Entry of the Lymphogranuloma Venereum Strain ofChlamydia trachomatisinto Host Cells Involves Cholesterol-Rich Membrane Domains

Abstract

Chlamydiae are bacterial pathogens which develop strictly inside the epithelial cells of their hosts. The mechanism used by chlamydiae to enter cells is not well characterized; however, it is thought to consist of a receptor-mediated process. In addition, the formation of clathrin-coated pits appears to be dispensable for chlamydiae to be internalized by host cells. Clathrin-independent endocytosis has recently been shown to occur through cholesterol-rich lipid microdomains, which are characterized by detergent insolubility. In the present study, we investigated whether these lipid domains play a role in Chlamydia trachomatis serovar L2 internalization by host cells. Our results show that after binding to HeLa cells, chlamydiae are associated with detergent-resistant lipid microdomains (DRMs), which can be isolated by fractionation of infected HeLa cells and flotation on a sucrose gradient. After internalization by HeLa cells, chlamydiae were still found in DRMs. In addition, extraction of plasma membrane cholesterol inhibited infection of HeLa cells by C. trachomatis. Many of the proteins associated with DRMs are glycosylphosphatidylinositol (GPI)-anchored proteins; however, our results could not identify a role for GPI-anchored proteins in the entry process. The same results were obtained for Chlamydia psittaci strain GPIC. We propose that cholesterol-rich domains participate in the entry of chlamydiae into host cells. Chlamydia binding to cholesterol-rich domains may lead to coalescence of the bacterial cells, which could trigger internalization by host cells.Chlamydiae are gram-negative bacterial pathogens which proliferate strictly inside host cells and are responsible for a wide range of diseases in animals (33). In humans, various strains of Chlamydia trachomatis are the causative agents of prevalent sexually transmitted bacterial diseases and are the main causes of preventable blindness worldwide. Chlamydiae are characterized by two distinct morphological forms: the small metabolically inactive elementary body (EB) (diameter, 0.3 m) and the larger metabolically active reticulate body (RB) (diameter, 1 m). EBs initiate infection by binding to host epithelial cells. Several hours following their internalization, EBs differentiate into noninfectious replicative RBs, which proliferate within a membrane-bound vacuole termed the inclusion. Within 2 days of infection, newly produced RBs have differentiated into EBs, which are released from the cell and can begin a new cycle of infection.The identity of the host cell receptor which binds EBs has remained elusive. Heparan sulfate glycosaminoglycans appear to be involved in the attachment process (42, 48), although the dependence on glycosaminoglycans for attachment may vary among chlamydial species and strains (12). Other putative chlamydial ligands used for attachment to host cells include the major outer membrane protein (MOMP) (43), the outer membrane protein OmcB (45), a heat-labile chlamydial cytadhesin (18), and heat shock proteins (29). Internalization of ...

Lipid Raft-Mediated Entry Is Not Required for Chlamydia trachomatis Infection of Cultured Epithelial Cells

“…To further investigate this hypothesis, we carefully tested whether C. trachomatis serovars L2, D, E, and K are associated with lipid rafts, caveolin, or GM1 during entry. In contrast to some published studies (18,25,36), we were unable to find evidence that C. trachomatis entry is associated with or requires lipid rafts.We first confirmed that acute depletion of plasma membrane cholesterol by the cholesterol-chelating agent M␤CD inhibits C. trachomatis entry. C. trachomatis was propagated as previously described (40).…”

Comparison of Chlamydia trachomatis serovar L2 growth in polarized genital epithelial cells grown in three-dimensional culture with non-polarized cells

“…This intriguing difference between strains may possibly be due to the recognition of and binding to different receptor molecules on host cell surfaces that, for serovar E, may be enriched or clustered in some regions or microdomains of polarized epithelial cell apical membranes, such as lipid rafts or caveolae, proposed to be involved in serovar E but not in serovar L2 entry [19,20]. Although their role in chlamydial entry is still controversial [21][22][23], it is an interesting possibility, as many pathogens are known to interact with these membrane microdomains and that receptor molecules, such as membrane-associated estrogen receptors that locate to caveolae, have been implicated in serovar E attachment/entry [11,24]. In contrast, initial interactions of serovar L2 EB with host cell surfaces may occur via recognition of a broader range of host molecules or molecules widely represented throughout cell surfaces, such as heparan sulfate proteoglycans [25], and/or via Tarp-mediated pedestallike formation [26,27], a phenomenon shown to be more prevalent for serovar L2 than with serovar D [26] or serovar E [28].…”

Chlamydial Intracellular Survival Strategies

“…The actin rearrangements that occur during entry are transient and may be terminated by secreted chlamydial effectors such as CT166, which glucosylates Rac1 (Thalmann et al 2010), or CT694, which interacts and colocalizes with AHNAK, an actin-binding protein (Hower et al 2009). Additional host factors that contribute to uptake into nonphagocytic cells include clathrin (Boleti et al 1999;Hybiske and Stephens 2007a) and cholesterol-rich microdomains (Norkin et al 2001;Jutras et al 2003;Stuart et al 2003;Gabel et al 2004). …”