Structure-Function Analysis of the Shigella Virulence Factor IpaB

Abstract

Infection by the gram-negative bacterium Shigella flexneri results in dysentery, an acute inflammatory disease of the colon. Essential events in the pathogenesis of Shigella infections include bacterial invasion of epithelial cells, escape from the phagosome, and induction of apoptosis in macrophages. The Shigella virulence factor invasion plasmid antigen B (IpaB) is required for all of these processes. Induction of apoptosis is dependent on IpaB binding to the cysteine protease caspase-1 (Casp-1). The activation of this enzyme triggers both apoptosis and release of the proinflammatory cytokine interleukin-1␤. Several IpaB mutants were generated to correlate function with protein subdomains. We determined that the N-terminal portion of IpaB is necessary for stable expression of IpaB. A putative amphipathic ␣-helical domain preserves the structure of IpaB. We found 10 consecutive residues within the amino terminus of the hydrophobic region that play a critical role in invasion, phagosomal escape, and cytotoxicity. An IpaB mutant carrying a mutation in this region binds to Casp-1 yet is not cytotoxic, even following direct delivery to the macrophage cytoplasm. These results indicate that the association between IpaB and Casp-1 is only a step in the activation of macrophage apoptosis.Infections with enterobacteria of the genus Shigella cause dysentery, a severe bloody diarrhea. Dysentery is characterized by an acute inflammation of the colon with mucosal erosion (11, 23). Development of shigellosis requires bacterial penetration across the intestinal epithelial barrier via M cells. Upon reaching the underlying lymphoid follicles, the bacteria are engulfed by resident macrophages (30,38). Once inside a macrophage, Shigella escapes from the phagosome into the cytoplasm and kills this cell by inducing apoptosis (42). The dying macrophage releases mature interleukin-1␤ (IL-1␤) (40) and IL-18 (32), two cytokines important in the initiation of inflammation (34). Shigella also invades epithelial cells through pathogen-directed endocytosis (23). Invasion of enterocytes and bacterial cell-to-cell spread enhance tissue damage (20,33).The Shigella invasion plasmid antigens B (IpaB), IpaC, and IpaD are required for epithelial cell entry and phagosome escape (14, 23). However, IpaB alone is sufficient to activate macrophage apoptosis (5). The Ipa proteins interact with host cells upon being secreted by a type III secretion apparatus (26). In the macrophage cytoplasm, IpaB binds to caspase-1 (Casp-1; also called IL-1␤ converting enzyme [ICE]), a proapoptotic and proinflammatory cysteine protease that cleaves IL-1␤ and IL-18 to their biologically active forms (6, 37). The activation of Casp-1 leads to macrophage apoptosis by an as yet ill-defined pathway (15, 40). Casp-1-deficient macrophages' resistance to Shigella-induced cell death demonstrates that Casp-1 is essential for this process (16).IpaB contains a hydrophobic region (amino acids [aa] 310 to 430) that contains two putative membrane-spanning domains (aa 313 to 346 and 4...

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

“…This localization and architecture of IpaB aggregates might be relevant for the activation of caspase-1 by IpaB, which is a hallmark of S. flexneri-triggered macrophage death. Previously, it has been shown that in vitro IpaB directly binds caspase-1 (Chen et al, 1996;Hilbi et al, 1997Hilbi et al, , 1998Guichon et al, 2001) and in infected macrophages partially co-localizes with activated caspase-1 on the bacterial surface or host-cell membranes, and in the cytoplasm (Schroeder & Hilbi, 2006).…”

“…This localization and architecture of IpaB aggregates might be relevant for the activation of caspase-1 by IpaB, which is a hallmark of S. flexneri-triggered macrophage death. Previously, it has been shown that in vitro IpaB directly binds caspase-1 (Chen et al, 1996;Hilbi et al, 1997Hilbi et al, , 1998Guichon et al, 2001) and in infected macrophages partially co-localizes with activated caspase-1 on the bacterial surface or host-cell membranes, and in the cytoplasm (Schroeder & Hilbi, 2006).The protonophore CCCP efficiently blocked type III secretion of S. flexneri and cell death in infected macrophages, even if added post-infection (Figs 2B, C, 3A, B), suggesting that after escape of S. flexneri from the phagosome, prolonged type III secretion in the macrophage cytoplasm is required for maximum induction of cell death. In line with these findings, the Mxi-Spa T3SS is functionally required after initial invasion of epithelial cells, and effector proteins are present in the bacterial cytoplasm prior to and after uptake by these cells (Ménard et al, 1994a;Page et al, 1999;Rathman et al, 2000;Schuch et al, 1999).…”

“…Subsequently, S. flexneri escapes the phagosome and, dependent on IpaB, triggers a pro-inflammatory type of apoptosis in macrophages (Zychlinsky et al, 1994b). IpaB is sufficient to induce apoptosis, co-localizes with caspase-1 [interleukin (IL)-1b converting enzyme] in the macrophage cytoplasm and directly binds caspase-1, but not caspase-2 or -3 (Chen et al, 1996;Guichon et al, 2001;Hilbi et al, 1998;Thirumalai et al, 1997). The apoptotic pathway is characterized by the requirement and proteolytic activation of caspase-1 but not other family members such as caspase-3 or caspase-11 (Chen et al, 1996;Hilbi et al, 1997Hilbi et al, , 1998.…”

Clostridium difficile Toxin B Causes Apoptosis in Epithelial Cells by Thrilling Mitochondria

“…As an example, the IpaB protein of Shigella spp. is implicated in the internalization of the bacterium into the host intestinal cell but also possesses the ability to trigger apoptosis in macrophages by activating caspase 1 (63).…”

O papel da flagelina e do sistema de secreção de Escherichia coli enteroinvasora na resposta imune inata dos macrófagos

“…A caspase-1 é produzida como um zimogênio, denominada pro-caspase-1, que é clivada em 2 subunidades -uma com 20 kDa (p20) e outra com 10 kDa ( Estudos vêm demonstrando o envolvimento da ativação de caspase-1 dos macrófagos no controle das infecções. Na infecção por Shigella, foi demonstrado que a caspase-1 nos macrófagos desempenha um duplo papel: a clivagem das citocinas pró-IL-1β e pró-IL-18 em suas formas ativas (HILBI et al, 1998;GUICHON et al, 2001) e a indução da morte celular por piroptose (SUZUKI et al, 2007). A morte celular dependente de caspase-1, denominada piroptose, é caracterizada por induzir uma intensa resposta inflamatória, crucial para controlar as infecções microbianas (BORTOLUCI & MEDZHITOV, 2010;MIAO et al, 2010a).…”