Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells

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Experimental Cell Research 287 (2003) 67–78

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Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells Elizabeth S. Stuart,* Wilmore C. Webley, and Leonard C. Norkin* Department of Microbiology, University of Massachusetts, 203 Morrill Science Center IVN, Amherst, MA 01003-5720, USA Received 17 July 2002, revised version received 5 January 2003

Abstract Obligate intracellular bacterial pathogens of the genus Chlamydia are reported to enter host cells by both clathrin-dependent and clathrin-independent processes. C. trachomatis serovar K recently was shown to enter cells via caveolae-like lipid raft domains. We asked here how widespread raft-mediated entry might be among the Chlamydia. We show that C. pneumoniae, an important cause of respiratory infections in humans that additionally is associated with cardiovascular disease, and C. psittaci, an important pathogen in domestic mammals and birds that also infects humans, each enter host cells via cholesterol-rich lipid raft microdomains. Further, we show that C. trachomatis serovars E and F also use these domains to enter host cells. The involvement of these membrane domains in the entry of these organisms was indicated by the sensitivity of their entry to the raft-disrupting agents Nystatin and filipin, and by their intracellular association with caveolin-1, a 22-kDa protein associated with the formation of caveolae in rafts. In contrast, caveolin-marked lipid raft domains do not mediate entry of C. trachomatis serovars A, 36B, and C, nor of LGV serovar L2 and MoPn. Finally, we show that entry of each of these chlamydial strains is independent of cellular expression of caveolin-1. Thus, entry via the Nystatin and filipin-sensitive pathway is dependent on lipid rafts containing cholesterol, rather than invaginated caveolae per se. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Chlamydia; Lipid rafts; Caveolae; Caveolin; Membrane domains; Nystatin; Filipin

Introduction Chlamydiae are gram-negative obligate intracellular bacterial parasites that enter host cells by endocytosis. Their replication occurs entirely within the segregated, membrane-bound compartment which forms. These compartments enlarge and at later times during the replicative cycle are called inclusions [1]. The initial endocytic vesicles and subsequent inclusions do not intersect the host cell endosomal compartment. Consequently, fusion with lysosomes is prevented [2]. Chlamydiae initially enter cells as metabolically inert elementary bodies (EBs) that differentiate into replicating metabolically active reticulate bodies (RBs).

* Corresponding authors. Elizabeth S. Stuart, Fax: ⫹1-413-545-1578; Leonard C. Norkin, Fax: ⫹1-413-545-1578. E-mail addresses: [email protected] (E.S. Stuart), lnorkin@ microbio.umass.edu (L.C. Norkin).

Later, the RBs reorganize back into EBs and these infectious forms are released from the host cell. The genus Chlamydia comprises three major species, C. trachomatis, C. pneumoniae, and C. psittaci. C. trachomatis is primarily a pathogen of humans. The species is subdivided into the trachoma biovariant (biovar), which contains serological variants (serovars) A to K, and the lymphogranuloma venereum (LGV) biovar, which contains serovars L1, L2, and L3. C. trachomatis also includes a third biovar, the mouse pneumonitis (MoPn) agent. C. trachomatis is the most common sexually transmitted bacteria in the United States, and causes ocular trachoma worldwide. Serovars A, B, and C primarily infect the conjunctiva, whereas serovars D through K primarily infect the urogenital tract. The LGV biovar causes chronic sexually transmitted disease that differs clinically from that caused by the trachoma biovar (see below). C. pneumoniae is another human pathogen. It is an important cause of respiratory tract infection and additionally has been implicated in atherosclerosis [3,4]. The natural

