Chlamydia Host Cell Interactions - Wiley Online Library

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Traffic 2004; 5: 561–570 Blackwell Munksgaard

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Blackwell Munksgaard 2004

doi: 10.1111/j.1600-0854.2004.00207.x

Review

Chlamydia – Host Cell Interactions: Recent Advances on Bacterial Entry and Intracellular Development Alice Dautry-Varsat*, Marı´a Eugenia Balan˜a´ and Benjamin Wyplosz Unite´ de Biologie des Interactions Cellulaires, Institut Pasteur, URA CNRS 2582, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France *Corresponding author: A. Dautry-Varsat, [email protected] Bacteria of the Chlamydiales order are very successful intracellular organisms that grow in human and animal cells, and even in amoebae. They fulfill several essential functions to enter their host cells, establish an intracellular environment favorable for their multiplication and exit the host cell. They multiply in a unique organelle called the inclusion, which is isolated from the endocytic but not the exocytic pathway. A combination of host cell factors and of proteins secreted by the bacteria, from within the inclusion, contribute to the establishment and development of this inclusion. Here we review recent data on the entry mechanisms and maturation of the inclusion. Key words: GTPase, host–pathogen interaction, intracellular bacteria, lipid microdomain, phagocytosis, raft Received 10 May 2004, revised and accepted for publication 17 May 2004

Pathogenic bacteria have evolved various strategies to create a safe niche for replication inside the host. For instance, intracellular bacterial pathogens that grow in nonprofessional phagocytes have evolved specific mechanisms to penetrate their host cells and to avoid subsequent degradation along the endocytic pathway (1). Some, such as Shigella or Listeria, rapidly escape to the cytoplasm after uptake. Others, such as Salmonella, Legionella pneumophila, Brucella abortus or Chlamydia spp., remain within membrane-bound vesicles and determine the properties and fates of the vesicles they occupy. Chlamydiae are obligate intracellular parasites of eukaryotic cells. These gram-negative bacteria constitute an important group of pathogens responsible for a variety of acute and chronic diseases of humans and are also of veterinary importance. The main species pathogenic for humans are Chlamydia trachomatis and Chlamydia pneumoniae. In developed countries, infection with

several Chlamydia trachomatis strains is the most common sexually transmitted bacterial disease and severe sequelae in women include pelvic inflammatory disease, ectopic pregnancy and sterility. A subset of Chlamydia trachomatis strains causes trachoma, a leading cause of preventable blindness in the developing world (reviewed in (2)). C. pneumoniae is estimated to cause an average of 10% of community-acquired pneumoniae and 5% of bronchitis and sinusitis cases in adults. Furthermore, C. pneumoniae is of intense interest due to the accumulating evidence that chronic infection with this pathogen might contribute to cardiovascular disease (recently reviewed in (3,4)). Chlamydia psittaci, which is common in avian species, can also cause rare but severe pneumonia in humans. Despite the difficulties imposed by their intracellular life style, by the absence of cell free growth conditions and the lack of a gene transfer system for these bacteria, Chlamydiae are important pathogens, with unique properties, clearly worth studying. All species share a unique developmental cycle, during which bacterial multiplication occurs only within an eukaryotic host cell. Chlamydiae undergo this entire cycle within a vacuole, termed an inclusion (Figure 1). The acute infection developmental cycle consists of infectious and noninfectious stages, which exhibit distinct biological and morphologic properties. The infectious elementary body (EB) is small, about 0.3 mm in diameter, and is metabolically inactive. After entering epithelial host cells, EBs differentiate within a few hours into larger, about 1 mm, more pleiomorphic, reticulate bodies (RBs), which are metabolically active. These proliferate, giving rise to 1000 or more progeny per host cell. The infectious cycle ends after 2 or 3 days, depending on the strain, when bacteria have differentiated back to EBs and are released in the extracellular medium. In addition to the acute chlamydial development cycle, Chlamydia can also persist. Chlamydial persistence represents a viable but noncultivable growth stage and results in a long-term relationship with the infected host cell (reviewed in (5)). During persistent infection, the cycle is altered and enlarged, nondividing RBs are generated that do not mature into EBs. Persistence can be induced in vitro by a variety of stimuli, including interferon gamma treatment and antibiotics. A body of evidence has accumulated for the existence of persistence of C. trachomatis and C. pneumoniae in vivo from clinical data from human disease and experimental animal infections (5). 561

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Figure 1: Chlamydia developmental cycle. Picture of the infection cycle as drawn in 1938. Source: Archives of the Pasteur Institute in Algiers. Historical note: at the time, the causative agent of trachoma, C. trachomatis, was named Rickettsia trachomatis. The productive cycle lasts about 2–3 days depending on the strain. EBs enter the host cell, differentiate into RBs and multiply. The infection progresses and the RBs differentiate back into EBs that are finally released from the host cell to begin another round of infection. 1, 2: early inclusion. 3: dividing cell containing an inclusion. 5, 6, 7, 8: the inclusion is expanding and is perinuclear.

