The Zebrafish as a New Model for the In Vivo Study of Shigella flexneri Interaction with Phagocytes and Bacterial Autophagy

Abstract

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.

Sep(t)arate or not – how some cells take septin-independent routes through cytokinesis

“…More interestingly, intracytosolic Shigella is entrapped by cage-like septin structures and delivered to autophagosomes for degradation (Mostowy et al, 2010). Although these initial findings originate from epithelial cells, similar septin-cage formation has also been observed in phagocytic cells, suggesting a common role whereby septins deliver phagocytic cargo to the autophagy pathway (Gong et al, 2012;Mostowy et al, 2013). Moreover, septins colocalize with phagosomes and are crucial for FcγR-mediated phagocytosis in macrophages (Huang et al, 2008).…”

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore‐forming toxins

“…Cell culture media and supplements were from Lonza. Wild-type AB zebrafish were from the Zebrafish International Resource Center (Eugene), and the transgenic line Tg (mpeg1:G/UmCherry) was previously described [52,53]. Embryos were raised at 28°C in E3 medium with 0.003% 1-phenyl-2-thiourea (Sigma) to prevent pigmentation for microscopy.…”

“…For injection of zebrafish larvae, washed bacteria were resuspended in phosphate-buffered saline (PBS; Lonza). Anaesthetized zebrafish larvae, 3 days post-fertilization (dpf), were microinjected in the hindbrain ventricle or tail muscle with 0.5-2 nl of bacterial suspension as described [52]. Real inoculum was verified by plating at least three independent larvae onto BHI agar.…”

Deciphering and Imaging Pathogenesis and Cording of <em>Mycobacterium abscessus</em> in Zebrafish Embryos

“…The increasing success of this alternative vertebrate model relies on major and unique opportunities that motivated and validated its use for a better understanding of numerous viral and bacterial infections 19,22,23,24,25,26,27,28,29 . As opposed to most other animal models, zebrafish embryos are optically transparent, allowing non-invasive fluorescence imaging 30 .…”