Results Salmonella Association with Polarized MDCK Cell Monolayers Requires Bacterial Protein Synthesis and Causes Loss of Host MicroviUi

Many intracellular parasites are capable of penetrating host epithelial barriers. To study this process in more detail we examined the interactions between the pathogenic bacteria Salmonella choleraesuis and polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells grown on membrane filters. Association of bacteria with the MDCK cell apical surface was an active event, requiring bacterial RNA and protein synthesis, and was blocked by low temperatures. Salmonella were internalized within a membrane-bound vacuole and exhibited penetration through, but not between MDCK cells. A maximum of 14 Salmonella per MDCK cell crossed the monolayer per hour to the basolateral surface yet the monolayer remained viable and impermeable to Escherichia coli. Apical S. choleraesuis infection resulted in an increase in paracellular permeability but the MDCK intercellular contacts were not significantly disrupted. Basolateral S. choleraesuis infection was inefficient, and only small numbers of S. choleraesuis penetrated to the apical medium. I NTRACELLULAR parasites use a variety of routes to gain entry into susceptible hosts (Moulder, 1985). One such route is passage through the host's epithelial barrier, allowing the pathogen access to underlying tissue, blood, and the reticuloendothelial system. Pathogenic bacteria which are facultative intracellular parasites commonly use this method of entry and these include species of Yersinia, Salmonella, Chlamydia, Shigella, Neisseria, and Brucellae. The underlying mechanisms used by these microorganisms to penetrate host epithelia are not well understood. Part of the difficulty has been the lack of suitable in vitro methods to study epithelial cell penetration. Rather, most of our current knowledge has accumulated from animal studies. A comprehensive descriptive study of Salmonella tissue penetration was published by Takeuchi (1967). Transmission electron microscopy was used to examine the intestinal epithelium of guinea pigs orally infected with Salmonella typhimurium. Takeuchi observed that as these bacteria came into close proximity to the brush border, the epithelial microvilli began to degenerate. Most of the bacteria entered directly into the epithelial cells and resided within membrane bound cavities. A few bacteria also appeared to enter at intercellular junctions and remained surrounded by a vacuole composed of neighboring epithelial cells. Neither bacterial exit from the epithelial cells nor entry into the underlying lamina propria was observed. Using murine ileal loops infected with Salmonella typhi, Kohbata et al. reported that ileal M cells, a type of intestinal epithelial cell, may be the target ofS. typhi (1986). These authors found that M cells lost their microvilli, and suffered serious cytopathic effects. In vitro tissue culture models have also been used to examine initial interactions and internalization of S. typhimurium and S. typhi into HeLa and HEp-2 cells (Giannella et al., 1973; Kihlstrom and Nilsson, 1977; Yabuuchi et al., 1986; Yokoyama et al., 1987). Internalization of Salmonellae into these cells appeared to proceed by a similar mechanism to that observed with the guinea pig model. We have demonstrated that Salmonella replicates inside vacuoles within epithelial cells, and bacterial entry and intracellular replication does not require endosome acidification (Finlay and Falkow, 1988). In this study we have focused our attention on Salmonella choleraesuis, a common pathogen of swine which is also a highly invasive and serious human pathogen. Unlike most other nontyphoid Salmonellae, S. choleraesuis rarely causes gastrointestinal symptoms; instead it is considered the prototype of Salmonella which cause invasive diseases (Rubin and Weinstein, 1977). S. choleraesuis infections often lead to serious bacteremia, with a fatality rate 2-3-fold greater than typhoid fever and other clinical manifestations include osteomyelitis, meningitis and metastatic lesions. To investigate the events that lead to Salmonella invasion, we examined the interaction of this pathogen with MadinDarby canine kidney (MDCK) cells, a polarized epithelial cell line isolated from the kidney of a cocker spaniel (ATCC, 1981). When grown on permeable supports, these cells will form a polarized epithelial monolayer which is impermeable to ions, has a measurable transepithelial electrical resistance, and has several defined apical and basolateral surface markers (reviewed by Simons and Fuller, 1985). Physiologists have long used this system for studying epithelial transport © The Rockefeller University Press, 0021-9525/88/07/221/10 $2.00 The Journal of Cell Biology, Volume 107, July 1988 221-230 22l on July 9, 2017 jcb.rress. org D ow nladed fom while cell biologists have used it to study cell polarity development and maintenance, and transcytosis (Simons and Fuller, 1985). Several workers have infected polarized monolayers with viruses and demonstrated that viral infections and maturation can be a polar event (Fuller et al., 1984). Bacterial infection of polarized cells has not been reported, however. In the present study we characterized the interactions which occur between S. choleraesuis and polarized MDCK cell monolayers. This experimental system permitted us to measure the initial interactions which occur between bacteria and host ceils such as adherence and invasion, and led to the remarkable observation that this bacterium penetrated through viable MDCK monolayers. Materials and Methods