Interestingly, despite their common growth properties, Chlamydiacae constitute a rather diverse family of bacteria, with different host ranges, morphologies, biological properties and diseases they cause. They have small genomes (ffi 1 Mb); at least six complete genome sequences from three species are available (6–8). The number of ORFs varies, 894 for C. trachomatis to 1073 for C. pneumoniae, and many have no known or proposed functions (15–30% depending on the species). Also, the level of similarity for individual proteins encoded by C. trachomatis and C. pneumoniae spans a wide spectrum (22–95% amino acid identity; average 62% between orthologues from the two species) (9). Chlamydia-related endosymbionts in free living amoebae have been recently discovered. One of them has a genome twice the size of the previously known Chlamydiae (10). Comparative analysis 562

shows that, in total, about 700 coding sequences are shared among all chlamydial genomes representing the core gene set of Chlamydiae. It has been proposed that ancient Chlamydiae may be the originators of mechanisms for the exploitation of eukaryotic cells (10). In the past few years, the information coming from genome sequences of several strains has opened new areas of research concerning strain comparison, metabolism, developmental cycle regulation and various aspects of the interactions of Chlamydiae with their host ((11) and references therein). Despite the wealth of information provided by these postgenomic approaches, the progress regarding topics such as bacteria attachment, entry mechanism and import Traffic 2004; 5: 561–570

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of components from the host cell to the inclusion has come from cell biology approaches. Recent results concerning Chlamydiae entry in epithelial cells and the development of the inclusion will be summarized here. Important previously obtained results on this topic can be found in excellent reviews (12–15).

rather confusing. This may simply reflect that Chlamydiae, as other intracellular pathogens such as Listeria or Yersinia are likely to have adapted several receptors and means of entry. In addition, different species likely also use different receptors. Furthermore, as discussed below, Chlamydiae concentrate in specialized membrane microdomains at the cell surface and their adhesion to host cells may thus be mediated by numerous weak ligand–receptor interactions, involving molecules concentrated in these domains.

Chlamydia Entry into Host Cells Adhesins and host cell receptors Chlamydiae survive and replicate in epithelial cells. Like other intracellular pathogens, they have evolved very efficient means to invade their host cells. The ability to attach and enter susceptible host cells is indeed an essential requirement. Presently, a number of distinct surface molecules have been proposed to function as adhesins including the major outer membrane protein, heat shock protein 70, Omc B, heparan sulfate-like glycosaminoglycans ((12,14,15) and references therein) (16). The heparan sulfate-like molecule on the surface of C. trachomatis EBs binds host cell fibronectin (17). Whether the presence of fibronectin on EBs has biological significance in mediating host cell interactions remains to be elucidated. Recently, a member of the family of 21 polymorphic membrane proteins (Pmps) of C. pneumoniae (PmpD) was proposed to mediate the early interaction of EBs with host cells, based on inhibition of chlamydial infectivity by antibodies against the N-terminal domain of PmpD (18). This protein is processed in two parts and the N-terminal domain translocates to the EB surface where it binds other components of the outer membrane. From the perspective of the host cell, cell surface heparanlike glycosaminoglycans are involved in the attachment of some Chlamydiae, as is the case for other viral and bacterial pathogens. In addition, a number of reports point to the receptor being a protein or glycoprotein still unknown, with the exception of a possible role for the estrogen receptor complex (19) (reviewed in (12)). The isolation of mutants resistant to C. trachomatis infection suggested that indeed attachment to host cell surfaces could be dissected in at least two steps: an initial, reversible step, likely mediated by electrostatic interactions with surface glycosaminoglycans, followed by binding to an unknown cell surface receptor that mediates the actual entry process (20). In another study, CHO mutant cell lines resistant to C. trachomatis infection were selected. Most of them were also resistant to C. pneumoniae attachment, suggesting that there is one host receptor or cofactor utilized by both of these species to mediate attachment (21) and that C. trachomatis and C. pneumoniae share an attachment mechanism. Furthermore, they appear to have different requirements for postadherence interactions that lead to productive infection. In conclusion, attempts to define specific receptor–ligand interactions mediating bacterial attachment and entry are Traffic 2004; 5: 561–570

Sites of entry into the cells: Lipid microdomains involvement In the last few years, it has become apparent that interactions between a number of bacteria or viruses do not occur randomly at the plasma membrane but rather in defined areas or microdomains. Lipid microdomains, or ‘rafts’, are characterized by a high cholesterol and glycosphingolipid content (22). They are enriched in molecules involved in signal transduction events, suggesting that localization to lipid microdomains plays an important role in signaling mechanisms (23). Lipid microdomains are thought to be highly dynamic and small (about 40 nm diameter) (24). Considering the size, they could only contain a limited number of proteins, but coalescence of rafts could bring different raft-associated molecules together. Several pathogenic bacteria that invade mammalian cells do so via a raft-dependent pathway (reviewed in (25)). These include mycobacteria, Shigella flexneri, Fim H-expressing E. coli, Brucella spp. and Chlamydia spp. Thus, C. trachomatis serovar L2 and Chlamydia caviae GPIC are associated with lipid microdomains on the host cell surface (26). (C. caviae GPIC, formerly called C. psittaci GPIC, infects guinea pigs and is a good experimental model for sexually transmitted C. trachomatis infection in humans). Disruption of these lipid rafts by a cholesterol depleting drug inhibited bacterial entry but not attachment (26). Furthermore, after 5 h infection, when the bacteria are intracellular, they were still associated with lipid rafts suggesting that the inclusion, at least at this early infection time, is enriched in lipid rafts. These rafts might confer specific properties to the early inclusion. Caveolin, which is concentrated in lipid microdomains, was colocalized with inclusions of C. pneumoniae and some, but not all, C. trachomatis serovars. However, caveolin itself was not essential for bacterial entry. In addition, entry of these bacteria was sensitive to cholesterol depletion by filipin and nystatin (27,28). It has been proposed that entry of bacteria via a raft-dependent pathway may prevent interactions with the endocytic degradative pathway (29). Although there are counter-examples, this hypothesis applies to Chlamydia since the inclusion avoids intersection with the endocytic pathway (see below). Considering the small size of lipid rafts, it is likely that Chlamydia binding leads to coalescence of these lipid rafts, which would then trigger internalization by host cells. Interestingly, signaling molecules that are known to be involved in bacterial infections have been found in rafts. 563

