Abstract: The bacterial flagellum is a whip-like structure that protrudes from the cell membrane and is one of the most complex and dynamic biological molecular machines that propels bacteria to swim toward beneficial environments and the sites of infection. It is composed of a basal body, a hook, and a long filament. The flagellar filament contains thousands of copies of the protein flagellin (FliC) monomer arranged helically and ending with a filament cap composed of oligomer protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases are even completely absent. Apart from its role in locomotion, the filament is critically important in several other aspects of bacterial survival, reproduction, and pathogenicity, such as adhesion to surfaces, secretion of effector molecules, penetration through tissue structures, and biofilms formation.
Bacterial flagellin is an important pathogen-associated molecular pattern (PAMP), which can activate both innate and adaptive immunity. Previous in vitro studies indicate that TLR5 is a major extracellular receptor for flagellin that mediates flagellin-induced production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-12, and tumor necrosis factor α (TNFα). Flagellin can also induce inflammasome activation through its intracellular receptor, the NLR family apoptosis inhibitory protein (NAIP) 5 and 6, leading to the generation of cytokines IL-1β and IL-18, as well as pyroptosis. Here, we found that inflammasome activation and subsequent pyroptosis, but not TLR5-mediated signal transduction, is responsible for flagellin-induced IL-6 and TNFα generation in vivo. Flagellin was fused to the cytosolic translocation domain of anthrax lethal factor (LFn) to enable efficient cytosolic delivery. LFn binds to anthrax protein protective agent (PA), which delivers the LFn-flagellin fusion protein into the cytoplasm through receptor-mediated endocytosis. Injection of LFn-flagellin with PA, but not LFn-flagellin alone by i. v., increased plasma concentrations of IL-1β, IL-6, and TNFα. LFn-flagellin/PA induced IL-1β, IL-6, and TNFα release was abolished in mice deficient in NAIPs, caspase-1, or GSDMD, but not TLR5. Depletion of monocytes and macrophages using clodronate inhibited LFn-flagellin/PA induced cytokine release. In addition, injection of the LFn fusion of another virulent factor, the T3SS rod protein EprJ from E. coli, together with PA also induced generation of IL-1β, IL-6, and TNFα in a caspase-1 and GSDMD dependent manner. Our data indicate that inflammasome activation leads to the generation of a broad range of inflammatory cytokines in vivo through pyroptosis, suggesting an important role of pyroptosis in cytokine storm.
Flagellin is a widespread bacterial virulence factor sensed by the membrane-bound Toll-like receptor 5 (TLR5) and by the intracellular NAIP5/NLRC4 inflammasome receptor. Bacterial flagellin is composed of highly conserved D0 and D1 domain, as well as hypervariable D2 and D3 domain. It has been reported that deletion of the D0 domain of flagellin completely abrogates the activation of TLR5. D0 domain of flagellin alone can bind NAIP5/6, leading to activation of the NLRC4 inflammasome, while whether the D1 domain of flagellin plays any functional role in NAIP5/NLRC4 inflammasome activation remains elusive. Besides, flagellins from S.typhimurium, Yersiniosis enterocolitica, and Pseudomonas aeruginosa can bind to NAIP5/6 and activate inflammasome NLRC4, those from enteropathogenic E. coli, enterohaemorrhagic E. coli, Shigella flexneri, and Burkholderia thailandensis cannot interact with NAIP5/6 and are unable to activate the inflammasome NLRC4. Replacement of the C-terminal D1 domain of flagellin from P. aeruginosa with the C-terminal D1 domain of E. coli flagellin diminished inflammasome activation. These data reveal that the D1 domain also plays an important role in flagellin-induced NAIP/NLRC4 inflammasome activation.
KEYWORDS: bacteria flagellin, virulence factors, PAMP, TLR5, NAIP/NLRC4 inflammasome activation, pyroptosis, cytokines