Chlamydia pneumoniae entry into epithelial cells by clathrin-independent endocytosis Juha T. Korhonen a, b, c, * , Mirja Puolakkainen d, e , Anu Haveri e , Anne Tammiruusu e , Matti Sarvas e , Riitta Lahesmaa a a Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland b Institute of Biomedicine, Faculty of Medicine, University of Turku, Turku, Finland c Drug Discovery Graduate School, Finland d Department of Virology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki, Finland e National Institute for Health and Welfare, Helsinki, Finland article info Article history: Received 7 June 2011 Received in revised form 8 December 2011 Accepted 14 December 2011 Available online 21 December 2011 Keywords: Chlamydia pneumoniae Lipid rafts Clathrin SR-BI abstract A gram-negative obligate intracellular bacterium, Chlamydia pneumoniae, is a common respiratory pathogen. Here, we examined the invasion and attachment of C. pneumoniae K6 into nonphagocytic HL epithelial cell line by manipulating host plasma membranes by using cholesterol-depleting methyl-beta- cyclodextrin (MbCD) and cholesterol-loading MbCD complexed cholesterol (chol-MbCD). The invasion was attenuated by MbCD-treatment while chol-MbCD augmented the attachment and invasion. In addition, the invasion was inhibited by cholesterol sequestering reagents, nystatin and filipin. Further- more, exposure of host cells to sphingomyelinase inhibited the invasion. RNA interference was used to assay the role of clathrin and human scavenger receptor B, type I (SR-BI) in the entry of C. pneumoniae into A549 lung epithelial adenocarcinoma cells. In contrast to Chlamydia trachomatis L2, the entry of C. pneumoniae was found to be independent of clathrin. In addition, the entry was found to be SR-BI- independent, but interestingly, the chlamydial growth was attenuated in the SR-BI-silenced cells. These findings suggest that the attachment and invasion of C. pneumoniae into nonphagocytic epithelial cells is dependent on the formation of cholesterol- and sphingomyelin-rich plasma membrane micro- domains, and the entry is a clathrin-independent process. In addition, our data indicate that SR-BI supports the growth of C. pneumoniae in epithelial cells. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Chlamydia pneumoniae, a gram-negative obligate intracellular bacterium, is a common cause of respiratory tract infections [1]. Like the other human pathogenic Chlamydia species (Chlamydia trachomatis and Chlamydia psittaci), C. pneumoniae has a tendency to cause chronic infections. The chronic C. pneumoniae infection has been suggested to associate with chronic vascular inflammation and atherosclerosis, but the exact role of these bacteria in the development of atherosclerotic lesions has remained elusive [2]. Owing to the fact that Chlamydiae are able to replicate only within eukaryotic cells, attachment, entry and lysosomal avoidance are fundamental steps in the chlamydial infection cycle and pathogenesis [3e5]. Therefore, these steps are attractive targets for chemotherapeutic interventions in Chlamydia infection. Great efforts have been made to understand the Chlamydia invasion, but unfortunately there have been only little consensus on mechanisms by which different Chlamydia species enter into their host cells (for a review see [6]). Cholesterol- and sphingolipid-rich lipid rafts [7,8] and caveolae [9] have been suggested to act as platforms for entry of many microbial pathogens [10e14]. It has been suggested that some intracellular pathogens avoid fusion with lysosomes and intracel- lular destruction by exploiting the lipid raft-mediated endocytosis [15]. There is a body of evidence supporting this hypothesis, and lipid rafts are also shown to be important platforms for bacterial virulence factors [16]. It has been suggested that some, but not all, pathogens of the genus Chlamydia invade the host by lipid raft-mediated pathway [17e19]. In contrast to this, however, inconsistent results have been reported with C. trachomatis, and the idea of the lipid raft and * Corresponding author. Turku Centre for Biotechnology, Tykistokatu 6, FI-20520 Turku, Finland. Tel.: þ358 2 333 8601; fax: þ358 2 333 8000. E-mail address: juha.korhonen@utu.fi (J.T. Korhonen). Contents lists available at SciVerse ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath 0882-4010/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2011.12.002 Microbial Pathogenesis 52 (2012) 157e164