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.

Figure 1. Characterization of zebrafish cells and infection with Shigella.

A. Western blots of larval extracts, using antibodies against SEPT7 or elongation factor 1-α (EF1α). See Fig. S1B for complete lane of SEPT7 blot and antibody specificity. B. Immunofluoresence microscopy of ZF-AB cells. F-actin (green), SEPT7 (red), DAPI (blue). Top: uninfected cell; note colocalization of actin and SEPT7 filaments (arrows). Bottom: Shigella-infected cell; note septin cage-like structure surrounding DAPI-labeled Shigella (arrow). Scale bar, 4 µm. C. Scheme of a 72 hpf larva (length ∼3.5 mm), showing the duct of Cuvier (the wide vessel flowing over the yolk; black arrowhead), also called common cardinal vein, and the iv injection site (red arrowhead) in the tail vein just caudal to the urogenital opening (ugo). D. Survival curves of 72 hpf larvae injected with various doses of S. flexneri and incubated at 28°C for 48 hpi. The effective inoculum, quantified a posteriori, was classified as low (<10³ CFU, open circles; effective range: 1.5–9.9×10² CFU), medium (∼4×10³ CFU, open triangles; effective range: 1.3–6.0×10³ CFU) or high (∼10⁴ CFU, open squares; effective range: 7.8–34.2×10³ CFU). Mean±SEM of n = 10–24 larvae per group from 3 or more independent experiments per inoculum class. E. Enumeration of live bacteria in homogenates from individual larvae at various times post infection measured by plating onto LB. Low dose inoculum = open circles. Medium or high dose inoculum = open squares. Of note, dead larvae at 24 or 48 hpi (overwhelmed with bacteria, as determined by fluorescence stereomicroscopy) were not included here, and only larvae having survived the infection (thus far) were included here (and in this case, statistics are in grey). Mean±SEM also shown (horizontal bars). Significance testing performed by Student's t test. ns, P>0.05; ***, P<0.001. F. Distribution of bacteria (GFP-Shigella) determined by live imaging using a fluorescence stereomicroscope at various times post injection of a low, medium or high dose inoculum. Overlay of transmission image (grey) and GFP fluorescence (green).

Figure 2. Macrophage and neutrophil depletion upon Shigella infection.

A. mpeg1:G/U:mCherry (red macrophages) and lyz:dsRed (red neutrophils) 3 dpf larvae were infected with sublethal or lethal GFP-Shigella inocula, fixed 4 and 24 hpi and leukocyte and bacteria labeled using anti-dsRed (red) and anti-GFP (green) antibodies. 4 and 24 hpi uninfected (CTRL), sublethal and lethal inoculated mpeg1:G/U:mCherry and lyz:dsRed fish are shown. Fixed, labeled whole larvae were imaged using a fluorescent stereomicroscope. Overlay of green and red fluorescence. B. Macrophage (left) and neutrophil (right) counts in uninfected (control, C) or upon sublethal (S) or lethal (L) Shigella injections. Macrophages and neutrophils were counted from images using ImageJ and plotted as specified in Material and Methods. Mean±SEM also shown (horizontal bars). Significance testing performed by ANOVA with Bonferroni posttest. ns, P>0.05; ***, P<0.001.

Figure 3. In vivo Shigella-phagocyte interactions.

A–E: Frames extracted from in vivo time-lapse confocal imaging sessions of 3 dpf larvae injected in the bloodstream and in the adjacent mesenchyme with sublethal inocula of GFP-Shigella. Caudal area, rostral to bottom right, dorsal to top right. Overlay of green (Shigella) and red (macrophages or neutrophils) fluorescence; transmission image (grey) is also overlaid on the first frame in A, B and E as an anatomical guide. Scale bars: A–B, 50 µm; C–E, 10 µm. A. mpeg1:G/U:mCherry larva (red macrophages); first frame at 20 mpi. By 20 mpi, Shigella have already adhered to or have been engulfed by macrophages (red cells, white arrows). They persist in macrophages over time (last frame at 2h30pi). Note that neutrophils (highlighted in green by the engulfed GFP-Shigella, #1,2,3 yellow arrows), that have engulfed bacteria in the mesenchyme have a similar bacterial load to that of macrophages, and efficiently kill the engulfed bacteria (as indicated by the diffuse GFP staining over time in these neutrophils). Maximum intensity projection from 25 planes every 2 µm. See also Video S1. B. lyz:dsRed larva (red neutrophils), first frame at 20 mpi. Neutrophils are rapidly recruited to engulf and degrade GFP-Shigella in the mesenchyme. By contrast, note the macrophage that remains strongly decorated by GFP-Shigella over time (white arrow), highlighting bacterial persistence inside macrophages. Maximum intensity projection from 25 planes every 2 µm. See also Video S4. C. mpeg1:G/U:mCherry larva, first frame at 20 mpi. A red macrophage harboring GFP-Shigella over time (white arrow) eventually undergoes cell death (loss of red label). Note in the bottom right corner of the field a group of neutrophils that have engulfed the bacteria in the mesenchyme and appear engorged by them, is able to control the engulfed GFP-Shigella over time (diffuse and decreasing in intensity GFP signal). Maximum intensity projection of 6 planes every 2 µm. See also Video S5. D. lyz:dsRed larva, first frame at 4h30pi. Neutrophils collect dying infected macrophages. GFP-Shigella proliferate inside a macrophage (white arrow), which then undergoes cell death, and is quickly engulfed by dsRed+ neutrophils (yellow arrows). Single confocal plane. See also Video S6. E. lyz:dsRed larva, first frame at 7 hpi. Shigella are able to invade other cell types and replicate in the cytoplasm, causing their death, and subsequent engulfment by neutrophils. The white line delineates a muscle fiber invaded by GFP-Shigella. Shortly before 6 h of time-lapse, the muscle cell bursts and is engulfed by red neutrophils (yellow arrows). Maximum intensity projection of 4 planes every 2 µm. See also Video S7.