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These include proteins of the Rac and Cdc42 dependent pathway (30) and proteins involved in phosphoinositide signaling (31). A raft requirement does not in itself preclude the involvement of clathrin-mediated uptake, but the conventional clathrin cage (about 100 nm diameter) cannot accommodate even the smallest bacteria such as Chlamydia elementary bodies. Furthermore, it has been shown by using dominant negative mutants of clathrin-mediated endocytosis, Eps 15 and dynamin 1 mutants, that C. trachomatis serovar L2 and C. caviae GPIC entry in HeLa cells was not affected. This demonstrates that clathrin-coated vesicles are not involved in Chlamydia entry (32); this comes as no surprise considering the size of Chlamydia EBs – much too large to fit a coated pit or vesicle. The apparent contradictions in previous reports about clathrindependent endocytosis of Chlamydia have been reviewed elsewhere (12,15). The possibility remains, however, that clathrin could participate in some way in bacterial entry by some other mechanism. Phagocytosis by macrophages of latex beads and even of pathogens, Leishmania donovani and Salmonella typhimurium, has been shown to involve the rapid recruitment of endoplasmic reticulum (ER) to the plasma membrane at the site of entry (33). We could not detect the presence of ER at the sites of entry of C. trachomatis serovar L2 and C. caviae GPIC by electron microscopy (I. Jutras, M. Desjardins, A. Dautry-Varsat, unpublished results). Therefore, ERmediated phagocytosis does not appear to be used for Chlamydia entry.

Actin cytoskeleton reorganization during Chlamydia entry The role of the actin cytoskeleton is critical in endocytosis and phagocytosis in a variety of cell types. A number of studies have shown that the actin cytoskeleton can be manipulated by microbial pathogens and that different pathogens have evolved various and very subtle ways to do so (34). Chlamydiae are no exception. Entry of C. trachomatis (serovar L2 and D), of C. caviae GPIC and of C. pneumoniae is inhibited by depolymerizing actin with cytochalasin D (32,35,36). Furthermore, association of EBs of these two C. trachomatis serovars and of C. caviae GPIC induced local actin polymerization (37,38). Upon bacterial attachment, lipid microdomains clustered within minutes and actin polymerization was rapidly initiated at these sites in a transient manner (Figure 2). Small guanosine 50 -triphosphatases (GTPases) of the Rho family (Rho, Rac, Cdc42) function as molecular switches that cycle between an active GTP-bound state and an inactive guanosine 50 -diphosphate (GDP)-bound state, and control actin filament assembly, organization and, thus, phagocytosis (39,40). The involvement of Rho GTPase family members was demonstrated by using the clostridial 564

Figure 2: Actin polymerization upon Chlamydia entry. HeLa cells were infected with FITC-coupled bacteria for 5 min before fixation. Actin filaments are visualized with fluorescently labeled phalloidin (red). The arrow points to a bacterium (green) associated with a locus of intense actin polymerization. A medial confocal microscopy section is shown. Bar ¼ 5 mm.

toxin B, a broad inhibitor of these GTPases, which inhibited C. trachomatis serovar L2 (37), C. caviae GPIC and C. pneumoniae entry (B. Wyplosz, unpublished). The participation of Rho family members was further analyzed. Rac is activated within minutes of C. trachomatis and C. caviae GPIC entry and this activation is transient. It is rapidly recruited to the site of entry where actin polymerizes. In addition, a Rac dominant negative mutant inhibited bacterial entry (37,38). The small GTPase Rho is not involved in this process, as shown using a dominant negative mutant and a specific toxin (EDIN) (38). However, these two species differ in their requirement for Cdc42, which is not involved in C. trachomatis (37), but is clearly implicated in C. caviae GPIC entry. In the latter case, endogenous Cdc42 is transiently activated within minutes, with kinetics similar to those of Rac. Furthermore, bacterial entry was inhibited in cells expressing a Cdc42 dominant negative mutant, although somewhat less than in cells expressing the corresponding Rac mutant. Bacterial entry was also inhibited by overexpression of the domain of WASP that interacts with the GTP bound form of Cdc42 (38). Thus, the small GTPases Rac and Cdc42, in the case of C. caviae GPIC, are activated and recruited at the site of bacterial entry, where lipid rafts also coalesce. These GTPases participate in local and transient actin cytoskeleton remodeling (Figure 3). Thus Chlamydiae join the evergrowing list of microorganisms that use the activity of small GTPases of the Rho family to invade eukaryotic cells (reviewed in (41)). Traffic 2004; 5: 561–570

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Figure 3: Initial events upon Chlamydia entry. Chlamydiae bind to an unidentified host cell receptor and are localized in cholesterol-rich membrane domains (yellow). After binding, specific host cell signaling pathways leading to bacteria internalization, are triggered. Putative effector molecule(s) secreted by a type III mechanism could contribute to this activation. The Rho family of small GTPases, which controls actin polymerization, is necessary for the entry of Chlamydia spp. to the host cell. Rac 1 and Cdc42 are both activated after C. caviae GPIC infection, but Rho does not seem to be implicated. Rac1, but not Cdc42, is activated upon C. trachomatis entry. Infection leads to the rapid phosphorylation of host cell proteins that accumulate at the bacteria entry sites. In particular, C. pneumoniae and C. caviae GPIC activate at least two distinct signaling pathways involving PI-3 kinase and MEK and ERK kinases. PI-3 kinase is not required for C. trachomatis serovar L2 entry. One of the early phosphorylation substrates identified during C. pneumoniae infection was the focal adhesion kinase (FAK), which interacts with the p85 regulatory subunit of PI-3 kinase. A few minutes after bacterial attachment, actin polymerization is initiated (right panel).

