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 bac- terial parasites that enter host cells by endocytosis. Their replication occurs entirely within the segregated, mem- brane-bound compartment which forms. These compart- ments 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 endoso- mal compartment. Consequently, fusion with lysosomes is prevented [2]. Chlamydiae initially enter cells as metaboli- cally inert elementary bodies (EBs) that differentiate into replicating metabolically active reticulate bodies (RBs). Later, the RBs reorganize back into EBs and these infec- tious 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 subdi- vided into the trachoma biovariant (biovar), which contains serological variants (serovars) A to K, and the lymphogran- uloma 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 dif- fers 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 addition- ally has been implicated in atherosclerosis [3,4]. The natural * Corresponding authors. Elizabeth S. Stuart, Fax: +1-413-545-1578; Leonard C. Norkin, Fax: +1-413-545-1578. E-mail addresses: esstuart@microbio.umass.edu (E.S. Stuart), lnorkin@ microbio.umass.edu (L.C. Norkin). R Available online at www.sciencedirect.com Experimental Cell Research 287 (2003) 67–78 www.elsevier.com/locate/yexcr 0014-4827/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4827(03)00059-4