Different injection sites of zebrafish larvae used to study Shigella infection. Main attributes of different injection sites used for the study of Shigella infection of zebrafish larvae (3 days postfertilisation). To study systemic infection and Shigella-phagocyte interactions, intravenous injection of Shigella into the circulation is performed via the caudal vein/posterior blood island or Duct of Cuvier (highlighted in red). To study compartmentalised infection and a directed leukocyte response to Shigella, injection of Shigella into the hindbrain ventricle, or subcutaneous/intramuscular injection of Shigella into epithelial cells of the tail muscle, is used (highlighted in green). The dashed line boxes indicate the aorta-gonad-mesonephros (AGM) and caudal hematopoietic tissue (CHT) where emergency granulopoiesis (see Glossary, Box 1) takes place.

The hallmarks ofShigellainfection. (A) Trigger-mediated entry by Shigella. HeLa cells were infected with Shigella (blue), fixed for fluorescent microscopy, and labelled with antibodies to SEPT9 (red) and phalloidin for F-actin (green) to highlight septin recruitment at the site of Shigella entry. Scale bar: 1 µm. (B) The Shigella actin tail. HeLa cells were infected with Shigella (blue; white arrowhead indicates a motile bacterium) for 3 h, fixed for fluorescent microscopy, and labelled with antibodies to SEPT2 (red) and phalloidin for F-actin (green) to highlight septin ring formation around the actin tails. Scale bar: 1 µm. (C) The Shigella-septin cage in vivo. SEPT7 (red) assembles into cage-like structures around S. flexneri (green). Zebrafish larvae were infected with green fluorescent protein (GFP)-Shigella for 4 h, fixed, labelled with antibodies to SEPT7 and imaged by confocal microscopy. The inset shows a higher magnification view of the boxed region in C, showing Shigella entrapped within a septin cage. Scale bar: 5 µm. (D) An autophagosome sequestering cytosolic Shigella in vivo. Zebrafish larvae were infected in the tail muscle with GFP-Shigella for 4 h and fixed for electron microscopy. The inset shows a higher magnification view of the boxed region in D, showing the double membrane, a hallmark of autophagosomes. Scale bar: 0.25 µm. Images adapted from Mostowy and Cossart (2009) (A), Mostowy et al. (2010) (B) and Mostowy et al. (2013) (C,D).

Interaction of the predatory bacteria Bdellovibrio with Shigella in the zebrafish. (A) Wild-type zebrafish larvae were injected in the hindbrain ventricle (HBV) at 3 days postfertilisation with >5×10³ colony forming units (CFUs) of GFP-S. flexneri (green), followed by hindbrain injection of either PBS or 1-2×10⁵ plaque forming units (PFUs) of mCherry-Bdellovibrio (red), 30-90 min after the initial Shigella infection. Representative images of the HBV in PBS- or Bdellovibrio-treated zebrafish larvae infected with Shigella are shown. The dashed line box shows the region of interaction between fluorescent Bdellovibrio and Shigella. For both treatments, the same larva was imaged over time. Scale bar: 100 µm. hpi, hours postinfection. (B) Representative images of Shigella predation by Bdellovibrio in vivo imaged by high-resolution confocal microscopy. Frames captured over time show stages of Bdellovibrio (red) invasive predation and rounding of Shigella (green). Scale bar: 2.5 µm. mpi, minutes postinfection. Adapted from Willis et al. (2016).