropinocytosis (Pak1, Rac1, and Arp2). Comparative quantitative PCR analysis was then performed on small interfering RNA-transfected HeLa cells to determine the effect on C. trachomatis entry. Structural and regulatory factors associated with clathrin-mediated endocytosis were found to be involved in Chlamydia entry, whereas those for caveolin-mediated endocytosis, phagocytosis, and macropinocytosis were not. Thus, clathrin and its coordinate accessory factors were required for entry of C. trachomatis, although additional, uncharacterized mechanisms are probably also utilized.

For details of the chlamydial endosome, see membrane recycling.

[Comment: The chlamydial entry process is complex but important. Even within the single family Chlamydiaceae there are a variety of different entry mechanisms and there are probably also host cell-determined different entry routes which depend on the cell surface membrane receptors that are displayed and their links to the underlying cell transport machinery. The entry route is also likely to influence key post entry events but we have hardly begun to understand these processes. Chlamydial entry in all its complexity is likely to be important in explaining some of the observed differences in the pathobiology of different chlamydial species and C. trachomatis biovars].

[MEW] December 29th 2007

Index: Biology index.

NEXT: Membrane recycling.

References

Birkelund, S., Johnsen, H. & Christiansen, G. (1994). Chlamydia trachomatis serovar L2 induces tyrosine phosphorylation during uptake by HeLa cells. Infection and Immunity 62, 4900 - 4908.

Carabeo, R. A., Greishaber, S. S., Hasenkrug, A., Dooley, C. & Hackstadt, T. (2004). Requirement for the Rac GTPase in Chlamydia trachomatis Invasion of Non-phagocytic Cells. Traffic. 5(6): 418 - 425.

Carabeo, R. A., Dooley, C. A., Grieshaber, S. S., and Hackstadt, T. (2007). Rac interacts with Abi-1 and WAVE2 to promote an Arp2/3-dependent actin recruitment during chlamydial invasion. Cellular Microbiology 9(9), 2278 - 2288.

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

Conant, C. G. & Stephens, R. S. (2007). Chlamydia attachment to mammalian cells requires protein disulfide isomerase. Cellular Microbiology 9(1) 222 - 232. Epub 2006 Aug 22.

Coombes, B.K. & Mahony, J. B. (2002). Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells. Cellular Microbiology 4, 447 - 460.

Dehio, C., Prevost, M_C. & Sansonetti, P. J. (1995). Invasion of epithelial cells by Shigella flexneri induces tyrosine phophorylation of cortactin by a pp60c-src-mediated signalling pathway. EMBO Journal 14, 2471 - 2482.

Escalante-Ochoa, C., Ducatelle, R. & Haesebrouck, F. (2000). Optimal development of Chlamydophila psittaci in L929 fibroblast and BGM epithelial cells requires the participation of microfilaments and microtubule-motor proteins. Microbial Pathogenesis 28, 321 - 333.

Fawaz, F. S., van Ooij, C., Homola, E., Mutka, S. C. & Engel, J. N. (1997). Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or localization of several host cell proteins including cortactin. Infection and Immunity 65, 5301 - 5308. Full article  

Finlay, B. B. & Cossart, P. (1997). Exploitation of mammalian host cell functions by bacterial pathogens. Science 276, 718 - 725. [Review]

Friis, R. R. (1972). Interaction of L cells and C. psittaci: entry of the parasite and host response to its development. Journal of Bacteriology 110, 706 - 721.

Gabel, B. R., Elwell, C., van Ijzendoorn, S. C. & Engel, J. N. (2004). Lipid raft-mediated entry is not required for Chlamydia trachomatis infection of cultured epithelial cells. Infection and Immunity 72(12) 7367 - 7373. Full article

Grieshaber, S. S., Grieshaber, N. A. & Hackstadt, T. (2003). Chlamydia trachomatis uses host cell dynein to traffic to the microtubule-organizing center in a p50 dynamitin-independent process. Journal of Cell Science 116, 3793 - 3802.