Actin remodeling induced by other bacteria such as Salmonella or Shigella relies on bacterial factors secreted by a type III mechanism (42,43). Chlamydiae also possess a type III apparatus (see below), which is present on EBs (44), and might use a similar activation mechanism (see note added in proof). Alternatively, host cell receptors could be activated by bacterial attachment, triggering intracellular signaling events, which would eventually activate the small GTPases. The different requirement for Cdc42 between Chlamydia species is interesting. As discussed previously, the receptors for cell attachment are unknown and could differ between species. Also, several proteins secreted by the type III mechanism are species-specific or have little sequence homology (A. Subtil and A. DautryVarsat, unpublished). Both characteristics could account for the different requirements for Rho family members. These differences are not surprising. Indeed, even in the case of phagocytosis by macrophages, the Rho family small GTPases involved are not the same in the case of Fc receptor or C3 receptor phagocytosis (39). Finally, the different requirement for Cdc42 illustrates the numerous tricks that intracellular pathogens have evolved to use cellular functions for invasion. Traffic 2004; 5: 561–570

Signaling in the host cell Intracellular pathogen entry into host cells is initiated by their binding, and rapidly followed by signaling events, as demonstrated for several intracellular bacteria (reviewed in (45)). Actin reorganization at the sites of entry results from this signaling. Chlamydiae are probably no exception, especially as they concentrate in rafts, which are signaling platforms, but the signal cascades are still poorly understood. We discuss here only recent results concerning early signaling events. Phosphorylation of unidentified host cell proteins was observed starting 15 min and 2 h after infection of HeLa cells by two serovars of C. trachomatis and increased with time (46,47). These phosphorylations were not detected at early infection times and are likely to be secondary signaling events, taking into consideration that Rac and Cdc42, for instance, are activated in less than 5 min (37,38). Indeed, tyrosine-phosphorylated proteins accumulated around C. caviae GPIC EBs within 5 min of infection (38). This is in agreement with the protein phosphorylation observed after 5 min infection with C. pneumoniae EBs 565

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(36). In this elegant study, it was shown that infection rapidly induced (starting at 5 min) two distinct signaling pathways involving phosphoinositide 3-kinase (PI 3-kinase) and MEK-ERK kinases, which did not appear to be fully redundant for invasion. ERK 1/2 was also activated within 5 min of C. caviae GPIC infection (M. E. Balan˜a´, unpublished). PI 3-kinase is an upstream and downstream effector of several members of the Ras superfamily of GTPases involved in actin polymerization, and inhibition of MEK and PI 3-kinase could antagonize microvilli formation induced by C. pneumoniae (36). Following PI 3-kinase activation, Akt was phosphorylated in a PI 3-kinase dependent manner. Entry of C. caviae GPIC also required PI 3-kinase (38) but, interestingly, this did not seem to be the case for C. trachomatis serovar L2 (35) (Figure 3). The results obtained with C. pneumoniae and C. caviae support the notion that early host signaling may function to facilitate cytoskeleton rearrangements leading to bacterial uptake. One of the early phosphorylation substrates identified during C. pneumoniae infection is the focal adhesion kinase (FAK). When phosphorylated, FAK presents docking sites for SH2 domains on other signaling molecules, and interacts with the p85 regulatory subunit of PI 3-kinase (36).

membrane, as observed by electron microscopy (48). Neither the early endosome marker EEA1 nor late endosome markers such as LAMP1 were found to localize to the chlamydial inclusion in epithelial cells. Other early and late endosome markers, Rab 5, 7 and 9, were not associated with the inclusion. However, markers of the recycling pathway, Rab 4 and Rab 11, were associated with the inclusion, as observed by confocal microscopy, with Rab 11 starting to be detected at 1 h post infection. The significance of this association is not yet established (49). Consistent with the absence of early and late endosome markers, the lumen of the inclusion is not acidified and inhibitors of vacuolar acidification do not affect C. trachomatis replication ((13) and references therein). As the infection cycle progresses, the properties and cellular interactions of the inclusion evolve. The molecular mechanisms involved are unknown, but they require early chlamydial transcription and translation. Even when prevented from modifying the properties of the inclusion by incubation in the presence of protein synthesis inhibitors, vesicles containing elementary bodies are very slow to acquire lysosomal characteristics. This implies a two-stage mechanism for chlamydial avoidance of lysosomal fusion: an initial phase of delayed maturation to lysosomes due to an intrinsic property of elementary bodies, followed by an active modification of the vesicular interactions of the inclusion requiring chlamydial protein synthesis (48).

