Virulent Shigella flexneri Causes Damage to Mitochondria and Triggers Necrosis in Infected Human Monocyte-Derived Macrophages

Abstract

Shigella flexneri is a gram-negative bacterium that causes bacillary dysentery in humans that is characterized by an acute inflammatory response of the colon. The fate of phagocytes that are infected in vitro with virulent Shigella has been the subject of some investigation and debate. In this study we found that virulent Shigella caused a rapid increase in the cell membrane permeability of infected human monocyte-derived macrophages (HMDM) but not in the cell membrane permeability of monocytes, as demonstrated by the uptake of fluorescent vital dyes. Within 2 h of infection, 59% ؎ 6% of the HMDM and <4% of the monocytes were stained with propidium iodide. Treatment of the cells with the inhibitors of caspases YVAD and zVAD, the antioxidants N-acetyl-L-cysteine and butylated hydroxyanisole, or an inhibitor of NADPH oxidase, diphenyleniodonium, did not alter the infection outcome. Importantly, we found that virulent Shigella caused a rapid drop in the ATP level to about 50% in infected HMDM. Furthermore, using a combination of fluorescent vital dyes and mitochondrial membrane potential-sensitive dyes, we observed that cells that exhibited a permeable cell membrane were not stained by the mitochondrion-specific dyes, indicating that the mitochondrial membrane potential was lost in these cells. We also observed infected cells that were not stained with either type of dye, indicating that the loss of the mitochondrial membrane potential preceded the increase in cell membrane permeability. Taken together, our studies showed that virulent Shigella flexneri targets the host cell mitochondria for destruction. This activity may account for the necrotic cell death precipitated by these pathogens.Shigella flexneri is a gram-negative enteric pathogen that causes dysentery, an acute inflammatory disease of the colon. Worldwide, the number of Shigella cases exceeds 150 million per year (22). Young children and infants are particularly vulnerable to Shigella, and more than 65% of the casualties are in this age group. Despite the fact that Shigella was discovered almost a century ago (35), the mechanisms underlying Shigellainduced disease is still an ongoing area of research.Shigella enters the intestinal epithelium via specialized epithelial cells, known as M cells, that overlie lymphoid follicles in the colon (18). The M cells selectively bind and deliver pathogens to the resident macrophages, T and B cells that are present in the mucosal lymphoid layer directly beneath them (25,26). The response of the phagocytes to the bacterial challenge is therefore central to progression of the disease. The fate of phagocytes infected with virulent Shigella has been the subject of some investigation and debate. Rapid changes in the cell membrane permeability were noted within a couple of hours following infection of J774 cells, a murine macrophage cell line (5, 42). However, since the cells also displayed apoptotic features that included nucleus condensation and DNA fragmentation, it was concluded that Shigella triggered apoptosis in J77...

Shigella flexneri Induces Cell Death by Damage to and Autophagy of Host Cell Mitochondria: An Electron-Microscopic Study of Infected Human Monocyte Derived Macrophages

“…In necrotic cells infected for 2 hrs, the mitochondria were extremely swollen and some ruptured (Fig 3). Occasionally, the intracellular virulent bacteria were found in phagosomes surrounded by multilamellar membranes composed of ring-shaped mitochondria and ER (Fig 4).These results support our observation that Shigella kills HMDM by damaging their mitochondria [1]. Our data also suggest that the multilamellar structures, resembling autophagosomes [2, 3], were frequently derived from mitochondria damaged by virulent Shigella.…”

“…Virulent Shigella can escape the host phagocytes to replicate in epithelial cells, ultimately resulting in tissue destruction. In a recent study we reported that virulent Shigella induced a necrotic death in infected human monocyte-derived macrophages (HMDM), which was associated with a damage of the host-cell mitochondria [1]. The current study focused on the morphological alterations induced by Shigella in infected HMDM.…”

“…In a recent study we reported that virulent Shigella induced a necrotic death in infected human monocyte-derived macrophages (HMDM), which was associated with a damage of the host-cell mitochondria [1]. The current study focused on the morphological alterations induced by Shigella in infected HMDM.Monocytes were isolated from the peripheral blood of healthy donors and cultured for a minimum of 7 days before infection as described [1]. The cells were fixed and processed for conventional transmission electron microscopy after infection with virulent or avirulent bacteria.…”

“…Monocytes were isolated from the peripheral blood of healthy donors and cultured for a minimum of 7 days before infection as described [1]. The cells were fixed and processed for conventional transmission electron microscopy after infection with virulent or avirulent bacteria.…”

Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death

“…6B), (iii) anti-apoptotic proteins targeting the mitochondrial death pathway (Bcl-2, Bcl-X L ) do not protect macrophages from S. flexneri (Hilbi et al, 1998), and (iv) during S. flexneri-triggered caspase-1-dependent macrophage cell death, the prototypic effector caspase-3 of mitochondrial apoptosis is neither required (Hilbi et al, 1998) nor activated, as its substrate poly-ADPribose polymerase (PARP) is not cleaved (Chen et al, 1996). While mitochondria are not required to trigger S. flexneri-induced macrophage cell death, virulent S. flexneri causes damage to these organelles in infected human monocyte-derived macrophages (Koterski et al, 2005). Mitochondrial damage triggers the intrinsic apoptotic pathway, which thus in turn might contribute to S. flexneri-induced macrophage death.…”