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hosts for C. psittaci are birds but this bacterium also can be transmitted to humans, causing psittacosis (parrot fever). Based largely on analysis of signature sequences in ribosomal RNA genes, the genus Chlamydia recently was subdivided into the Chlamydia (containing C. trachomatis), and the Chlamydophila (containing C. pneumoniae and C. psittaci) [5]. For convenience, the C. pneumoniae and C. psittaci strains used in this study are referred to as Chlamydia. Recently, we reported that C. trachomatis (serovar K) enters host cells via particular lipid microdomains in the host cell plasma membrane that are known as detergentinsoluble glycolipid-rich domains, or lipid “rafts” [6]. Rafts form in the membrane by the preferential packing of sphingolipids and cholesterol, and they move within the fluid bilayer [reviewed in 7]. The raft domains of most cell types contain invaginations called caveolae. These are distinguished from clathrin-coated pits by their distinctive size (70 –100 nm), flask-like shape, and lack of a visible coat in thin sections. Caveolae are believed to form within the rafts by the self-association of the hairpin-shaped protein, caveolin-1, which thus serves as a characteristic caveolae marker. Since caveolae form within rafts, they too are enriched for sphingolipids and cholesterol. The functions of caveolae/ rafts are not entirely clear, although they have been implicated in sorting and trafficking through endocytic and secretory pathways, and in organizing signal transduction pathways. A variety of infectious agents, including viruses and intracellular bacterial parasites, recently were found to enter host cells via caveolae or rafts [reviewed in 8,9]. The term “caveolae” is sometimes used synonymously with lipid rafts. Alternatively, caveolae sometimes are referred to as a specialized form of lipid raft that contains caveolin-1 and is invaginated [10]. We find the latter more restrictive usage to be preferable since one purpose of this study, as discussed below, was to determine whether raftmediated chlamydial entry might depend on caveolin-1 and invaginated caveolae per se. Despite intensive investigation of the mechanisms by which Chlamydia enter host cells, those mechanisms remain poorly understood. This is partly because of conflicting reports of Chlamydia entering via clathrin-dependent, as well as via clathrin-independent, pathways that also are not well understood [11–16]. Thus, a major goal of this study was to determine which Chlamydia biovars and serovars, in addition to C. trachomatis serovar K, might enter via lipid rafts. We reported earlier that caveolin-1 is associated with the compartment that internalizes C. trachomatis serovar K [6]. That observation was one line of evidence we presented to show that serovar K entry indeed is mediated by rafts or caveolae. Typical caveolae are 70 to 100 nm in diameter, whereas EBs are several fold larger at 300 nm. Therefore, we suggested that entry of these organisms is mediated by their association with lipid rafts, rather than by their enclosure within caveolae per se. However, we had no additional

experimental evidence to support that conjecture. Moreover, even if invaginated caveolae were not necessary for chlamydial entry, caveolin-1 might nevertheless be necessary for subsequent stages in the establishment of a parasitic state by the organism. Thus, an additional goal of the present study was to determine whether caveolin-1 might function in chlamydial entry and during later stages as well.

Materials and methods Cells and organisms Stock chlamydial organisms were grown in HeLa 229 cells. The organisms used were C. pneumoniae AR39 (Cpn), C. psittaci, guinea pig inclusion conjunctivitis (GPIC strain), C. trachomatis serovars A/Har-13, Har-36B, C/TW-3, E/VW-KX, F, K/VR887, mouse pneumonitis agent, and lymphogranuloma venereum (LGV 434). HeLa 229 cells were obtained from the American Type Culture collection. Fischer rat thyroid (FRT) wt cells and FRT-TrA cells were kindly provided by Dr. Michael P. Lisanti (Albert Einstein College of Medicine, Bronx, NY). These cell lines, and the transformation of FRT using a construct containing gD1-DAF with a Rous sarcoma virus promoter, have been described previously [17,18], along with the activation of gene expression by treatment with sodium butyrate. Assays of sensitivity of entry to Nystatin and filipin using indirect immunofluorescence Cells were grown to confluence in 24-well culture plates (Becton; Dickinson Labware, Franklin Lakes, NJ) on 12-mm coverslips in Richter’s improved MEM insulin (IMEMZO, Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum [FBS] (Atlantic Biologicals, Norcross, GA) at 37°C and 5% CO2. The cells were treated with 10, 20, or 30 ␮g/ml of Nystatin or 2, 4, and 6 ␮g/ml of filipin (Sigma Chemical Co., St. Louis, MO) in IMEMZO for 30 min. The organisms were then diluted in growth media with the appropriate concentrations of each drug (as above) to provide a multiplicity of infection (MOI) of 3.0 when 200 ␮l was added to each monolayer. After 2 h, the media with drugs were removed and fresh complete cycloheximide overlay media (Bio-Whittaker, Walkersville, MD) containing 10% FBS, 1X L-glutamine (CCOM) were added. At 36 h the cells were fixed with 70% cold methanol (MEOH) for 10 min. Specific treatment controls, to assess drug effects on EBs, used a concentrated inoculum of EBs from serovar K or serovar A. One-half of each inoculum was incubated for 30 min with either 30 ␮g/ml of Nystatin, or 6 ␮g/ml of filipin; the other halves were incubated in drug-free media. These drug-treated inocula, and the matched untreated aliquots, were then mixed with drug-free complete overlay medium. This reduced the drug concentration (1.25 ␮g filipin, 6.25 ␮g Nystatin) and provided an