The Chlamydial Inclusion Chlamydiae multiply strictly within their membrane-bound inclusion. As the RBs divide and proliferate, the inclusion expands so that it occupies most of the cytoplasm of the infected cell. It does so by receiving components both from the host cell and from the bacteria that grow within the inclusion. The bacteria secrete proteins to the inclusion membrane and into the cytosol by a type III secretion mechanism and participate in giving the inclusion its unique properties. Recent results concerning the development of the inclusion will be presented here. An excellent and extensive review of the inclusion properties can be found in (13). Inclusion properties The characteristics of the inclusion, including its membrane and lumenal components, are unique and largely undetermined. They certainly evolve with time as the chlamydial cycle progresses. Following bacterial internalization in epithelial cells, Chlamydia containing compartments do not appear to interact with the endocytic pathway. Indeed, markers for the endocytic pathway have not been detected in the chlamydial inclusion, but transferrin, for instance, appears to be located in vesicles surrounding the inclusion in some cases. Even very early on, when bacteria enter, tubular endosomes containing transferrin are closely associated with nascent inclusions but do not fuse with the inclusion 566

In contrast, shortly after their internalization by dendritic cells or a monocytic cell line, Chlamydiae colocalize with LAMP1, a marker of late endosomes/lysosomes. This may be due to the rapid phagosomal/lysosomal maturation in these specialized antigen-presenting cells, in which the entry pathway seems to be different from the one in host epithelial cells (50,51). As the inclusion expands, the actin binding protein, cortactin, was found associated with the C. trachomatis serovar L2 inclusion, from 4 to 20 h post infection. At 20 h, actin itself was no longer associated with the inclusion. Proteins phosphorylated on tyrosine residues were also associated with the inclusion (47). The physical environment within the C. trachomatis mature inclusion has been further characterized. Determination of Hþ, Naþ, Kþ and Ca2þ concentrations indicated that all ions assayed within the lumenal space of the inclusion approximated the concentrations within the cytoplasm. In addition, the chlamydial inclusion appears to be freely permeable to cytoplasmic ions. This study also suggests that some small molecules, precursors such as amino acids, sugars and nucleotides may enter the lumen of the inclusion by simple diffusion, where they would then be available for chlamydial transporters (52). Shortly following the entry of Chlamydia in cells, nascent inclusions migrate to the microtubule organizing center in Traffic 2004; 5: 561–570

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the perinuclear region, where they remain close to the Golgi apparatus (53,54). The process of translocation of C. trachomatis serovar L2 depends on the minus-end directed microtubule motor complex dynein and the p150(Glued) associated subunit. This inclusion migration is inhibited when chlamydial protein synthesis is inhibited with chloramphenicol. Taken together, this suggests that the bacteria secrete unknown proteins associated to the inclusion membrane, possibly by type III secretion, which directly interact with dynein to support migration of the early inclusion along microtubules to the perinuclear region.

Contribution from the host cell to the inclusion development The development of the inclusion does not appear to intersect with the endocytic pathway and does not depend on de novo eukaryotic protein synthesis. The rapid expansion of the inclusion membrane during the infection cycle requires an important source of lipids. In a series of very elegant studies, T. Hackstadt’s group has shown that host cell lipids are transported to the inclusion (for review see (13) and references therein). Sphingolipids are trafficked to inclusions containing C. trachomatis, C. pneumoniae or C. caviae GPIC within a few hours following infection (55,56). This occurs by vesicular transport and requires bacterial protein synthesis. Sphingomyelin is then incorporated into the chlamydial cell wall. In addition, cholesterol, a lipid not normally found in prokaryotes, is also delivered to the C. trachomatis serovar L2 inclusion and to the bacteria (57). This delivery is brefeldin-A sensitive and microtubuledependent, and also requires bacterial protein synthesis. Thus, it has been proposed that cholesterol and sphingomyelin may be cotransported from the Golgi to the inclusion. The chlamydial inclusion also acquires eukaryotic glycerophospholipids, including phosphatidylinositol and phosphatidylcholine (58). The possibility also exists that some lipids are transported to the inclusion by nonvesicular mechanisms.

of C. caviae GPIC (H. Boleti and A. Dautry-Varsat, unpublished results), C. trachomatis serovar L2 but not C. pneumoniae (49).

Modification of the inclusion membrane by chlamydial proteins Many chlamydial proteins associated with the inclusion membrane have now been identified; these have been termed Inc proteins. Although these proteins show little sequence homology, similarities in their hydropathy profiles indicate a structural homology, which has served to define the Inc family of proteins. Inc proteins comprise a large bilobed hydrophobic region of 40–70 amino acids. Over 40 candidate Inc proteins have been identified from C. trachomatis and C. pneumoniae genomes (59,60). Chlamydial genomes contain orthologs to secretion apparatus from other intracellular bacteria, but secreted proteins are difficult to identify (61). Using a heterologous secretion system, C. pneumoniae IncA-C, as well as three other proteins exhibiting the characteristic hydrophobic domain, have been shown to undergo type III-dependent secretion in Shigella flexneri (62). These results strongly support a model whereby Inc proteins are inserted in the inclusion membrane through a type III secretion system (63). Interestingly, some Incs are expressed and secreted by 2 h after infection, whereas the expression of type III-specific genes is not detected until 6–12 h (44,64). Components of the type III apparatus were found in both purified EB and RB extracts, a finding consistent with the hypothesis that type III secretion may occur very early in infection and participate in the maturation of the inclusion. So far, IncA is the only Inc protein for which a function has been proposed, namely a role in the homotypic fusion of inclusions during C. trachomatis infection (65–67).

In contrast, no host membrane protein has been shown to be diverted to the inclusion, which might reflect that they are excluded from vesicles en route to the inclusion or that their localization to the inclusion is transient.

