Work in the SM laboratory is supported by a Wellcome Trust Research Career Development Fellowship [WT097411MA].

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The authors declare that they have no competing financial interests.

When monitoring autophagy and the cytoskeleton in vitro using tissue culture cells, the protocols described in sections 1 and 2 can be applied to a wide variety of tissue culture cell types. Moreover, to follow autophagy (e.g., ATG8/LC3+ve autophagosomes) and cytoskeleton (e.g., actin tails, septin cages) dynamics in real-time during Shigella infection using live imaging, tissue culture cells can be transiently transfected using GFP-, RFP- or CFP-tagged constructs as previously described7,8. To increase the percentage of cells infected by Shigella (i.e., generally desirable for real-time analysis considering that Shigella can invade 5-30% of HeLa cells at 100:1 MOI), directly add 400 &#181;l of Shigella (OD600 = 0.3-0.6) to cells in 2 ml MEM (serum-starved) and wait at least 1.5 hr post infection for sufficient bacterial entry, escape from the phagosome, replication, autophagy recognition and septin caging. Alternatively, one may use the Shigella M90T AfaI strain that expresses the adhesin AfaE and have much higher invasion abilities in epithelial cells compared to the M90T strain28. Of note, the M90T AfaI strain has not yet been tested in vivo using zebrafish. Plates of Shigella colonies can be kept at 4 &#176;C for 2-3 days and used for several experiments. However, over time, colonies of Shigella that have lost the virulence plasmid can also absorb the Congo Red and appear to have retained their virulence plasmid. For this reason we recommend to use fresh bacterial stocks when possible.
When monitoring the cell biology of infection in vivo, protocols described in sections 3 and 4 use wildtype AB line zebrafish. To monitor Shigella-leukocyte interactions, transgenic zebrafish lines can be used, e.g., mpx:GFP or lyz:DsRed to visualize neutrophils19,29,30 or mpeg1:mcherry to visualize macrophages19,31. To visualize autophagy in vivo, the GFP-Lc3 zebrafish transgenic line19,24 can be used as described in section 3.8.
To perturb autophagy in vivo, the effective morpholino oligonucleotide dose has to be assessed experimentally based on its efficiency to inhibit transcript splicing or protein translation. It is advisable to perform a titration experiment and to confirm the depletion by RT-PCR (for splice morpholino oligonucleotide) or by SDS-PAGE (for translational morpholino oligonucleotide)32. RNA isolation from zebrafish embryos or larvae can be performed using guanidinium thiocyanate-phenol-chloroform extraction. To extract protein from zebrafish larvae (8 to 15 larvae/tube), mechanically homogenize using a pestle in 200 &#181;l lysis buffer (1 M Tris, 5 M NaCl, 0.5 M EDTA, 0.01% octylphenol ethylene oxide condensate, and protease inhibitor). Centrifuge tubes at 19,000 x g at 4 &#176; C for 15 min and transfer the supernatant to a new tube. Add Laemmli buffer and heat the sample at 95 &#176; C for 15 min. Lysates can be stored at -80 &#176; C until needed, and can be evaluated by Western blotting as described in section 2.3.
The zebrafish is an excellent model for in vivo drug application. Analysis using morpholino oligonucleotides can be complemented with established drugs to manipulate autophagy (e.g., rapamycin and bafilomycin). Uninfected and/or infected larvae can be treated with rapamycin (1.5 &#181;M) or bafilomycin (80 nM) diluted in E2 and autophagic flux can be evaluated by Western blotting as described in 25,33. The consequence of autophagy manipulation on the outcome of the infection and survival of the infected larvae can be evaluated as described in section 3.5.
In addition to studying host cell determinants, in vitro and in vivo protocols can be applied to assess bacterial determinants required for autophagy recognition, using bacterial mutant strains that are differentially recognized by autophagy, e.g., Shigella&#916;icsA (the Shigella protein IcsA recruits N-WASP and then Arp2/3 for actin tail and septin cage formation; in its absence there can be no actin tails, no septin cages) and Shigella&#916;icsB (Shigella avoids the autophagic response via the bacterial effector protein IcsB, which prevents the recruitment of autophagy machinery to IcsA; in its absence there can be more septin cages, more autophagy)7,8.
