The enteric bacterium and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide. Shigella is acquired by ingestion of contaminated food or water; upon reaching the colon, the bacteria invade colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. There is no animal model that faithfully recapitulates human disease; thus, cultured cells have been used to model Shigella pathogenesis. However, the use of transformed cells in culture does not provide the same environment to the bacteria as the normal human intestinal epithelium. Recent advances in tissue culture now enable the cultivation of human intestinal enteroids (HIEs), which are derived from human intestinal stem cells, grown ex vivo, and then differentiated into "mini-intestines." Here, we demonstrate that HIEs can be used to model Shigella pathogenesis. We show that Shigella flexneri invades polarized HIE monolayers preferentially via the basolateral surface. After S. flexneri invades HIE monolayers, S. flexneri replicates within HIE cells and forms actin tails. S. flexneri also increases the expression of HIE proinflammatory signals and the amino acid transporter SLC7A5. Finally, we demonstrate that disruption of HIE tight junctions enables S. flexneri invasion via the apical surface.
Shigella is an enteroinvasive human pathogen that infects the colonic epithelium and causes Shigellosis, an infectious diarrheal disease. There is no vaccine for the prevention or treatment of Shigellosis and antibiotic-resistant strains of Shigella are increasing, emphasizing the need for a deeper understanding of Shigella pathogenesis in order to design effective antimicrobial therapies. Small animal models do not recapitulate Shigellosis, therefore tissue-cultured cells have served as model systems to study Shigella pathogenesis. Here, protocols to enumerate Shigella invasion, cell-cell spread, and plaque formation in the tissue-cultured cell lines Henle-407 and CoN-841 are described. Additionally, a new method to study Shigella invasion in primary intestinal enteroids is described. These protocols can be used to examine different aspects of Shigella virulence. © 2018 by John Wiley & Sons, Inc.
Arthrobacter crystallopoietes growing exponentially as cocci were changed to rods by adding succinate to the medium. Cells were sampled before, during, and after this transition for Gram-staining and ultrastructural studies. Cells were Gram stained by the standardized method of Bartholomew, and all samples were fixed and prepared for thin sectioning in an identical manner. Cocci were gram positive, and thin sections demonstrated a gram-positive type of cell wall having an average thickness of 31 nm. Cells sampled during morphogenesis appeared as cocci with most having a single rodlike projection. The coccus portion of these transition cells was gram positive and bound by a gram-positive type of wall having an average thickness of 29 nm. The rodlike projection of the transition cells appeared to be gram negative; it was also surrounded by a gram-positive type of wall, but its average thickness was only 22 nm. Gram-negative rods of the type species, Arthrobacter globiformis, were also examined and found to produce a gram-positive type of wall with a 19-nm average thickness. Evidence for the trilaminar region, characteristic of most gram-negative bacterial cell walls, was totally lacking in both species. These results suggest that variations in cell wall thickness may be an important contributing factor to the variable Gram-staining characteristics of this genus.
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