Other proteins which do not belong to the Inc family may be secreted to the inclusion membrane or in the host cell cytosol during infection and participate to the maturation of the inclusion (68,69). A systematic genomic study was undertaken using heterologous secretion by S. flexneri as a screen to search for putative proteins of unknown function secreted by the type III mechanism and revealed new secreted proteins (A. Subtil and A. Dautry-Varsat, in preparation).

A dynamin 1 dominant negative mutant was shown to inhibit the development of inclusions containing C. trachomatis serovar L2 and C. caviae GPIC, but not the entry of these bacteria, supporting the importance of some intracellular trafficking pathway regulated by dynamin for the inclusion development (32). Interestingly, the small GTPase Rab1 involved in ER to Golgi and intra-Golgi transport was localized to chlamydial inclusions of all species. In contrast, Rab6, which is involved in retrograde trafficking from the Golgi to the ER or in endosome to Golgi trafficking, was colocalized with the inclusions

A different approach consisted of comparing C. pneumoniae and C. trachomatis proteins from whole lysates of infected cells to profiles of proteins from purified bacteria (70). One protein was identified, the chlamydial proteaselike activity factor (CPAF), which had already been identified as a translocated protein able to cleave an eukaryotic transcription factor (71). The mechanism of CPAF translocation is not known. It is separated from its production, as it is inhibited in culture conditions mimicking persistent infection (72), and it does not seem to occur via a type III mechanism (A. Subtil, unpublished result).

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Conclusion The developmental cycle of Chlamydiae has been known for a long time; however, many important and basic cell biology questions still remain:

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How do the bacteria induce entry in the host cells? To what extent does it depend on the Chlamydia species and the host cells?;

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How does the inclusion avoid fusion with endocytic compartments? Which secreted bacterial proteins are involved and how?;

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What signal transduction pathways are activated at the different steps of the development cycle?.

We know that the inclusion receives lipids, coming from the Golgi. What pathway do they follow? Which host proteins are involved? Despite the difficulties due to the lack of genetic tools to analyze Chlamydia infection, many fundamental questions concerning these pathogen–host interactions can be approached using cell biology, especially now that a wealth of information is coming in from the sequencing of Chlamydia genomes. Note added in proof. A chlamydial protein secreted by the type III mechanism has just been reported by Dr T. Hackstadt’s group (Clifton, D.R. et al., 2004, Proc Natl Acad Sci USA, June 15, 10.1073/pnas.0402829101). This protein is rapidly phosphorylated at the sites of bacterial entry when exposed to the cell cytoplasm. This protein can recruit actin and thus appears to function in signaling of cytoskeletal rearrangements at the entry sites.

Acknowledgments Owing to the limitations on page length and on the number of references, many papers of value in the field could not be cited. Our apologies to the authors whose papers are not quoted in this review. Marı´a Eugenia Balan˜a´ was supported by a postdoctoral fellowship from the ‘Fondation pour la Recherche Me´dicale’. We thank Dr. Andre´s Alcover for the confocal microscopy presented in Figure 2. We are grateful to Dr. Agathe Subtil for useful discussions and to Drs Isabelle Derre´ and Agathe Subtil for reading this manuscript.

References 1. Knodler LA, Celli J, Finlay BB. Pathogenic trickery: deception of host cell processes. Nat Rev Mol Cell Biol 2001;2:578–588. 2. Stephens RS. The cellular paradigm of chlamydial pathogenesis. Trends Microbiol 2003;11:44–51. 3. Campbell LA, Kuo CC. Chlamydia pneumoniae – an infectious risk factor for atherosclerosis? Nat Rev Microbiol 2004;2:23–32.

