Escape of Actively Secreting Shigella flexneri from ATG8/LC3-Positive Vacuoles Formed during Cell-To-Cell Spread Is Facilitated by IcsB and VirA

Abstract

The enteropathogenic bacterium Shigella flexneri uses a type 3 secretion apparatus (T3SA) to transfer proteins dubbed translocators and effectors inside host cells, inducing bacterial uptake and subsequent lysis of the entry vacuole. Once in the cytoplasm, the outer membrane protein IcsA induces actin polymerization, enabling cytoplasmic movement and cell-to-cell spread of bacteria. During this infectious process, S. flexneri is targeted by ATG8/LC3. The effector IcsB was proposed to inhibit LC3 recruitment by masking a region of IcsA recognized by the autophagy pathway component ATG5. The effector VirA, a GTPase-activating protein (GAP) for Rab1, was also shown to prevent LC3 recruitment. However, the context of LC3 recruitment around S. flexneri is not fully understood. Here, we show that LC3 is recruited specifically around secreting bacteria that are still present in vacuoles formed during entry and cell-to-cell spread. While LC3 recruitment occurs around a small proportion of intracellular wild-type bacteria, the icsB, virA, and icsB virA mutants display incremental defaults in escape from LC3-positive vacuoles formed during cell-to-cell spread. Our results indicate that IcsB and VirA act synergistically to allow bacteria to escape from LC3-positive vacuoles by acting at or in the immediate vicinity of the vacuole membrane(s). We also demonstrate that LC3 is recruited around bacteria still present in the single-membrane entry vacuole, in a manner akin to that seen with LC3-associated phagocytosis. Our results indicate that LC3 recruitment occurs around bacteria still, or already, in membrane compartments formed during entry and cell-to-cell spread, and not around bacteria free in the cytoplasm.

How Do the Virulence Factors of Shigella Work Together to Cause Disease?

“…A single icsB − mutant is capable of escaping the entry vacuole, visualized by actin comet tail formation (Allaoui et al, 1992, Figure 8A). Therefore, the lack of escape from a single membrane entry vacuole by the double icsB − virA − mutant (Campbell-Valois et al, 2015, Figure 4) can be attributed to loss of VirA function. To confirm this, EM analysis of a single virA − mutant is required.…”

“…Electron microscopy (EM) 3 h after cell infection visualized icsB − mutants remaining trapped in a double membrane, with several bacteria in one vacuole (Allaoui et al, 1992). Galectin-3 had then been used to show that an icsB − mutant has only a 53% disruption of the double membrane vacuole, compared to 70% disruption mediated by the wild type (Campbell-Valois et al, 2015). However, on closer inspection, we think the EM images (Allaoui et al, 1992, Figure 8B) were misinterpreted, and the double membrane interpreted as the secondary entry vacuole was actually a starting autophagosome wrapping around the icsB − mutant unable to inhibit autophagy.…”

“…A virA − mutant leads to reduced Shigella intercellular persistence, but does not greatly reduce Golgi fragmentation, as IpaJ is more potent in fragmenting the Golgi (Figure 6) (Dong et al, 2012; Burnaevskiy et al, 2013). VirA, similarly to IcsB, has been implicated in the disruption of the secondary vacuole after intercellular spread following membrane protrusion formation (Figure 1, step 10) (Campbell-Valois et al, 2015). However, like for IcsB, this may have been misinterpreted.…”

“…In our view, the evidence indicates that VirA is involved in lysis of the single membrane entry vacuole (Figure 1, step 8). Indeed, Lysosomal Associated Membrane Protein 2 (LAMP2), a marker for lysosomal fusion with the entry vacuole, has been localized by confocal microscopy to entry vacuoles containing a single virA − mutant and a double icsB − virA − mutant (Campbell-Valois et al, 2015, Figures 2B, 3C) (Dong et al, 2012, Figure S3). A single icsB − mutant is capable of escaping the entry vacuole, visualized by actin comet tail formation (Allaoui et al, 1992, Figure 8A).…”

Bacterial Pathogens versus Autophagy: Implications for Therapeutic Interventions

“…Shigella flexneri avoids recognition by autophagy factors while it replicates in the cytosol of epithelial cells and fibroblasts by expressing the effector IcsB, which masks a region of the S. flexneri surface protein IcsA that is recognized by ATG5. The effector VirA, a GTPase-activating protein (GAP) for Rab1, also prevents LC3 recruitment to S. flexneri [32–35]. These shielding strategies allow these bacteria to replicate undetected by the autophagy machinery, thus subverting this type of immune defense.…”