Shiga toxins induce autophagy leading to differential signalling pathways in toxin-sensitive and toxin-resistant human cells

Abstract

Summary The bacterial virulence factors Shiga toxins (Stxs) are expressed by Shigella dysenteriae serotype 1 and certain Escherichia coli strains. Stxs are protein synthesis inhibitors and induce apoptosis in many cell types. Stxs induce apoptosis via prolonged ER stress signaling to activate both extrinsic and intrinsic pathways in human myeloid cells. Studies have shown that autophagy, a lysosome-dependent catabolic process, may be associated with activation of pro-survival or death processes. It is currently unknown if autophagy contributes to apoptosis or protects cells from Stxs. To study cellular responses to Stxs, we intoxicated toxin-sensitive cells (THP-1 and HK-2 cells), and toxin-resistant cells (primary human monocyte-derived macrophages) and examined toxin intracellular trafficking and autophagosome formation. Stxs translocated to different cell compartments in toxin-resistant versus toxin-sensitive cells. Confocal microscopy revealed autophagosome formation in both toxin-resistant and toxin-sensitive cells. Proteolytic cleavage of Atg5 and Beclin-1 play pivotal roles in switching non-cytotoxic autophagy to cell death signaling. We detected cleaved forms of Atg5 and Beclin-1 in Stx-treated toxin-sensitive cells, while cleaved caspases, calpains, Atg5 and Beclin-1 were not detected in toxin-resistant primary human monocytes and macrophages. These findings suggest that toxin sensitivity correlates with caspase and calpain activation, leading to Atg5 and Beclin-1 cleavage.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1β and Apoptotic Cell Death

“…Extracts were collected and cleared by centrifugation at 15,000 ϫ g for 20 min. Cell lysates were prepared as previously described (18,33). Protein concentrations of each extract were determined using the DC protein assay (Bio-Rad, Hercules, CA).…”

“…Primary human monocytes were cultured in 6-well tissue culture plates (Thermo Fisher Scientific, Waltham, MA) in RPMI 1640 medium containing 10% human serum (Thermo Fisher Scientific). Culture of human monocyte-derived macrophages (hMDM) was performed as previously described (33). The medium was replaced every 3 days, and adherent cells were used for experiments within 11 days of plating.…”

“…Stxs trigger signaling pathways in a cell type-specific manner; consequently, Stx treatment leads to terminal executioner caspase-8/3 activation only in toxin-sensitive THP-1 or HK-2 cells, but not in toxinrefractory primary human monocyte-derived macrophages (hMDM) (33). To determine whether inflammasome activation is critical for the susceptibility of host cells to the cytotoxic activity of Stxs, we investigated the ability of toxins to produce active Casp-1 p20 and mature IL-1␤ in both toxin-sensitive (D-THP-1 and UD-THP-1 cells) and toxin-resistant cell types (hMDM and T84 epithelial cells).…”

Regulation of Cytokine and Chemokine Expression by the Ribotoxic Stress Response Elicited by Shiga Toxin Type 1 in Human Macrophage-Like THP-1 Cells

“…apoptosis or autophagy, and toxin B subunits lacking enzymatic activity have been shown to induce apoptosis in some cell types (16,31,37). Thus, the induction of programmed cell death may be dissociated from protein synthesis inhibition.…”

The role of autophagy in bacterial infections

“…Furthermore, it is known that the Shiga toxin induces autophagy in THP-1 cells and human macrophages and promotes death of kidney epithelial cells through a mechanism dependent on autophagy. In toxin-sensitive cells, toxins are translocated to the endoplasmic reticulum, and calpain, caspase-3 and caspase-8 are activated, resulting in the cleavage of ATG5 and beclin-1 (83). Moreover, it is known that Shigella encodes another effector protein, VirA.…”