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MOI of 3.0 when 200 ␮l was added to each of the paired HeLa cell monolayers. The subsequent steps followed the standard protocol described above. To visualize chlamydial inclusions, samples were incubated for 1 h at 37°C with a rabbit anti-chlamydia antibody, elicited by immunization with 15 different chlamydial serovars. The antibody was diluted 1:500 in phosphate-buffered saline [PBS]. Following 3 rinses in PBS, bound rabbit IgG was detected using a 1:50 dilution in PBS of FITC-conjugated goat anti-rabbit H & L chain secondary antibody. Unless otherwise specified, in all instances the secondary antibodies used in these studies were obtained from Jackson Immuno Research (West Grove, PA). The secondary antibody was incubated for 1 h at 25°C. Samples were examined at 400⫻ magnification. For each sample, the chlamydial inclusions in 20 fields were counted for each of the quadruplicate sample replicates of each drug concentration. Inclusion numbers were then graphed using Sigma Plot 2000 software. Western blot FRT cells were grown to confluence at 37°C in 25-cm2 flasks in IMEMZO ⫹ 10% FBS. A 10 mM sodium butyrate (Sigma Chemical Co. MO), the gene-regulating agent, was added to the culture flask of the FRT-TrA cells overnight to induce expression of the transfected caveolin-1 gene. The cells were harvested by scraping and lysed by vortexing with 3-mm sterile glass beads (Fisher Scientific, Pittsburgh, PA). Sample lysates were mixed 1:1 with 2X sodium dodecyl sulfate (SDS) sample buffer with 2-␤-mercaptoethanol, boiled for 5 min, and electrophoresed through an 8 –16% Tris-glycine gradient gel under denaturing conditions (Invitrogen Life Technologies, Carlsbad, CA). The proteins were transferred to PVDF membrane (Millipore Corp., Bedford, MA), presoaked with 70% MEOH, using a Bio-Rad transfer apparatus (Bio-Rad Laboratories, Hercules, CA). After 1 h transfer at 80 V using 1X transfer buffer, membranes were blocked with 5% nonfat dry milk and then incubated with a mouse anti-caveolin-1 monoclonal antibody (Transduction Laboratories, Lexington, KY) diluted 1:500 in 0.1% bovine serum albumin in PBS (BSA, Sigma Chemical Co.). After incubation for 3 h at room temperature (RT) with shaking and then appropriate rinses, membranes were incubated with gentle shaking for 1 h at 25°C in alkaline phosphatase [AP]-conjugated goat antimouse secondary antibody diluted 1:1000 in 0.1% BSA/ PBS. Following 4 rinses in 0.1% BSA/PBS, the localization of bound secondary antibody was visualized by addition of the Sigma Fast BCIP/NBT alkaline phosphatase substrate (Sigma Chemical Co.). Immunostaining for localization of Chlamydia and caveolin-1 HeLa 229 or FRT cells were infected with the various chlamydial strains at an MOI of 3.0 for 48 h at 37°C and 5%

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CO2. Cells then were fixed as described above and stained with a rabbit anti-chlamydia polyclonal antibody plus the monoclonal mouse anti-caveolin-1 antibody. As a control for gene activation, HeLa cell monolayers also were treated overnight with 10 mM sodium butyrate and then incubated with infectious inoculum and inclusion formation monitored by immunostaining. The secondary antibodies were TRITCconjugated goat anti-rabbit and FITC-conjugated goat antimouse antibody. Coverslips were mounted using Fluoromount-G (Southern Biotechnology Associates Inc., Birmingham, AL). Slides were examined at 600X using a Bio-Rad MRC-600 laser confocal microscope system. Images were captured and, as relevant, merged using the Confocal Assistant version 4.02 Image Processing Software. Attachment and entry of Chlamydia into FRT cells FRT-wt and FRT-TrA cells were infected with each of the different biotypes and species of Chlamydia as described and listed above, using an MOI of 4.0. The infections were allowed to continue for 10, 24, 36, and 72 h at 37°C and 5% CO2. At 10 and 24 h post infection, cells were detached from the coverslips by treatment with a trypsinversene mixture (Bio Whittaker, Walkersville, MD) for 10 min at 37°C to remove any external EBs. Following centrifugation at 1000 rpm for 5 min to remove the trypsincontaining media, the cells were resuspended in IMEMZO ⫹ 10% FBS growth media and allowed to re-adhere to new coverslips. Upon attachment, the cells were rinsed with sterile PBS, fixed, and immunostained using a 1:500 dilution of rabbit anti-chlamydia polyclonal antibody and a 1:50 dilution of FITC-conjugated goat anti-rabbit secondary antibody. For the remaining time points, cell monolayers were rinsed in PBS, fixed, and immunostained. Fixation and immunostaining protocols and confocal microscopy were as described above

