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

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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.

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). 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 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 detergent-insoluble 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 raft-mediated 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], [12], [13], [14], [15], [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.

Section snippets

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

Results

We began by asking how widespread raft- or caveolae-mediated 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

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, sensitivity to Nystatin and filipin is a general indicator that endocytosis of particular

Acknowledgements

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

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