Shigella is not a natural pathogen of zebrafish and grows optimally at 37 &#176;C. However, work has shown that virulence factors required for Shigella invasion, escape from the phagocytic vacuole and replication in the cytosol can be expressed and are functional in zebrafish larvae at 28 &#176;C19. 28 &#176;C is the most commonly used temperature for zebrafish rearing and standard temperature to ensure normal zebrafish development23. Strikingly, the major pathogenic events that lead to shigellosis in humans (i.e., macrophage cell death, invasion and multiplication within epithelial cells, cell-to-cell spread, inflammatory destruction of the host epithelium) are faithfully reproduced in the zebrafish model of Shigella infection19.
Autophagy and cytoskeleton genes are ubiquitously expressed and have a wide range of biological functions. Mouse studies have shown that knockout of essential autophagy26 or septin genes5 are embryonic lethal, and it is likely that some of these genes will also be essential for zebrafish development (although this problem may be reduced by the fact that zebrafish have multiple paralogous genes33). If so, there are several alternatives to overcome this issue, including (i) the use of pharmacological reagents to regulate autophagy and the cytoskeleton, (ii) morpholinos can be titrated down, (iii) knockout of genes can be designed for only specific cell types, and/or (iv) genes involved in autophagic recognition that are not essential for animal development (e.g., p62) may be targeted.
While the zebrafish is an ideal model system to investigate autophagy and the cytoskeleton during Shigella infection, molecular tools are currently lacking. The field needs to generate new tools and drive cell specific expression of the proteins of interest. To knock down expression of autophagy/cytoskeleton genes, new morpholino sequences are required, and novel methods for genome engineering (e.g., TALEN, CRISPR/Cas9) can also be used. In the meantime, several tools previously generated for human or mouse studies may equally work for zebrafish.
The intracellular bacteria S. flexneri has emerged as an exceptional model organism to address key issues in biology, including the ability of bacteria to be recognized by the immune system1,2. The host cell employs septins to restrict the motility of S. flexneri and target them to autophagy, a critical component of cell autonomous immunity7,8. These observations suggest a new molecular framework to study autophagy and its ability to degrade cytosolic bacteria. A major issue is now to fully decipher the underlying molecular and cellular events, and to validate these events analyzed in vitro during bacterial infection in vivo using relevant animal models. To this end, the zebrafish has been established as a valuable new host for the analysis of S. flexneri infection19. Interactions between bacteria and host cells can be imaged at high resolution, and the zebrafish model should prove useful for understanding the cell biology of Shigella infection in vivo. Zebrafish larvae can be used to investigate the role of bacterial autophagy in host defense, and work has shown that that the perturbation of autophagy can adversely affect host survival in response to Shigella infection19.
The observations generated from study of Shigella, septin caging and autophagy in vitro using tissue culture cells and in vivo using zebrafish larvae might provide fundamental advances in understanding host defense. They could also suggest the development of new strategies aimed at combating infectious diseases.
A critical aim of this report is to make sense of the molecular and cellular events analyzed in vitro (i.e., autophagy, actin tails, septin caging) during bacterial infection in vivo in the context of an entire organism, using zebrafish larvae. If not familiar with zebrafish biology and handling, one may refer to in depth protocols for proper zebrafish husbandry23 and in vivo analysis of zebrafish infection19,35.

Shigella flexneri, a Gram-negative invasive enteropathogenic bacterium, can escape from phagosomes to the cytosol, and polymerize the host actin cytoskeleton to evade cytosolic immune responses and promote intra- and intercellular movement1,2. Despite the understanding of actin-based motility in vitro3,4, the mechanisms restricting bacterial dissemination in vivo have not been fully defined. This is critical for a more complete understanding of innate immunity and host defense.