Results We began by asking how widespread raft- or caveolaemediated entry might be among the Chlamydiae. For this purpose we examined the entry of C. psittaci (GPIC) and C. pneumoniae (AR 39), as well as other C. trachomatis serovars and biovars. If rafts or caveolae mediated the intracellular entry of those organisms, then impairment of raft function should result in decreased entry. The antibiotics Nystatin and filipin each selectively disrupt raft and caveolae function by precipitating cholesterol in the plane of the host cell plasma membrane. Clathrin-coated pits and other submembraneous structures are not affected by these treatments [19,20]. To verify that these drugs specifically impede raft- or caveolae-mediated chlamydial entry, we showed that Nystatin and filipin each inhibits the normal entry pathway of C. trachomatis serovar K, but not entry of the same organism when it occurs by an antibody-mediated

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opsonic process [6]. Additionally, these drugs did not impair entry of BSA-coated microspheres [6], and earlier work had demonstrated that the same treatments had no effects on internalization of transferrin, a standard marker of clathrinmediated endocytosis [21]. Using procedures similar to ones we described earlier [6], we found that the C. psittaci and C. pneumoniae strains, GPIC and AR39, respectively, enter HeLa cells by a pathway that indeed is sensitive to each of the cholesterolchelating agents (Fig. 1). Also, we confirmed our earlier finding that C. trachomatis serovar K infection is sensitive to both drugs. In addition, infections by C. trachomatis serovars E and F were sensitive to Nystatin and filipin treatments. In contrast, we found infections by C. trachomatis serovars A, B, and C, the MoPn agent, and LGV (L2) were not sensitive to the cholesterol-chelating drugs. For each strain, sensitivity to one of the raft-disrupting agents correlated completely with sensitivity to the other. Despite the clear dose dependency, some cells were infected as shown by inclusion formation, thus suggesting the drug was not toxic to the EBs. Specific controls in which EBs, but not the HeLa cell monolayers, were treated with either of the drugs, demonstrated levels of infection and inclusion formation comparable to matched, untreated inoculum controls (data not shown). Thus, even using treatment with the maximum drug concentrations, the EBs clearly retained their infectivity and the observed dose-dependent decreases in inclusion formation thus could not be attributed to toxic effects on the EBs. Note that whereas we are examining 30-h samples in the above experiment, our data do not depend upon inclusion development. Indeed, undeveloped EB-containing vacuoles are readily visible by immunostaining throughout these experiments and are counted. For example, see the experiments in FRT cells described below. The host protein caveolin-1 is found in host cell plasma membranes exclusively in caveolae. We previously found that caveolin-1 is associated with C. trachomatis serovar K inclusions at both early and late stages of infection [6]. This finding implies that at least some of the inclusion-associated caveolin-1 was acquired quite early, perhaps at entry. We next asked whether caveolin-1 might be associated with inclusions of the other Chlamydia serovars and biovars that we tested here for sensitivity to Nystatin and filipin. Furthermore, we asked whether the association of caveolin-1 with the inclusions of a particular chlamydial strain might correlate with the sensitivity of infection by that strain to inhibition by Nystatin and filipin. HeLa cells were infected with each of the chlamydial strains for 18, 24, 36, 48, and 72 h. At these times they were fixed and examined by confocal immunofluorescence to assess caveolin-1 distribution, relative to the bacterial inclusions. Inclusions of AR39, GPIC, and C. trachomatis serovars E and F, as well as serovar K, indeed were associated with caveolin-1 at each of the above times. In addition, optical sections through the Z-axis clearly demonstrated the caveolin-1 as “packets” apparently beside, or

associated with, the vacuolar membrane. In contrast, for inclusions formed by MoPn, LGV (L2), and ocular serovars of C. trachomatis, A, B, and C, confocal microscopy did not indicate a similar localization of caveolin-1 in the same focal plane as the actual inclusions these serovars developed. Representative 48-h samples of the caveolin-1-positive and caveolin-1-negative inclusions are shown in Fig. 2A and B, respectively. A comparison of these findings with those for drug sensitivity reported above (Fig. 1) shows that there was complete concordance between the data demonstrating caveolin-1 association with the inclusions of a particular strain and the data defining the sensitivity of infection by that strain to inhibition by both Nystatin and filipin. In addition, samples also were fixed and immunostained at early times post infection. At 3 h postinfection, internalized EBs can readily be recognized and have not yet accumulated at a perinuclear position within the HeLa cells. However, the diameter of the early EB-containing vacuoles precludes defining a precise relationship between caveolin-1 and the vacuolar membrane by optical sectioning. Nevertheless, even at this early time, dual immunostaining clearly demonstrated the presence of caveolin-1 and this result is consistent with acquisition at entry, as a component in lipid rafts (data not shown). We next asked whether caveolin-1 or invaginated caveolae per se actually might be required for chlamydial binding or entry. For this purpose we used Fischer rat thyroid cells, which neither express caveolin-1 nor form invaginated caveolae [18,22,23]. We confirmed by Western blotting that the FRT cells do not express detectable caveolin-1 (Fig. 3, WT). Next, we used FRT cells to assess each of the above Chlamydia strains for binding, entry, and inclusion formation. The FRT cells were incubated with each of the strains for 10, 24, and 72 h at 37°C. To release EBs that were adherent but external, the 10 and 24-h samples were treated with trypsin and then allowed to reattach to new coverslips. All samples were immunostained and then examined by confocal immunofluorescence microscopy. These confocal images demonstrated that each of the chlamydial strains entered the caveolin-1-negative FRT cells, regardless of whether their entry into HeLa cells was sensitive to the cholesterol-chelating agents and their inclusions were marked by caveolin-1 (representative 24- and 72-h samples are shown in Fig. 4). To ascertain that chlamydial entry into the FRT cells yet depends on lipid rafts though these cells do not contain caveolae, we asked whether pathogen entry into FRT cells is sensitive to Nystatin and filipin. Entry into FRT cells of C. trachomatis serovars E, F, and K, C. psittaci (GPIC), and C. pneumoniae was sensitive to the raft-disrupting drugs (Fig. 5). In contrast, entry into FRT cells of C. trachomatis serovars A, B, and C, LGV (L2), and MoPn was not affected by drug treatment (Fig. 5). Thus, the Nystatin/filipin effect on chlamydial entry into caveolin-1-negative FRT cells exhibits the same strain-specific sensitivity as is exhibited by chlamydial entry into HeLa cells. This demon-