Hackstadt, T. (1998). The diverse habitats of obligate intracellular parasites. Current Opinion in Microbiology 1, 82 - 87. [Good review]

Hodinka, R. L., Davis, C. H., Choong, J. & Wyrick, P. (1988). Ultrastructural study of endocytosis of C. trachomatis by McCoy cells. Infection and Immunity 56, 1456 - 1463.

Hybiske, K. & Stephens, R. S. (2007). Mechanisms of Chlamydia trachomatis entry into nonphagocytic cells. Infection and Immunity 75(8):3925-34. Epub 2007 May 14.

Jewett, T. J., Fischer, E. R., Mead, D. J. & Hackstadt, T. (2006). Chlamydial TARP is a bacterial nucleator of actin. Proc Natl Acad Sci USA 103(42):15599-604. Epub 2006 Oct 6.  Full article

Jutras, I., Abrami, L. & Dautry-Varsat, A. (2003). Entry of the Lymphogranuloma Venereum Strain of Chlamydia trachomatis into Host Cells Involves Cholesterol-Rich Membrane Domains. Infection and Immunity 71, 260 - 266. Full article 

Majeed, M. & Kihlstrom, E. (1991). Mobilization of F-actin and clathrin during redistribution of C. trachomatis to an intracellular site in eucaryotic cells. Infection and Immunity 59, 4465 - 4472.

Majeed, M., Krause, K. H., Clark, R. A., et al., (1999). Localization of intracellular Ca2+ stores in HeLa cells during infection with Chlamydia trachomatis. Journal of Cell Science 112, 35 - 44. Full article     

Murray, A. & Ward, M. E. (1984). Control mechanisms governing the infectivity of C. trachomatis for HeLa cells: the role of calmodulin. Journal of General Microbiology 130, 193 - 201.

Norkin, L. C., Wolfrom, S. A. & Stuart, E. S. (2001). Association of caveolin with Chlamydia trachomatis inclusions at early and late stages of infection. Experimental Cell Research 266, 229 - 238. [Innovative study linking entry with membrane recycling and trafficking]

Prain, C. J. & Pearce, J. H. (1989). Ultrastructural studies on the intracellular fate of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) and C. trachomatis (strain lymphogranuloma venereum 434): modulation of intracellular events and relationship with endocytic mechanism. Journal of General Microbiology 135, 2107 - 2123.

Reynolds, D. J. & Pearce, J. H. (1990). Characterization of the cytochalasin D-resistant (pinocytic) mechanisms of endocytosis utilized by chlamydiae. Infection and Immunity 58, 3208 - 3216.

Stuart, E. S., Webley, W. C. & Norkin, L. C. (2003). Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells. Experimental Cell Research 287(1), 67 - 78.

Swanson, K. A., Crane, D. D. & Caldwell, H. D. (2007). Chlamydia trachomatis species-specific induction of ezrin tyrosine phosphorylation functions in pathogen entry. Infection and Immunity 75, 5669-77.

Ward, M. E. & Murray, A. (1984). Control mechanisms governing the infectivity of C. trachomatis for HeLa cells: mechanisms of endocytosis. Journal of General Microbiology 130, 1765 - 1780.

Ward, M. E. & Salari, H. (1982). Control mechanisms governing the infectivity of C. trachomatis for HeLa cells: modulation by cyclic nucleotides, prostaglandins and calcium. Journal of General Microbiology 128, 639 - 650.

Webley, W. C., Norkin, L. C. & Stuart, E. S. (2004). Caveolin 2 associates with intracellular chlamydial inclusions independently of caveolin 1. Biomed Central Infectious Diseases 4, 23 - 35. [Open access journal: HTML full version;  PDF full version  _; Pre-publication history].

Wyrick, P. B., Choong, J., Davis, C. H., et al., (1989). Entry of genital C. trachomatis into polarized human epithelial cells. Infection and Immunity 57, 2378 - 2389.

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