Cellular Microbiology (2000) 2(4), 275±282
Microreview Intracellular survival by Chlamydia Priscilla B. Wyrick Department of Microbiology and Immunology, University of North Carolina School of Medicine, CB#7290, 804 M. E. Jones, Chapel Hill, NC 27599-7290, USA. Summary Chlamydiae are obligate intracellular bacterial pathogens whose entry into mucosal epithelial cells is required for intracellular survival and subsequent growth. After a seemingly stealthy entry, chlamydiae quickly modify their vacuole (i) for exit from the endosomal pathway to the exocytic pathway and (ii) to permit fusion with intercepted endoplasmic reticulum- and Golgi-derived vesicles carrying glycerophospholipids and sphingolipids for chlamydiaecontaining vacuole membrane expansion. Chlamydiae possess novel hollow proteinaceous structures, termed projections, which they use to pierce the inclusion membrane, possibly to acquire from the epithelial cytoplasm nutrients they cannot synthesize; whether or not these truncated flagellar-like structures serve a dual exchange function for secretion of molecules to programme host cell signalling is unknown. Despite the accumulation of some 500±1000 progeny in the enormously enlarged inclusion, host cell function is surprisingly little disrupted, and progeny escape can be unobtrusive. This elegant adaptive pathogen strategy, which leads to silent, chronic human infection, is fascinating from a cellular microbiology perspective. Introduction Chlamydiae are among the most widespread bacterial pathogens in the world, and complications from infections represent an economic burden in billions of dollars annually. Chlamydia trachomatis and C. pneumoniae are human pathogens and well recognized as agents of Received 15 February, 2000; revised 5 April, 2000; accepted 12 April, 2000. ²Present address: Department of Microbiology, Box 70579, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37684, USA. *For correspondence. E-mail
[email protected]; Tel. (11) 423 439 6294; Fax (11) 423 439 8044. Q 2000 Blackwell Science Ltd
the blinding eye disease trachoma, sexually transmitted infections and their sequelae, and a variety of respiratory infections. As C. pneumoniae can also grow in alveolar macrophages, persist in circulating monocytes and has been isolated from vascular plaques, there has been an accumulating strong association of this species with coronary artery disease and atherosclerosis. C. psittaci and C. pecorum are primarily bird/animal pathogens, although zoonotic transmission of C. psittaci to humans causes important diseases with serious complications. For example, the respiratory affliction psittacosis, acquired from sick pet birds, can lead to endocarditis, and pregnant female farmers assisting in lambing have aborted in their third trimester with C. psittaci ovine strain isolated from the abortafacient material. Despite differences in species and syndromes, all chlamydia share common biological features: (i) they are obligate intracellular bacteria; (ii) their primary host cell is a mucosal epithelial cell; (iii) growth in the host epithelial cell is termed a `developmental cycle', which involves two distinct morphological and functional forms ± the elementary body (EB) and the reticulate body (RB) ± which transition from EB ! RB, RB to RB and, finally, RB ! EB; and (iv) the developmental cycle occurs within a membrane-bound vacuole, termed an `inclusion'. The entire process can occur with little apparent damage to the infected epithelial cell, leading to the characterization of chlamydia as a `stealth pathogen' and explaining, in part, why chlamydial infections are mostly chronic and why the diseases and sequelae are immune mediated. The cellular microbiological aspects of this pathogen± epithelial interaction are intriguing and will be summarized using C. trachomatis as the prototype. Important details as well as differences among the species, which account for variations in biological phenomena and, ultimately, probably disease outcome, can be found in excellent reviews (McClarty, 1994; 1999; Raulston, 1995; Bavoil et al., 1996; Hatch, 1996; 1999; Hackstadt et al., 1997; Sinai and Joiner, 1997; Stephens et al., 1998; Hackstadt, 1999). Entry As Chlamydia is an obligate intracellular bacterium, entry into host mucosal epithelial cells is not simply an option
276 P. B. Wyrick for chlamydiae; it is essential for their intracellular survival and growth as well as a prerequisite for causing disease. Therefore, chlamydiae appear to use all avenues available for entry, including receptor-mediated endocytosis in clathrin-coated pits, especially in polarized epithelial cells (Wyrick et al., 1989), pinocytosis in non-clathrin-coated pits (Prain and Pearce, 1989) and phagocytosis (Byrne and Moulder, 1978; Ward and Murray, 1984). Several adhesins for mediating receptor-mediated endocytosis and pinocytosis have been proposed, but the data are controversial; the details can be found in excellent recent reviews by Bavoil et al. (1996) and Hackstadt (1999). There is no evidence for macropinocytosis of chlamydia, whereby a `triggered' entry (Finlay and Cossart, 1997) induces actin activation, rearrangement and membrane ruffling, as described for Salmonella typhimurium (Francis et al., 1993), or evidence of a major upheaval of host apical membrane, such as microvillus effacement seen with enteropathogenic Escherichia coli (EPEC) (Jerse et al., 1990). The ligands mediating a circumferential `zipper' function remain unidentified. In any event, these many options and strategies provide flexibility for chlamydiae in different mucosal environments to attach to and enter epithelial cells with vastly different physiological functions, which may require several protein and carbohydrate adhesins and be reflected in a different array of surface receptors involved in either high-affinity interactions or multiple low-affinity events. Early intracellular fate, 0±4 h Specific contact of infectious EB with the epithelial apical surface triggers a series of early events, which programs the chlamydia and primes the host cell for productive chlamydial infection. Several events happen rapidly. First (Fig. 1A), the EB endosome pH only drops to 6.2 before stabilizing at 6.6, possibly as a result of the retention of the endosomal Na1/K1 ATPase (Schramm et al., 1996), akin to that described for Mycobacterium (SturgillKoszycki et al., 1994); fusion with or maturation of EB endosomes to lysosomes does not occur. Based on confocal microscopy, co-localization of EB-containing endosomes with transferrin receptor endosomes has been reported (van Ooij et al., 1997). However, higher resolution transmission electron microscopy shows the tubular endosomes containing transferrin-conjugated horseradish peroxidase juxtaposed with, but not fused to, vesicles containing EB. By $ 2 h after infection, the EB-containing vesicle is devoid of transferrin receptor, mannose-6-phosphate receptor, LAMP1, cathepsin D and vacuolar H1 ATPase, markers typifying early and late endosomes and lysosomes respectively; further, this vesicle does not accumulate fluid-phase markers, such
Fig. 1. Early intracellular fate of chlamydiae in epithelial cells. A. Inhibition of maturation of EB-containing endosomes to lysosomes. B. Entry signals local actin accumulation, annexin-induced homotypic fusion of EB-containing vesicles with one another and escape from the endocytic to the exocytic pathway by translocation of EB vesicles on microtubules to the peri-Golgi/nuclear hof region. C. Early chlamydial protein synthesis results in EB-containing vacuole modification to ensure appropriate trafficking and translocation.
as lucifer yellow. Secondly (Fig. 1B), EB receptor signalling triggers tyrosine phosphorylation of epithelial proteins and possibly cortactin (Birkelund et al., 1994; Fawaz et al., 1997), which in turn results in rearrangement of the host cytoskeleton. There is an immediate local accumulation of F-actin and clathrin (Majeed and Kihlstrom, 1991), which serve as scaffolding for redistribution of EB endosomes to the perinuclear region, a translocation also aided by dynein motor movement (Clausen et al., 1997) of the EB vacuoles on microtubules (Schramm and Wyrick, 1995). Thirdly, Majeed et al. (1993; 1994) demonstrated that, as long as intracellular calcium levels ([Ca21]i) remain homeostatic, annexins III, IV and V can bind to specific endosomal membrane phospholipids to regulate and promote homotypic fusion of the EB-containing endosomes with one another but not with lysosomes. Q 2000 Blackwell Science Ltd, Cellular Microbiology, 2, 275±282
Intracellular survival by Chlamydia For C. trachomatis, homotypic fusion results in a single vacuole containing several EB, sometimes referred to as a nascent inclusion. This strategic manoeuvre may represent an early version of cell density-dependent quorum sensing to signal co-ordinate gene expression for vacuole modification. It may also provide an opportunity for genetic exchange, as double infection of host cells with two different C. trachomatis serovars, such as E and F, eventually results in the one inclusion containing both serovars (Ridderhof and Barnes, 1989; Suchland et al., 2000). In marked contrast, homotypic endosomal fusion does not occur with other Chlamydia species. C. psittaci and C. pneumoniae each give rise to multiple inclusions per infected cell, reflective of both the multiplicity of infection and early inclusion division with progeny replication. In host cells doubly infected with C. trachomatis and C. psittaci, the C. trachomatis endosomes fused with one another to form the single inclusion, but they did not fuse with any of the multiple C. psittaci inclusions in the same infected host cell (Matsumoto et al., 1991). These data indicate the important role of vacuole modification by chlamydia for their survival and that such modification is unique to each Chlamydia species. Lastly, fascinating studies by Hackstadt et al. (1997) (Fig. 1C) show that, concomitantly, early chlamydial gene expression results in vacuole modification to ensure that trafficking of EB is diverted from the endocytic pathway continuum to the exocytic pathway. In permissive cell lines, internalized vacuoles containing isolated chlamydial outer membrane complexes (COMCs) are much delayed in maturation to lysosomes. How much of this early pathway direction is a receptor-directed phenomenon and/or the result of mild acid-induced rearrangement of envelope components that might temporarily define the EB vesicle as an early endosome is controversial. It is agreed that chlamydial early intracellular fate is soon dependent on chlamydia-specific transcription and translation, as exposure of infected cells to rifampin and chloramphenicol results in the dispersion of EB vesicles throughout the epithelial cytoplasm and eventual accumulation of lysosomal markers (Scidmore et al., 1996) versus the normal vacuole-specific modification for homotypic fusion, escape to the exocytic pathway and congregation in the apical domain trans-Golgi region. Type III secretion Do the metabolically inert EB, on contact with the epithelial cell surface, deliver preformed effector proteins via the chaperone-dependent pathway (Galan and Collmer, 1999) to prime host cells for chlamydial entry and modulate epithelial signal transduction and trafficking pathways? Unique structural appendages, termed `projections', Q 2000 Blackwell Science Ltd, Cellular Microbiology, 2, 275±282