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Fig. 1. Sensitivity/resistance of infection of HeLa cells by different chlamydial strains to the raft-disrupting drugs, Nystatin and filipin. HeLa 229 cells were treated with media containing different concentrations of Nystatin (A), or filipin (B), for 30 min and then infected with Chlamydia in media containing the drug. After 2 h the inoculum was removed and replaced with cycloheximide overlay media. At 30 h post infection the cells were fixed and stained with a rabbit anti-chlamydia and FITC-conjugated secondary antibody. Twenty fields were counted at 400⫻ magnification in quadruplicate for each point on the graph. Error bars represent ⫾2 SEM.

strates that those chlamydial strains and species that enter HeLa cells via rafts similarly depend on the integrity of raft domains for entry into caveolin-1-negative FRT cells. A comparison of the samples in Fig. 4 demonstrates that despite the successful entry into the caveolin-1negative FRT cells, and survival by all the above chla-

mydial strains, only two of the strains developed large inclusions in those cells. These strains were GPIC, whose entry was sensitive to Nystatin and filipin treatment, and MoPn, whose entry was not. We then asked whether those chlamydial strains that did not develop inclusions in the caveolin-1 negative FRT cells might generate in-

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Fig. 2. Localization of chlamydial inclusions and caveolin-1 in HeLa cells. HeLa 229 cells were infected with chlamydial strains for 48 h. (A) Inclusions of C. pneumoniae (AR39) Cpn, C. Psittaci (guinea pig inclusion conjunctivitis, GPIC strain), C. trachomatis serovars E/VW-KX, F, and K are seen to colocalize with caveolin-1. (B) Inclusions of C. trachomatis serovars, A/Har-13, Har 36B, C/TW-3, mouse pnuemonitis agent (MoPn) and lymphogranuloma venereum biovar (LGV 434) do not colocalize with caveolin-1. Cells were fixed and double-stained with a rabbit anti-chlamydia and a mouse anti-caveolin-1 antibody. Original magnification: 600⫻.

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Fig. 3. Western blot of FRT cell lysates showing that FRT-WT cells do not express detectable levels of the caveolin-1 protein, while the FRT-TrA cells express caveolin-1 upon induction of the transfected caveolin-1 gene. Fischer rat thyroid cell lysates were separated on a Tris-glycine gel under reducing conditions. The proteins were transferred to PVDF membrane and the blot was probed with a mouse anti-caveolin-1 antibody. Wild-type (WT) cells do not express caveolin-1, while the transfectant cells (FRTTrA) express caveolin-1 after overnight treatment with 10 mM sodium butyrate. MW is the molecular weight marker and CON is a human endothelial cell control, which expresses the caveolin-1 protein.

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clusions in FRT cells that expressed caveolin-1 from a transfected inducible caveolin-1 gene. These FRT-TrA cells carry an inducible gene for expression of caveolin-1 and were treated with 10 mM sodium butyrate as described under materials and methods. Successful induction of caveolin-1 was confirmed by Western blotting (Fig. 3, TrA). Moreover, caveolin-1 levels remained high in those cells throughout the course of infection, as indicated by Western blot analysis at both 12 and 48 h (data not shown). Nevertheless, those chlamydial strains that did not produce large inclusions in the caveolin-1-negative FRT cells remained unable to produce them in the caveolin-1-expressing transfectant FRT cells. As seen in Fig. 4, results obtained in the caveolin-1 expressing FRTTrA cells were identical to those obtained in the caveolin-1-negative FRT cells. Despite the seeming stasis in inclusion development displayed by all the infectious agents except GPIC and MoPn, inoculating HeLa monolayers with a lysate made from either FRT or FRT-TrA cells infected with these other serovar or species for 48 h demonstrated abundant infectious EBs were nevertheless present. A second control, treatment of HeLa cell monolayers with sodium butyrate followed by incubation with chlamydial EBs, demonstrated that these cells became infected. In addition, there was no evidence of stasis in inclusion development when the host cells were pretreated with sodium butyrate (data not shown).