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4. Belland RJ, Ouellette SP, Gieffers J, Byrne GI. Chlamydia pneumoniae and atherosclerosis. Cell Microbiol 2004;6:117–127. 5. Hogan RJ, Mathews SA, Mukhopadhyay S, Summersgill JT, Timms P. Chlamydial persistence: beyond the biphasic paradigm. Infect Immun 2004;72:1843–1855. 6. Stephens RS, Kalman S, Lammel C, Fan J, Marathe R, Aravind L, Mitchell W, Olinger L, Tatusov RL, Zhao Q, Koonin EV, Davis RW. Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 1998;282:754–759. 7. Read TD, Brunham RC, Shen C, Gill SR, Heidelberg JF, White O, Hickey EK, Peterson J, Utterback T, Berry K, Bass S, Linher K, Weidman J, Khouri H, Craven B, et al. Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucl Acids Res 2000;28:1397–1406. 8. Shirai M, Hirakawa H, Kimoto M, Tabuchi M, Kishi F, Ouchi K, Shiba T, Ishii K, Hattori M, Kuhara S, Nakazawa T. Comparison of whole genome sequences of Chlamydia pneumoniae J138 from Japan and CWL029 from USA. Nucl Acids Res 2000;28:2311–2314. 9. Kalman S, Mitchell W, Marathe R, Lammel C, Fan J, Hyman R, Olinger L, Grimwood J, Davis RV. Comparative genomes of Chlamydia pneumoniæ and C. Trachomatis Nature Genet 1999;21:385–389. 10. Horn M, Collingro A, Schmitz-Esser S, Beier CL, Purkhold U, Fartmann B, Brandt P, Nyakatura GJ, Droege M, Frishman D, Rattei T, Mewes HW, Wagner M. Illuminating the evolutionary history of chlamydiae. Science 2004;304:728–730. 11. Subtil A, Dautry-Varsat A. Chlamydia: five years A.G. (after genome). Curr Opin Microbiol 2004;7:85–92. 12. Hackstadt T, Stephens RS, ed. Chlamydia: Intracellular Biology, Pathogenesis, and Immunity. Washington, D.C.: American Society for Microbiology, 1999: 101–138. 13. Fields KA, Hackstadt T. The chlamydial inclusion: escape from the endocytic pathway. Annu Rev Cell Dev Biol 2002;18: 221–245. Epub . 14. Wyrick PB. Intracellular survival by Chlamydia. Cell Microbiol 2000;2:275–282. 15. Bavoil PM, Hsia R, Ojcius DM. Closing in on Chlamydia and its intracellular bag of tricks. Microbiology 2000;146:2723–2731S. 16. Raulston JE, Davis CH, Paul TR, Hobbs JD, Wyrick PB. Surface accessibility of the 70-kilodalton Chlamydia trachomatis heat shock protein following reduction of outer membrane protein disulfide bonds. Infect Immun 2002;70:535–543. 17. Kleba BJ, Banta E, Lindquist EA, Stephens RS. Recruitment of mammalian cell fibronectin to the surface of Chlamydia trachomatis. Infect Immun 2002;70:3935–3938. 18. Wehrl W, Brinkmann V, Jungblut PR, Meyer TF, Szczepek AJ. From the inside out – processing of the Chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells. Mol Microbiol 2004;51:319–334. 19. Davis CH, Raulston JE, Wyrick PB. Protein disulfide isomerase, a component of the estrogen receptor complex, is associated with Chlamydia trachomatis serovar E attached to human endometrial epithelial cells. Infect Immun 2002;70:3413–3418. 20. Carabeo RA, Hackstadt T. Isolation and characterization of a mutant Chinese hamster ovary cell line that is resistant to Chlamydia trachomatis infection at a novel step in the attachment process. Infect Immun 2001;69:5899–5904. 21. Fudyk T, Olinger L, Stephens RS. Selection of mutant cell lines resistant to infection by Chlamydia trachomatis and Chlamydia pneumoniae. Infect Immun 2002;70:6444–6447. 22. Brown DA, London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 1998;14:111–136. 23. Simons K, Toomre D. Lipid rafts and signal transduction. Nature Rev Mol Cell Biol 2000;1:31–39. 24. Kusumi A, Koyama-Honda I, Suzuki K. Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts. Traffic 2004;5:213–230.

Traffic 2004; 5: 561–570

Chlamydia – Host Cell Interactions 25. Lafont F, Abrami L, van der Goot FG. Bacterial subversion of lipid rafts. Curr Opin Microbiol 2004;7:4–10. 26. Jutras I, Abrami L, Dautry-Varsat A. Entry of the Lymphogranuloma Venereum strain of Chlamydia trachomatis into host cells involves cholesterol-rich membrane domains. Infect Immun 2003;71: 260–266. 27. Norkin LC, Wolfrom SA, Stuart ES. Association of caveolin with Chlamydia trachomatis inclusions at early and late stages of infection. Exp Cell Res 2001;266:229–238. 28. Stuart ES, Webley WC, Norkin LC. Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells. Exp Cell Res 2003;287:67–78. 29. Duncan MJ, Shin JS, Abraham SN. Microbial entry through caveolae: variations on a theme. Cell Microbiol 2002;4:783–791. 30. Gruenheid S, Finlay BB. Microbial pathogenesis and cytoskeletal function. Nature 2003;422:775–781. 31. Brumell JH, Grinstein S. Role of lipid-mediated signal transduction in bacterial internalization. Cell Microbiol 2003;5:287–297. 32. Boleti H, Benmerah A, Ojcius D, Cerf-Bensussan N, Dautry-Varsat A. Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth. J Cell Sci 1999;112:1487–1496. 33. Gagnon E, Duclos S, Rondeau C, Chevet E, Cameron PH, SteeleMortimer O, Paiement J, Bergeron JJ, Desjardins M. Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 2002;110:119–131. 34. Frischknecht F, Way M. Surfing pathogens and the lessons learned for actin polymerization. Trends Cell Biol 2001;11:30–38. 35. Carabeo RA, Grieshaber SS, Fischer E, Hackstadt T. Chlamydia trachomatis induces remodeling of the actin cytoskeleton during attachment and entry into HeLa cells. Infect Immun 2002;70:3793–3803. 36. Coombes BK, Mahony JB. Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells. Cell Microbiol 2002;4:447–460. 37. Carabeo RA, Grieshaber SS, Hasenkrug A, Dooley C, Hackstadt T. Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells. Traffic 2004;5:418–425. 38. Subtil A, Wyplosz B, Balan˜a´ ME, Dautry-Varsat A. Analysis of Chlamydia Caviae entry sites and involvement of Cdc42 and Rac activity. J. Cell Sci 2004;117:in press. 39. Caron E, Hall A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998;282: 1717–1721. 40. Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J 2000;348:241–255. 41. Boquet P, Lemichez E. Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis? Trends Cell Biol 2003;13:238–246. 42. Hardt WD, Chen LM, Schuebel KE, Bustelo XR, Galan JE. S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 1998;93:815–826. 43. Tran Van Nhieu G, Caron E, Hall A, Sansonetti PJ. IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells. Embo J 1999;18:3249–3262. 44. Fields KA, Mead DJ, Dooley CA, Hackstadt T. Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early-cycle development. Mol Microbiol 2003;48:671–683. 45. Ireton K, Cossart P. Interaction of invasive bacteria with host signaling pathways. Curr Opin Cell Biol 1998;10:276–283. 46. Birkelund S, Bini L, Pallini V, Sanchez-Campillo M, Liberatori S, Clausen JD, Ostergaard S, Holm A, Christiansen G. Characterization of Chlamydia trachomatis L2-induced tyrosine-phosphorylated HeLa cell proteins by two-dimensional gel electrophoresis. Electrophoresis 1997;18:563–567. 47. Fawaz FS, van Ooij C, Homola E, Mutka SC, Engel JN. Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or

Traffic 2004; 5: 561–570

48.