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were first detected by Matsumoto (1988) by scanning electron microscopy on purified C. psittaci EB about 25 years ago and have since been found on most species of Chlamydia cultured in vitro as well as visualized on chlamydiae in vivo. Approximately 13±30 projections are arranged in a hexagonal array on the EB surface (Fig. 2A), but they become randomly redistributed as EB transform into the larger, metabolically active RB. These supramolecular structures originate in the inner, cytoplasmic membrane, emerge from the outer membrane via holes surrounded by a `rosette' of nine subunits (Fig. 2E), extend some 30 nm from the chlamydia surface and are hollow. As scanning electron microscopy of isolated inclusions shows the projections piercing the inclusion, and transmission electron microscopy of infected cells shows the projections extending through the inclusion membrane into the host cell cytoplasm (Fig. 2C), it was suggested that the projections served as a conduit for the exchange of nutrients, and perhaps energy, between growing RB ± sequestered protectively in the membrane-bound inclusion ± and the host cytosol. Isolated projections from EB have a `nail-like' morphology (Fig. 2B) remarkably similar to that characterized for flagella. Further, information from the C. trachomatis serovar D genome sequence first revealed the existence of some flagella gene orthologues (Stephens et al., 1998; Fig. 2F). This was surprising, as chlamydiae are not motile; the rapid bouncing movement of chlamydiae inside the inclusion has been presumed, perhaps wrongly, to be Brownian motion. Closer scrutiny of the chlamydial flagellar gene product orthologues reflects some basal body structural and flagellar-associated transport functional equivalents. Hsia et al. (1997), on sequencing large fragments of DNA from the guinea pig strain of C. psittaci, discovered an operon containing four open reading frames (ORFs) whose encoded, predicted products were homologous to structural and regulatory components of the type III secretion system. The Yersinia homologue equivalents of the chlamydial gene products include: YscU, part of the cytoplasmic membrane anchor; LcrD, a regulator of the low Ca21 response; YopN, which senses host cells and assists in contact activation of the secretion pathway; and a chaperone or secretion/translocation pilot. Bavoil and Hsia (1998) then reasoned that the chlamydial projections could serve an alternative function as the type III secretion machinery and, with associated transport proteins and a cytoplasmic membrane energizing ATPase (YscN equivalent), deliver RB secreted molecules into the host cell cytoplasm to modulate the host±parasite interaction. This was an exciting conceptual advancement! The scenario can be taken one step further. Contrary to physics dogma, which predicts that, as EB drop down to mucosal surfaces, the projections should be oriented away from
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Fig. 2. Chlamydial projections and their orthologous/homologous relationship to flagellar and type III system components. A. Electron photomicrograph of freeze-etched EB reveals surface projections. B. `Nail-like' morphology of projections isolated from EB (permission by Jane E. Raulston). C. Projections extend from RB through the inclusion membrane and into the host cytoplasm. D. Projections on attaching EB can be oriented downwards into coated pits. (A, C and D copyright permission by P. B. Wyrick) E. Schematic diagram of projections on EB (adapted from Matsumoto, 1988). Approximately 13±30 projections are arrayed in a hexagonal pattern on EB. The projections, < 70±90 nm in length and < 5±10 nm diameter, extend from the cytoplasmic membrane through the outer membrane via an opening surrounded by a `rosette' composed of nine subunits. F. Schematic diagram of E. coli flagellar basal body and hook components and the type III secretion apparatus in Salmonella and Yersinia with orthologous/homologous chlamydial predicted products (pink) derived from chlamydial genome sequence analyses.
epithelial apical surfaces, the projections can be oriented downwards into coated pits (Fig. 2D). Perhaps contact activates CopN which, in combination with the encoded chaperone, enhances substrate routing via the projection machinery to modulate eukaryotic signal transduction in preparation for early chlamydia±vesicle trafficking and to introduce a blockade of caspase 3 activation to prevent apoptosis (Fan et al., 1998). Inclusion development, 6±40 h Soon after reductase activity at the epithelial surface and internalization of EB, the EB begin a complex and lengthy reorganization (Hatch, 1996; 1999), fuelled in part by stored ATP pools in EB and chlamydial protein synthesis. Virtually nothing is known about the biochemistry or kinetics of the transition of infectious EB (