Fig. 4. Chlamydiae do not require caveolin-1 for entry into FRT cells. FRT-WT and TrA cells were grown on 12-mm coverslips to confluence. FRT-TrA cells were treated to induce caveolin-1 expression and then infected with the various biovars/serovars of chlamydia. At 10 and 24 h the first sets of cells were trypsinized to remove any EBs that had not entered the cells. The cells were reseeded on new coverslips and after attachment, they were fixed and stained with a rabbit anti-chlamydia and FITC-conjugated secondary antibody. Another set of cells was incubated until 72 h, then fixed, and stained. Only MoPn and C. psittaci developed into mature inclusions at 72 h. The remaining serovars/biovars had EB-like material inside the cells at a size similar to that observed at 24 h. This was true in both the WT and the TrA FRT cells. The figure above is representative of the size and morphology seen in all the other serovars. Original magnification: 600⫻.

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Fig. 5. Sensitivity/resistance of infection of FRT cells by different chlamydial strains to the raft-disrupting drugs, Nystatin and filipin. FRT cells were treated with different concentrations of Nystatin or filipin, infected with chlamydia, incubated, fixed, immunostained, and counted as described in Fig. 1. Error bars represent ⫾ 2 SEM.

Discussion Lipid raft microdomains and caveolar invaginations that are contained within lipid rafts of most cell types are comprised of lateral assemblies of cholesterol and sphingolipids that float in the glycerophospholipid plasma membrane [7]. As expected, removal of plasma membrane cholesterol by agents such as Nystatin or filipin specifically impairs the ability of these domains to function [7]. Consequently, sen-

sitivity to Nystatin and filipin is a general indicator that endocytosis of particular ligands is mediated by caveolae or rafts. Caveolae are further characterized by the marker protein caveolin-1. Earlier, we reported that C. trachomatis serovar K enters host cells via a pathway that is sensitive to Nystatin and filipin, and that caveolin-1 is associated with the parasitecontaining vesicles and inclusions [6]. Based on that experimental evidence, we concluded that serovar K enters via a

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pathway that involves either cholesterol-rich lipid rafts or caveolar invaginations. In the current study, we used the same criteria of drug sensitivities, and association of caveolin-1 with vacuoles and inclusions, to ask how general this entry pathway might be among the members of Chlamydia genus. To facilitate discussion of our results, we note here and elaborate below that these criteria alone do not enable us to distinguish between entry that might be dependent on caveolae per se, from entry that might be mediated by lipid rafts regardless of the presence of caveolae. However, other data reported above and discussed later demonstrate that this chlamydial entry pathway is actually dependent on cholesterol-rich lipid rafts. Thus, in the following discussion we refer to this pathway as raft-mediated entry. Results in the current study show that in addition to C. trachomatis serovar K, serovars E and F also enter by means of lipid rafts. Moreover, C. pneumoniae strain AR39, and C. psittaci strain GPIC enter using this pathway as well. Therefore, raft-mediated entry is not limited to the trachomatis species of the Chlamydiae. In contrast however, C. trachomatis serovars A, B, and C, the C. trachomatis MoPn biovar, and LGV (L2) do not enter via lipid rafts. Thus, raft-mediated entry is not a property of all serovars and biovars of a particular Chlamydia species (i.e., C. trachomatis). Furthermore, these examples of strains that did not enter via rafts serve both as controls for the strains that did enter via rafts and as a demonstration that the experimental system did not inherently impose a lipid raft route of entry on the pathogens. Importantly, pretreatment of the infectious EBs, but not host cells, with the Nystatin or filipin resulted in seemingly normal infection and inclusion formation. This finding indicates that the inhibition observed when the cultures were treated with drugs was not caused by a toxic effect on EBs themselves. This result is not unexpected since despite a notable decrease in inclusion number as drug concentrations increased, some chlamydial inclusions clearly did develop. These findings also demonstrate strain-specific differences under constant, cell culture conditions. This fact is important since other studies demonstrate culture conditions, such as polarization of cells, change the route used for entry [24]. Regardless, the current findings indicate cholesterol-rich lipid rafts may account for much of the clathrin-independent chlamydial entry previously observed [11–16]. The use by a particular chlamydial strain of clathrin pit-mediated entry versus raft-mediated entry, or in some cases the use of both routes, may depend initially on the particular host molecule or molecules at the cell surface that EBs use as a receptor. Although the nature of such receptors is unknown, raft-associated receptors, for example, glycosylphospatidylinositol (GPI)-linked proteins [22,23], may channel entry via a raft-associated pathway, whereas clathrin-coated pit-associated receptors will channel entry via the clathrin-coated pit-mediated route. The route may also depend on the appropriate functionality of components that operate in the preferred pathway at postentry times.

