Manipulation of epithelial cell death pathways by Shigella

Shigella, a major cause of bacterial dysentery, knows when it is not wanted. To generate and maintain its niche within host cells, this unwelcome guest injects several dozen virulence factors via a type 3 secretion system (T3SS). In this issue, Ashida et al (2020) have elucidated the role of two such factors from Shigella flexneri—OspC1 and OspD3—that together counteract apoptotic and necroptotic death pathways in colonised epithelial cells. As a result, Shigella can replicate to high levels within the colonic epithelium, leading to the substantial epithelial damage in shigellosis and efficient bacterial release for faecal transmission.

New work uncovers secreted effectors of Shigella flexneri that prevent both apoptotic and necroptotic cell death, permitting pathogen replication in colonic epithelial cells.

Intestinal epithelial cell death is an important part of the innate immune response to bacterial invasion, allowing maintenance of an uncompromised barrier and elimination of the pathogen's intracellular niche. There are at least three means of regulated cell death in response to infection (Lacey & Miao, 2020). Pyroptosis, initiated by various inflammasome complexes, is a lytic inflammatory death resulting from caspase‐1/caspase‐4‐mediated generation of gasdermin D (GSDMD) pores. Apoptosis, mediated by activation of caspase‐8/caspase‐9, generates apoptotic bodies with maintained membrane integrity. Necroptosis results in phosphorylation and trimerisation of MLKL that causes membrane destabilisation and lytic death. Crosstalk between cell death pathways allows for backup responses to pathogens adapted to evade particular responses. In particular, loss of caspase‐8 in the apoptotic pathway leads to necroptotic cell death in the intestinal epithelial cells of mice (Gunther et al, 2011). In spite of these redundancies, bacterial pathogens exist that can seemingly subvert all forms of epithelial cell death. Intestinal epithelial cells infected with Shigella flexneri do not undergo programmed cell death. Prior work showed that during early infection of epithelial cells, Shigella uses the T3SS effector OspC3 to prevent caspase‐4‐dependent pyroptotic death (Kobayashi et al, 2013). Ashida et al (2020) have now demonstrated the T3SS‐dependent inhibition of the interconnected caspase‐8 and necroptotic pathways.

By screening mutant strains of S. flexneri that lack T3SS‐secreted effectors in HT29 human colon epithelial cells, Ashida et al (2020) identified OspD3 as an inhibitor of necroptosis. Further analysis showed that OspD3 is a protease that cleaves within the RHIM domain of essential necroptotic factors RIPK1 and RIPK3, targeting them for degradation. In searching for what triggered the necroptosis, the authors found that Shigella effector OspC1 can prevent caspase‐8 activity and apoptotic cell death. Typically, caspase‐8 drives apoptosis and inhibits necroptosis through disruption of MLKL activation by RIPK1 and RIPK3, possibly by cleaving RIPK1 (Newton et al, 2019). However, if caspase‐8 is blocked necroptosis proceeds, as interaction between RIPK1 and RIPK3 leads to the phosphorylation of RIPK3 and subsequent recruitment and phosphorylation of MLKL (Lacey & Miao, 2020) (Fig 1).

Figure 1. Shigella fights to maintain its replicative niche within epithelial cells by delivering T3SS effectors to inhibit three major programmed cell death pathways.

Early in the infection, OspC3 prevents caspase‐4‐dependent pyroptosis. In the later stages of infection, apoptosis is activated through an unknown mechanism, and OspC1 counters this, by preventing caspase‐8 activation. The host cell recognises this inhibition of caspase‐8 and triggers necroptosis as a backup death pathway, through RIPK1 and RIPK3 interaction and subsequent phosphorylation and activation of MLKL. The third effector, OspD3, blocks this pathway by cleaving RIPK1 and RIPK3, which targets them for degradation.

Knockout of ospC1 in Shigella lacking OspD3 inhibited the ability to induce necroptosis, and this was rescued by either ospC1 gene complementation or caspase‐8 inhibition. These results indicate that OspC1‐mediated inhibition of caspase‐8 can induce necroptosis. By time‐course analysis within the deletion mutants, Ashida et al beautifully demonstrated the successive activation of the three death pathways during Shigella infection. An OspC3 deletion showed that GSDMD cleavage was initiated prior to 2 h of infection. OspC1 and OspD3 deletions showed that caspase‐8 cleavage and phosphorylation of MLKL occur after 5 and 6 h postinfection, respectively. This indicates that during the course of infection, Shigella first uses OspC3 to prevent pyroptosis, followed by OspC1 to prevent apoptosis. The host cell can then recognise this blockade and trigger necroptosis, but this is inhibited by OspD3 (Fig 1).

The mechanisms of caspase‐8 activation and its inhibition by OspC1 during Shigella infection remain unknown. OspC1 is not related to known T3SS effectors from other pathogens (Ashida et al, 2020). Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) inhibit apoptosis through NleF that inhibits the activity of caspase‐8 and caspase‐3 and NleB that blocks recruitment of pro‐caspase‐8 by FADD as well as disrupting TNF‐induced necroptosis (Li et al, 2013; Pollock et al, 2017). While Shigella and E. coli apparently lack conserved effectors inhibiting apoptosis, Shigella OspD3 is a homologue of EPEC EspL, which also functions by degrading RHIM‐containing proteins, targeting the same C‐H‐D motif as OspD3 (Pearson et al, 2017). Activation of necroptosis must seemingly be combated universally by pathogens that attempt to create a niche by inhibition of caspase‐8. Similar battles have been observed between viral pathogens and host cells; herpes simplex virus (HSV) blocks caspase‐8 and this is sufficient to activate necroptosis, which the virus subsequently counteracts by inhibiting the interaction between RIPK1 and RIPK3 (Guo et al, 2015).

Shigella is thought to enter the lamina propria through M cells and then infect and kill macrophages prior to invasion of epithelial cells from the basolateral side (Ashida et al, 2015). Replication within the cytosol of epithelial cells and spread within the monolayer is a critical part of the pathogenesis and transmission of this organism. Early death of infected cells is a conservative and efficient host mechanism to eliminate the intracellular bacterial niche while flushing pathogens and debris out of the host. In normal homeostasis, intestinal crypt stem cells provide differentiated cells to replace most of the epithelium every 3–4 days, and minor infections can be coped with by extrusion of dying infected cells from the monolayer and coordinated division of stem cells to maintain the epithelial barrier. By injection of virulence factors such as OspC1, C3 and D3, Shigella delays the epithelial death long enough to permit replication to high levels, followed by invasion of neighbouring cells. With this delay, there is ultimately more catastrophic epithelial damage and inflammation, with release of large numbers of bacteria into faecal matter, facilitating transmission.

The EMBO Journal (2020) 39: e106202