Identification of Sphingomyelinase on the Surface of Chlamydia pneumoniae: Possible Role in the Entry into Its Host Cells.

We have recently suggested a novel mechanism, autoendocytosis, for the entry of certain microbes into their hosts, with a key role played by the sphingomyelinase-catalyzed topical conversion of sphingomyelin to ceramide, the differences in the biophysical properties of these two lipids providing the driving force. The only requirement for such microbes to utilize this mechanism is that they should have a catalytically active SMase on their outer surface while the target cells should expose sphingomyelin in the external leaflet of their plasma membrane. In pursuit of possible microbial candidates, which could utilize this putative mechanism, we conducted a sequence similarity search for SMase. Because of the intriguing cellular and biochemical characteristics of the poorly understood entry of Chlamydia into its host cells these microbes were of particular interest. SMase activity was measured in vitro from isolated C. pneumoniae elementary bodies (EB) and in the lysate from E. coli cells transfected with a plasmid expressing CPn0300 protein having sequence similarity to SMase. Finally, pretreatment of host cells with exogenous SMase resulting in loss plasma membrane sphingomyelin attenuated attachment of EB.

ABC-cassette transporter 1 (ABCA1) expression in epithelial cells in Chlamydia pneumoniae infection.

ATP-binding cassette transporter A1 (ABCA1) mediates reverse cholesterol transport and innate immunity response in different cell types. We have investigated the regulation of ABCA1 expression in response to intracellular Chlamydia pneumoniae infection in A549 epithelial lung carcinoma cells. C. pneumoniae infection decreased ABCA1 expression in A549 cells, and the activity of the ABCA1 promoter was decreased. The decreased promoter activity was dependent on its E-box and GnT-box elements of the promoter. Chlamydial growth was decreased in ABCA1-silenced epithelial lung carcinoma cells. These data indicate an important role for ABCA1 in intracellular bacterial infection.

Flotillin-1 (Reggie-2) contributes to Chlamydia pneumoniae growth and is associated with bacterial inclusion.

Chlamydiae are obligate intracellular pathogens replicating only inside the eukaryotic host. Here, we studied the effect of human flotillin-1 protein on Chlamydia pneumoniae growth in human line (HL) and A549 epithelial cell lines. RNA interference was applied to disrupt flotillin-1-mediated endocytosis. Host-associated bacteria were detected by quantitative PCR, and C. pneumoniae growth was evaluated by inclusion counts. C. pneumoniae attachment to host cells was unaffected, but bacterial intracellular growth was attenuated in the flotillin-1-silenced cells. By using confocal microscopy, we detected flotillin-1 colocalized with the inclusion membrane protein A (IncA) in the C. pneumoniae inclusion membranes. In addition, flotillin-1 was associated with IncA in detergent-resistant membrane microdomains (DRMs) in biochemical fractioning. These results suggest that flotillin-1 localizes to the C. pneumoniae inclusion membrane and plays an important role for intracellular growth of C. pneumoniae.

Chlamydia pneumoniae entry into epithelial cells by clathrin-independent endocytosis.

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 (MßCD) and cholesterol-loading MßCD complexed cholesterol (chol-MßCD). The invasion was attenuated by MßCD-treatment while chol-MßCD augmented the attachment and invasion. In addition, the invasion was inhibited by cholesterol sequestering reagents, nystatin and filipin. Furthermore, 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 microdomains, 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.