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As noted above, the results of our earlier study [6] did not permit distinguishing between entry mediated by caveolar invaginations versus entry dependent on raft domains. Nystatin and filipin disrupt caveolae. But, rafts are sensitive to those drugs regardless of whether they contain caveolin-1 and caveolar invaginations. If chlamydial EBs were to enter by a raft-mediated pathway that is not dependent on caveolae, then the association of caveolin-1 with the vacuoles could derive from caveolae that are in the raft domains of most cell types. We favored a raft-mediated process that is not dependent on caveolae since these invaginations typically are 70 to 100 nm in diameter, whereas at 300 nm, infectious chlamydial EBs are several-fold larger [1]. To account for the presence of caveolin-1 associated with the serovar K-containing vacuoles, we suggested that entry could involve a zipper-type process [11], potentially initiated by the binding of the bacteria to receptor molecules preferentially associated with rafts in the host cell plasma membrane. Internalization then might occur by the coalescence of rafts around the EB. An association of caveolin-1 with the vacuoles containing internalized EBs then would simply be incidental, occurring because caveolae are located in the rafts. To determine whether caveolin-1 and caveolar invaginations might actually be required for chlamydial entry, we used Fisher rat thyroid cells. These cells do not express caveolin-1, and consequently do not form invaginated caveolae [17,18,22]. Interestingly, all of our chlamydial strains entered the caveolin-1-negative FRT cells, irrespective of whether their entry into HeLa Cells was sensitive to Nystatin and filipin. Importantly, pathogen entry into the FRT cells exhibited the same strain-specific sensitivity to Nystatin and filipin as entry into HeLa cells, confirming that even in the caveolae-negative cells, entry occurs via lipid rafts. Moreover, the absence of caveolar invaginations does not cause pathogen strains that ordinarily enter via raft domains to enter by a raft-independent pathway if caveolae are not available. Research with the interleukin-2 receptor provides a precedent for lipid raft-mediated internalization in cells lacking caveolin-1 [25]. This finding has led to the belief that raft-mediated endocytosis of certain ligands can occur independent of caveolin-1 [25,26]. However, to the best of our knowledge, the current study is the first to demonstrate caveolin-1-independent raft-mediated entry by a microbial parasite and contrasts with findings for simian virus 40 (SV40). For this latter organism, biochemical analysis of membrane fractions [21], negative-stained images of isolated caveolae [27], and thin-sections of infected cells [28] demonstrated that SV40 unequivocally enters via caveolae. Further, its entry depended specifically on caveolin-1, as shown by sensitivity to the expression, of dominant-negative truncation mutants of caveolin-1 [29]. All of the chlamydial strains in this study entered the caveolin-1-negative FRT cells. However, only C. psittaci (GPIC) and C. trachomatis (MoPn) produced inclusions in those cells, indicating that one or more fundamental require-