49.

50.

51.

52.

53.

54.

55.

56. 57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

localization of several host cell proteins including cortactin. Infect Immun 1997;65:5301–5308. Scidmore MA, Fischer ER, Hackstadt T. Restricted fusion of Chlamydia trachomatis vesicles with endocytic compartments during the initial stages of infection. Infect Immun 2003;71:973–984. Rzomp KA, Scholtes LD, Briggs BJ, Whittaker GR, Scidmore MA. Rab GTPases are recruited to chlamydial inclusions in both a species-dependent and species-independent manner. Infect Immun 2003;71: 5855–5870. Ojcius D, Bravo de Alba Y, Kanellopoulos J, Hawkins R, Kelly K, Rank R, Dautry-Varsat A. Internalization of Chlamydia by dendritic cells and stimulation of Chlamydia-specific T cells. J Immunol 1998;160: 1297–1303. Ojcius D, Hellio R, Dautry-Varsat A. Distribution of endosomal, lyposomal, and major histocompatibility complex markers in a monocytic cell line infected by Chlamydia psittaci. Infect Immun 1997;65: 2437–2442. Grieshaber S, Swanson JA, Hackstadt T. Determination of the physical environment within the Chlamydia trachomatis inclusion using ionselective ratiometric probes. Cell Microbiol 2002;4:273–283. Clausen J, Christiansen G, Holst H, Birkelund S. Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection. Mol Microbiol 1997;25:441–449. Grieshaber SS, Grieshaber NA, Hackstadt T. Chlamydia trachomatis uses host cell dynein to traffic to the microtubule-organizing center in a p50 dynamitin-independent process. J Cell Sci 2003;116: 3793–3802. Hackstadt T, Rockey DD, Heinzen RA, Scidmore MA. Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane. EMBO J 1996;15:964–977. Wolf K, Hackstadt T. Sphingomyelin trafficking in Chlamydia pneumoniae-infected cells. Cell Microbiol 2001;3:145–152. Carabeo RA, Mead DJ, Hackstadt T. Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion. Proc Natl Acad Sci U S A 2003;100:6771–6776. Wylie JL, Hatch GM, McClarty G. Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis. J Bacteriol 1997; 179:7233–7242. Bannantine JP, Griffiths RS, Viratyosin W, Brown WJ, Rockey DD. A secondary structure motif predictive of protein localization to the chlamydial inclusion membrane. Cell Microbiol 2000;2:35–47. Fields KA, Fischer E, Hackstadt T. Inhibition of fusion of Chlamydia trachomatis inclusions at 32 °C correlates with restricted export of IncA. Infect Immun 2002;70:3816–3823. Subtil A, Blocker A, Dautry-Varsat A. Type III secretion system in Chlamydia species: identified members and candidates [letter]. Microb Infect 2000;2:367–369. Subtil A, Parsot C, Dautry-Varsat A. Secretion of predicted Inc proteins of Chlamydia pneumoniae by a heterologous type III machinery. Mol Microbiol 2001;39:792–800. Fields KA, Hackstadt T. Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism. Mol Microbiol 2000;38:1048–1060. Slepenkin A, Motin V, de la Maza LM, Peterson EM. Temporal expression of type III secretion genes of Chlamydia pneumoniae. Infect Immun 2003;71:2555–2562. Hackstadt T, Scidmore-Carlson MA, Shaw EI, Fischer ER. The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion. Cell Microbiol 1999;1:119–130. Suchland RJ, Rockey DD, Bannantine JP, Stamm WE. Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane. Infect Immun 2000;68:360–367. Rockey DD, Viratyosin W, Bannantine JP, Suchland RJ, Stamm WE. Diversity within inc genes of clinical Chlamydia trachomatis variant

569

Dautry-Varsat et al. isolates that occupy non-fusogenic inclusions. Microbiology 2002; 148:2497–2505. 68. Fling SP, Sutherland RA, Steele LN, Hess B, D’Orazio SEF, Maisonneuve JF, Lampe MF, Probst P, Starnbach MN. CD8þ T cells recognize an inclusion membrane-associated protein from the vacuolar pathogen Chlamydia trachomatis. Proc Natl Acad Sci USA 2001;98:1160–1165. 69. Belland RJ, Scidmore MA, Crane DD, Hogan DM, Whitmire W, McClarty G, Caldwell HD. Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes. Proc Natl Acad Sci U S A 2001;98:13984–13989.

570

70. Shaw AC, Vandahl BB, Larsen MR, Roepstorff P, Gevaert K, Vandekerckhove J, Christiansen G, Birkelund S. Characterization of a secreted Chlamydia protease. Cell Microbiol 2002;4:411–424. 71. Zhong G, Fan P, Ji H, Dong F, Huang Y. Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med 2001;193: 935–942. 72. Heuer D, Brinkmann V, Meyer TF, Szczepek AJ. Expression and translocation of chlamydial protease during acute and persistent infection of the epithelial HEp-2 cells with Chlamydophila (Chlamydia) pneumoniae. Cell Microbiol 2003;5:315–322.

Traffic 2004; 5: 561–570