The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis Kari Fine-Coulson, Barbara J. Reaves, Russell K. Karls, Frederick D. Quinn* Department of Infectious Diseases, University of Georgia, Athens, Georgia, United States of America Abstract Dynamic, cholesterol-dense regions of the plasma membrane, known as lipid rafts (LR), have been observed to develop during and may be directly involved in infection of host cells by various pathogens. This study focuses on LR aggregation induced in alveolar epithelial cells during infection with Mycobacterium tuberculosis (Mtb) bacilli. We report dose- and time- dependent increases in LR aggregation after infection with three different strains at multiplicities of infection of 1, 10 and 100 from 2–24 hr post infection (hpi). Specific strain-dependent variations were noted among H37Rv, HN878 and CDC1551 with H37Rv producing the most significant increase from 15 aggregates per cell (APC) to 27 APC at MOI 100 during the 24 hour infection period. Treatment of epithelial cells with Culture Filtrate Protein, Total Lipids and gamma-irradiated whole cells from each strain failed to induce the level of LR aggregation observed during infection with any of the live strains. However, filtered supernatants from infected epithelial cells did produce comparable LR aggregation, suggesting a secreted mycobacterial product produced during infection of host cells is responsible for LR aggregation. Disruption of lipid raft formation prior to infection indicates that Mtb bacilli utilize LR aggregates for internalization and survival in epithelial cells. Treatment of host cells with the LR-disruption agent Filipin III produced a nearly 22% reduction in viable bacteria for strains H37Rv and HN878, and a 7% reduction for strain CDC1551 after 6 hpi. This study provides evidence for significant mycobacterial-induced changes in the plasma membrane of alveolar epithelial cells and that Mtb strains vary in their ability to facilitate aggregation and utilization of LR. Citation: Fine-Coulson K, Reaves BJ, Karls RK, Quinn FD (2012) The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis. PLoS ONE 7(9): e45028. doi:10.1371/journal.pone.0045028 Editor: Olivier Neyrolles, Institut de Pharmacologie et de Biologie Structurale, France Received May 31, 2012; Accepted August 11, 2012; Published September 14, 2012 Copyright: 2012 Fine-Coulson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by research grants from the American Lung Association (R.K.) and the University of Georgia Faculty of Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: fquinn@uga.edu Introduction Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected an estimated one-third of the world’s population with potentially 60–90% of these individuals harboring the latent form of the disease [1]. While significant research has been focused on Mtb/host interactions, there remains a paucity of data defining attachment and internalization of bacilli with the epithelial cells that line the alveolus. Mtb bacilli have been shown to infect and replicate in alveolar epithelial cells both in vitro and in vivo [2–6]. It is believed that these interactions play an important role during both early and chronic infection by influencing the host immune response to the pathogen [7–9]. Therefore, it is imperative to better define the events that occur during Mtb infection of the alveolar epithelial cell. Over the last 15–20 years our understanding of eukaryotic cell membrane organization has changed dramatically. Areas of the plasma membrane, known as lipid rafts (LR), have been described as dynamic regions within the membrane enriched in cholesterol, glycosphingolipids, sphingomyelin, phospholipids with acyl chains, glycosylphosphatidylinositol (GPI)-linked proteins as well as other membrane proteins such as innate immune receptors [10–12]. Studies utilizing phototonic force microscopy and fluorescent resonance energy transfer have established the size of rafts in an unperturbed cell system to be approximately 5 nm-50 nm in diameter which would be undetectable by light microscopy [12– 14]. However, other studies have demonstrated that stimuli applied to the plasma membrane, such as bacteria and/or the toxins they produce, can induce aggregation of LR to a size observable by confocal microscopy [15–20]. Lipid raft aggregation on target host cells in response to interactions with infectious agents has yielded interesting results for viruses and bacteria alike. Influenza virus has been shown to associate with LR via hemagglutinin and neuraminidase, and examination of enveloped virions post-budding shows a signifi- cant number of raft domains within the viral envelope [21]. Further it has been demonstrated that LR are important for HIV-1 viral budding [22]. During bacterial infections, LR have been shown to induce important changes in lipid raft formation. Some bacterial proteins, produced during infection, help facilitate hijacking of the host cell. Binding of cholera toxin subunit B to ganglioside (GM1) found in LR is required for uptake of the toxin [23]. Further, various bacterial proteins have been shown to induce LR aggregation to promote host cell responses to the pathogen. For example, treatment of macro- phages with Listeriolysin-O (LLO) has been shown to induce large ‘‘super’’ aggregates of LR which facilitate signaling through receptor tyrosine kinase domains, suggesting LR aggregation may facilitate an innate immune response during infections with Listeria monocytogenes [15]. PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45028