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ments for development by these chlamydia was not being met. For experiments with FRT cells, FRT-TrA cells that express caveolin-1 would seem a logical control. Nevertheless, whereas the FRT-TrA cells expressed caveolin-1 and also formed caveolae [17,18], the postentry block to the developmental cycles of the other chlamydial strains was not reversed in these cells. Several possibilities might plausibly account for these experimental results. First, caveolin1-dependent processes may still be defective in FRT-TrA cells. In direct support of this possibility, SV40, which infects host cells via caveolae [21,27–30], is unable to enter FRT-TrA cells (L.C. Norkin, unpublished result). Also, GPI-linked proteins normally are preferentially transported to the apical plasma membrane of polarized epithelial cells, by a process involving their incorporation into caveolin-1containing lipid rafts in the Golgi apparatus. However, transfectant caveolin-1-expressing FRT cells fail to incorporate GPI-linked proteins into lipid rafts, or to properly transport them to the apical plasma membrane [23]. A second possibility is suggested by our finding that the guinea pig C. psittaci strain and the murine C. trachomatis (MoPn) strain, but not the human C. trachomatis strains, developed inclusions in the FRT cells, perhaps indicating a host range block. However, other research with rodentderived cell lines [31] and with in vivo murine models using C. trachomatis (i.e., serovars D, F and K) exclude this possibility [32–34] as does the demonstrated infectivity of progeny EBs derived from FRT and FRT-TrA infected cells, noted below. Thus, the basis for the block to inclusion development exhibited by some of the chlamydiae in FRT and FRT-TrA cells remains to be determined. We suggested earlier that the raft-mediated chlamydial entry pathway might be a factor that enables the phagosome to avoid intersecting the host cell endosomal/lysosomal compartment, in which degradation would occur [6]. This suggestion remains a viable possibility, at least for some strains. However, the current results indicate that in other instances, lysosomal avoidance is not dependent on host cell expression of caveolin-1 per se. For example, GPIC enters via rafts and normally has caveolin-1 associated with its inclusions (Fig. 1). Nevertheless, it generated inclusions in the caveolin-1-negative FRT cells. In addition, the human chlamydial genital serovars E, F, and K enter via rafts and normally have caveolin-1 associated with them. Nevertheless, they entered the caveolin-1-negative FRT cells, and their continued presence after 72 h indicates they avoid degradation. Furthermore, despite an inability to generate large inclusions in FRT cells or in FRT-TrA cells (Fig. 4), experiments using lysates from either of these infected cell types demonstrated the inocula did infect HeLa cells. Therefore the pathogen present in the FRT, or FRT-TrA cells, remained viable and infectious (data not shown). Since for both GPIC and MoPn, inclusions developed in the caveolin-1-negative cells, these pathogens do not require caveolin-1, and their developmental requirements may

differ from those of the other chlamydiae tested. Possibly the low levels [10 mM] of sodium butyrate, a global gene regulator required to initiate caveolin-1 expression by FRTTrA, might simultaneously alter expression of other host cell genes so as to interfere with inclusion development. However, identical sodium butyrate treatment of HeLa cells, followed by infection using various serovars, did not lead to arrested inclusion development but rather, to apparently normal inclusion formation. Chlamydia might have to stimulate a signal transduction pathway to promote their entry into nonprofessional phagocytes, including FRT cells [2,35,36] and caveolin-1 is implicated in organizing signal transduction complexes within rafts [37]. Thus, entry by Chlamydia in the absence of caveolin-1 indicates that the signaling pathways they induce in host cells does not require this protein to transmit the signal that promotes initial entry per se. Possibly, needed signal transmission occurs via lipid raft microdomains, which also could concentrate relevant signaling molecules [38]. Alternatively, some strains might use noncaveolar and nonraft membrane domains to transmit signals. Finally, although caveolin-1 might not be required for entry, some strains might require a signal dependent upon caveolin-1, to prime the host cell which then could provide support necessary for postentry events critical to the pathogen’s developmental cycle. In summary, we have shown here that some, but not all, pathogens of the genus Chlamydiae enter host cells by a route that involves cholesterol-rich lipid raft domains in the host cell plasma membrane. These domains were implicated in pathogen entry by demonstrating sensitivity of that entry to the raft-disrupting agents Nystatin and filipin, and by showing an association of intracellular bacteria with the caveolae marker protein, caveolin-1. For each of the 10 strains tested, there was a complete correlation between the sensitivities of their entry to each of the drugs, and their intracellular association with caveolin-1. A second important finding is that each of the 10 strains was able to enter caveolin-1-negative cells, regardless of whether they entered by the pathway that is sensitive to Nystatin and filipin. Thus, neither caveolin-1 proteins nor invaginated caveolae are necessary for initial entry using the route that clearly is sensitive to these drugs. Therefore, this entry pathway involves cholesterol-rich lipid rafts, regardless of whether they contain caveolin-1 and caveolae. However, our results leave open the possibility that caveolin-1 may yet play a critical role in the postentry developmental cycle of some of these pathogens. Finally, these findings may resolve some of the conflicting reports of Chlamydia entering by pathways that are independent of clathrin, versus entry by a coated pit-dependent pathway. Further examination of lipid raft domains in the entry and intracellular biology of the Chlamydiae should provide a fuller understanding of a basis for diversity of these pathogens.

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Acknowledgments We thank Michael P. Lisanti for providing wild-type FRT cells and FRT cells that express caveolin-1, Judy Whittum-Hudson for providing C. trachomatis serovars C and E and LGV (L2), and Roger G. Rank for providing C. trachomatis (MoPn). This work was supported by the U.S. Department of Agriculture Massachusetts Agricultural Experiment Station (MA00845). The University of Massachusetts Central Microscopy Facility is supported by a grant from the National Science Foundation (NSF BBS 8714235). Work carried out by Wilmore C. Webley was in partial fulfillment of the requirements for a Ph.D. from the Department of Microbiology, University of Massachusetts, Amherst. W.C.W. was supported in part by a Fulbright Scholarship.

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