| Abstract: | When nucleotide-binding oligomerization domain–like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN+/− mice were more responsive to inflammasome activation than those from GLMN+/+ mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN.Inflammasome activation is a key defense mechanism against bacterial infection that induces innate immune responses such as caspase-1 activation and inflammatory cell death (1–3). Although the mechanisms through which various bacterial activities promote infection remain incompletely understood, some bacterial pathogens stimulate inflammasome activity by delivering cytotoxins, type III secretion (T3SS)-mediated effectors, T3SS components, flagellin, or cytotoxins to the host cell membrane and cytoplasm. These foreign components modify the host cell-surface architecture, induce membrane damage, subvert cell signaling, reorganize the actin cytoskeleton, and alter cell physiology (4) through interactions with various cytoplasmic receptors, e.g., nucleotide-binding oligomerization domain–like receptors (NLRs)—including NLRP1, NLR family CARD domain-containing 4 (NLRC4), NLR family pyrin domain-containing 3 (NLRP3), AIM2, IFI16, and RIG-1—as pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) (2, 3, 5). Upon recognition of these PAMPs and DAMPs, NLRs induce the assembly of inflammasomes, which are composed of NLR, apoptosis-associated speck-like protein (ASC), and inflammatory caspases such as caspase-1. Inflammasome assembly ultimately results in the extracellular release of IL-1β and IL-18 and induces inflammatory cell death (called “pyroptosis”) (6). For example, NLRP3 senses membrane rupture that occurs during infection with Listeria monocytogenes, Shigella, Salmonella typhimurium, Staphylococcus aureus, Neisseria gonorrhoeae, and Chlamydia spp. and upon exposure to bacterial pore-forming toxins, leading to caspase-1 activation (7–10). NLRC4 detects L. monocytogenes and S. typhimurium infection and stimulates caspase-1 activation (11–14). NLRC4 also senses flagellin and the T3SS rod components of Legionella pneumophila, Pseudomonas aeruginosa, Shigella, and S. typhimurium (11, 15–20) and the T3SS needle components of Chromobacterium violaceum, S. typhimurium, enterohemorrhagic Escherichia coli, Burkholderia thailandensis, and Shigella (21). Therefore, NLR inflammasomes act as major cytoplasmic pattern-recognition receptors and as central platforms that transmit alarm signals to a variety of downstream innate immune systems.Some bacterial pathogens, such as S. typhimurium (22) and Yersinia pseudotuberculosis (23–25), can induce macrophage death after they have fully replicated, promoting the egress of bacteria from their replicative compartments and the subsequent dissemination of bacteria into new host cells. This causal relationship suggests that these pathogens may benefit from and exert control over host cell death and the inflammatory response. In the case of Shigella, the bacteria rapidly induce macrophage cell death at early stages of infection, which is accompanied by NLR inflammasome activation and inflammatory cell death through a T3SS-dependent mechanism (19, 22). Previous studies indicated that during replication in macrophages, LPS, peptidoglycan, and T3SS rod or needle components of Shigella are recognized by the NLRC4 and NLRP3 inflammasomes (8, 19–21). Interestingly, the mode through which NLRs recognize Shigella infections seems to vary across different infection conditions. At a low infectious dose e.g., a multiplicity of infection (MOI) of 10–25], bacteria induce rapid NLRC4–caspase-1–dependent pyroptosis at 2–3 h postinfection through the recognition of the T3SS components or some uncharacterized T3SS-delivered substance(s) (19, 22). However, at a high infectious dose (e.g., an MOI over 50) and at later time points (6 h postinfection), the bacteria induce NLRP3-dependent but caspase-1–independent necrosis-like cell death with inflammation (called “pyronecrosis”) (8). Because pyroptosis results in the release of intracellular contents, including proinflammatory cytokines and chemokines, and because, in the case of Shigella, macrophage death is a prerequisite for the subsequent infection of surrounding epithelial cells (19, 26), it remains unclear whether Shigella-mediated rapid cell death is beneficial to the pathogen or to the host. Nevertheless, these studies strongly suggest that the bacteria deploy multiple mechanisms to manipulate macrophage cell-death pathways in a T3SS-dependent manner.Shigella flexneri, e.g., the YSH6000 strain, possesses three invasion plasmid antigen H (ipaH) genes, ipaH9.8, ipaH7.8, and ipaH4.5, on a large virulence plasmid (27, 28). These IpaH proteins, which originally were identified in the S. flexneri M90T strain (29, 30), recently were found to act as enzyme 3 (E3) ubiquitin ligases (31) and were thus named “novel E3 ligases” (32). The ipaH cognate genes are distributed among various Gram-negative bacterial pathogens, including Shigella, Salmonella, Yersinia, Edwardsiella ictaluri, Bradyrhizobium japonica, Rhizobium sp. strain NGR234, Pseudomonas putida, Pseudomonas entomophila, Pseudomonas fluorescens, and Pseudomonas syringae (31). IpaH protein family members share structural and functional similarity; they are composed of an N-terminal leucine-rich repeat (LRR) and a highly conserved C-terminal region (CTR) (33, 34). The conserved CTR contains a Cys residue, which is critical for E3 ubiquitin ligase activity (31, 35, 36). Each of the IpaH family effectors characterized to date (e.g., Shigella IpaH9.8 and IpaH2077, Salmonella SlrP, SspH1, and SspH2, Yersinia YopM, and Rhizobium Y4fR and BIpM) has distinct host protein targets in different host cell types. Some act as effectors to attenuate host inflammatory responses, whereas others modulate host defense responses in plants (37, 38). The existence of multiple effectors with E3 ligase activity suggests that an array of E3 ligases is required to promote bacterial infection and antagonize host innate defense responses.Fernandez-Prada et al. (39) previously reported that Shigella lacking the ipaH7.8 gene are less capable than the WT strain of escaping the phagocytic vacuole of macrophages and that Shigella infection of macrophages induces apoptotic-like cell death. Paetzold et al. (40) subsequently showed that Shigella lacking the ipaH7.8 gene had no effect on phagosome escape compared with the WT strain, but bacterial-induced cytotoxicity was low compared with that of the WT strain. Although the biological significance of IpaH7.8 as an E3 ubiquitin ligase during Shigella infection remains to be elucidated, these studies suggested that IpaH7.8 is involved in inducing macrophage cell death.In this context we wished to clarify the pathological role of IpaH7.8 as an E3 ubiquitin ligase in Shigella infection of macrophages and the modality of cell death. Here we provide evidence that IpaH7.8 potentiates macrophage killing in an IpaH7.8 E3 ligase-dependent manner, in which E3 ligase activity triggers NLR inflammasome-mediated macrophage cell death by targeting glomulin/FAP68 (GLMN); this activity ultimately appears to benefit the pathogen. |
