Date Published: August 23, 2018
Publisher: Public Library of Science
Author(s): Shouwen Chen, Dahai Yang, Ying Wen, Zhiwei Jiang, Lingzhi Zhang, Jiatiao Jiang, Yaozhen Chen, Tianjian Hu, Qiyao Wang, Yuanxing Zhang, Qin Liu, Dana J. Philpott.
Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains undiscovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cell lines. Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion and gut inflammation in mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.
The host innate immune system can sense invading bacteria by detecting pathogen-associated molecular patterns (PAMPs) . Lipopolysaccharide (LPS), a component of the outer cell membrane of Gram-negative bacteria, is one of the strongest immune activators . Extracellular and endocytosed LPS is recognized by the transmembrane protein Toll-like receptor 4 (TLR4), leading to gene transcriptional regulation in response to infection . Recent studies showed that host can detect LPS in the cytosol via a second LPS receptor, caspase-11 in mice and caspase-4/5 in humans [4–6]. Caspase-11/4/5 directly binds cytosolic LPS , leading to its own activation, which thus cleaves gasdermin D to induce pyroptotic cell death and activate non-canonical activation of NLRP3 to release interleukin-1β (IL-1β) or IL-18 [7–8]. Therefore, compartmentalization of LPS receptors within cells allows host to respond differentially and sequentially to LPS at distinct subcellular locales, which function in concert to constitute host noncanonical inflammasome defenses.
Sensing of LPS in the cytosol by inflammatory caspase-11/4/5 has emerged as a central event of innate immune responses during Gram-negative bacterial infections [4–5,7]. Vanaja et al. reported that OMVs of extracellular Gram-negative bacteria can deliver LPS into the host cell cytosol from early endosomes; however, the mechanism of LPS translocation remains unclear. Biological membrane characteristics inherent to OMVs may permit them to fuse with endosomal membranes, leading to LPS cytosolic access . Recently, it was reported that mGBPs were involved in OMV-dependent non-canonical inflammasome activation [44–45]. Mechanistically, mGBPs didn’t promote the entry of OMVs into the cytosol, but directly target cytosolic OMVs and facilitate the interaction of LPS with caspase-11. However, GBPs are different in mice and human due to a loss of immunity-related GTPases (IRGs) in humans, and thus hGBP2 might not have the same action as mGBP2 . In addition to host factors, whether bacterial factors, such as OMV-associated bacterial components, are involved in promoting non-canonical inflammasome activation remains unknown. Here, we demonstrate that hemolysin binds OMVs and promotes the lysis of OMV-residing vesicles, which facilitates cytosolic release of OMV-LPS and eventually triggers significant non-canonical inflammasome signals (Fig 6). Our results suggest that hemolysin represents a biologically important mechanism for releasing endosome-constrained OMV-LPS to cytosolic sensors. In addition to hemolysin, whether other pore-forming proteins produced by bacteria  play roles in liberating pathogen-associated molecular patterns for immune detection requires further examination.