Figure 4. Shigella escape to the cytosol and induce septin caging and autophagy in zebrafish larvae in vivo.

A. Zebrafish larvae were infected in the tail muscle with GFP-Shigella for 4 (medium dose) or 24 h (low dose), fixed, labeled with antibodies against SEPT7 (red) and to GFP (green) and imaged by confocal microscopy. Scale bar, 5 µm. B. GFP-Lc3 zebrafish larvae were infected with mCherry-Shigella for 4 (medium dose), fixed, labeled with antibodies against mCherry (red) and to GFP (green) and imaged by confocal microscopy. Scale bar, 5 µm. Shown here is an example of a GFP-Lc3 positive cell controlling bacterial replication (better than its neighbouring cells) 4 hpi. See also Video S8 for live imaging observations of GFP-Lc3 recruitment to Shigella. C. Cytosolic Shigella are sequestered in autophagosomes in vivo. Zebrafish larvae were infected in the tail muscle with GFP-Shigella for 4 h (medium dose) and fixed for electron microscopy. C2 is an expanded region of boxed region in C1 (i.e., autophagosome sequestering bacteria), C3 is an expanded region of boxed region in C2 (i.e., double membrane, a hallmark of autophagosomes). Scale bar, 1 µm (C1) or 0.25 µm (C2). See also Video S9 for a tilt series of Shigella autophagosomes in vivo.

Figure 5. In vivo perturbation of bacterial autophagy via p62 depletion.

A. Western blots of extracts of larvae injected with control (CTRL) or p62 morpholinos (mo), using antibodies against elongation factor 1-α (EF1α; control) or p62. Representative blots using lysates from AB fish; p62 depletion has also been observed in p62 morpholino-treated mpeg:G/U:mCherry and lyz:dsRed fish (data not shown). B. Survival curves of zebrafish larvae treated with control (CTRL) or p62 morpholinos and infected with a sublethal dose of S. flexneri (+M90T) or not infected (no bact). Representative experiment of at least 3 independent ones; n = 12 or more larvae per group. Significance testing performed by Log Rank test. **, P = 0.01. C. Bacterial counts in zebrafish larvae treated with control (CTRL) or p62 morpholinos and infected with a sublethal dose of S. flexneri. Enumeration of live bacteria in homogenates from individual larvae at various times postinfection measured by plating onto LB. CTRL larvae = open circles. p62-depleted larvae = open squares. One representative experiment of at least 3 independent ones; 3 larvae per condition. Of note, dead larvae at 24 or 48 hpi (overwhelmed with bacteria, as determined by fluorescence stereomicroscopy) were not included here, and only larvae having survived the infection (thus far) were included here (and in this case, statistics are in grey). Mean±SEM also shown (horizontal bars). Significance testing performed by Student's t test. ns, P>0.05; **, P<0.01; ***, P<0.001. D. Leukocyte behaviour and infection progression upon Shigella inoculation in p62 knockdown larvae over time. lyz:dsRed p62-depleted 3 dpf larva inoculated with GFP-Shigella and live imaged from 20 mpi to 40 hpi. Maximum intensity projection of 35 sections spaced by 2 µm. Frames extracted at various time points (20 mpi, 5h30mpi, 16 hpi, 24 hpi, 32 hpi and 40 hpi) suggest that neutrophils are unable to restrict bacterial proliferation. The infected larva is progressively depleted in neutrophils and overwhelmed with Shigella. Scale bar, 50 µm. E. Zebrafish larvae treated with either control (CTRL; left image) or p62 (right image) morpholinos were infected with GFP-Shigella for 4 (medium dose), fixed, labeled with antibodies against SEPT7 (red) and to GFP (green), and imaged by confocal microscopy. Arrows highlight some examples of Shigella entrapped in septin cages (CTRL) or not (p62 depleted) a 4 hpi. Scale bar, 5 µm.

Figure 6. In vivo perturbation of autophagy via rapamycin stimulation.

A. Survival curves of zebrafish larvae treated with control (CTRL) or rapamycin (RAPA) and infected with a sublethal dose of S. flexneri (+M90T) or not infected (no bact). Representative experiment of at least 3 independent ones; n = 10 or more larvae per group. Significance testing performed by Log Rank test. ***, P = 0.001. B. Bacterial counts in zebrafish larvae treated with control (CTRL) or rapamycin (RAPA) and infected with a sublethal dose of S. flexneri. Enumeration of live bacteria in homogenates from individual larvae at various times postinfection measured by plating onto LB. CTRL larvae = open circles. RAPA-treated larvae = open squares. n = 3 larvae per treatment, pooling of 3 independent experiments. Of note, dead larvae at 24 or 48 hpi (overwhelmed with bacteria, as determined by fluorescence stereomicroscopy) were not included here, and only larvae having survived the infection (thus far) were included here (and in this case, statistics are in grey). Mean±SEM also shown (horizontal bars). Significance testing performed by Student's t test. ns, P>0.05; ***, P<0.001. C. Inflammatory response is not affected in rapamycin-treated larvae. Expression of il1b transcripts at 96 hpf in larvae injected with ∼10³ S. flexneri at 72 hpf (+M90T) or in uninfected controls, treated after infection with rapamycin (RAPA) or vehicle only (DMSO, i.e., CTRL). Il1b/ef1α transcript number ratios, normalized to the mean value in uninfected controls. Results for 5 pools of 3 larvae per group, mean±SEM also shown (horizontal bars).