Septins, a highly conserved family of proteins among metazoans, are guanosine triphosphate (GTP)-binding proteins that assemble into hetero-oligomeric complexes and form nonpolar filaments that associate with cellular membranes and the cytoskeleton5,6. Recent work has discovered that infected host cells can prevent Shigella actin based motility by compartmentalizing bacteria targeted to autophagy inside &#8216;septin cages&#8217;, revealing the first cellular mechanism that counteracts actin based motility7,8. A wide open field of investigation now lies in &#8216;septin biology and infection&#8217;. Septin assembly, induced by a variety of pathogens (e.g., Listeria monocytogenes7,9,10, Mycobacterium marinum7,8, Candida albicans11), may emerge as a key issue in host defense5,12.
Autophagy, a highly conserved intracellular degradation process, is viewed as a key component of cell-autonomous immunity because of its ability to deliver cytosolic bacteria to the lysosome13,14. However, the role of bacterial autophagy in vivo to restrict or promote bacterial replication remains poorly understood15,16. 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 functional17,18. Recent work has characterized the susceptibility of zebrafish larvae to S. flexneri, a paradigm for bacterial autophagy15, and has used the Shigella-zebrafish infection model to study the manipulation of autophagy for antibacterial therapy in vivo19.
This report provides new tools and assays to study S. flexneri interactions with autophagy and the cytoskeleton. In a first step, protocols to monitor autophagy-cytoskeleton interactions are described using Shigella infection of the human epithelial cell line HeLa. To assess the role of autophagy-cytoskeleton interactions on the Shigella infection process in vitro, methods to manipulate autophagy and cytoskeleton components (using siRNA or pharmacological reagents) are provided. New work has shown that by using Shigella infection of zebrafish larvae, similar assays can be applied to study the cell biology of infection in vivo. Protocols to prepare and infect zebrafish larvae are detailed, and to assess the host response to Shigella infection in vivo, protocols to determine host survival and bacterial burden of infected larvae are provided. Methods to monitor the recruitment of septin and autophagy markers to Shigella (using either fixed or living zebrafish larvae) and methods to test the role of these processes in vivo [using morpholino oligonucleotides (injected in 1-4 cell stage embryos) or pharmacological reagents (added directly to the zebrafish bath water)] are also discussed. This program&#160;of work is expected to provide insights into the mechanisms required for the control of infection by cytosolic host responses.

<table border="0" cellpadding="0" cellspacing="0" width="771">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Name of Material/ Equipment</td>
			<td style="width:240px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Bafilomycin A1</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td>B1793&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Blebbistatin</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td>B0560</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass microcapillars&#160;</td>
			<td>Harvard Apparatus</td>
			<td>30038</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coarse manual manipulator&#160;</td>
			<td>Narishige</td>
			<td style="width:119px;">M-152</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytochalasin D</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6762</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">D1306</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Dumont #5 fine tweezers</td>
			<td style="width:240px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forchlorfeneuron&#160;</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">32974</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Goat anti-mouse IgG (H+L) antibody</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">N/A&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Goat ant-rabbit IgG (H+L) antibody</td>
			<td style="width:240px;">Molecular Probes</td>
			<td style="width:119px;">N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">JetPEI transfection reagent</td>
			<td style="width:240px;">Polyplus transfection</td>
			<td style="width:119px;">101-01N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Latrunculin B</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5288</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">LC3 antibody</td>
			<td style="width:240px;">Novus Biologicals</td>
			<td style="width:119px;">NB100-2220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Low melting agarose</td>
			<td style="width:240px;">Promega</td>
			<td style="width:119px;">V2111</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">MatTek glass bottom dish</td>
			<td style="width:240px;">MatTek corporation</td>
			<td style="width:119px;">P35G-1.0-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM plus L-alanyl-L-glutamine&#160;</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">41090028</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">MEM non-essential amino acids solution</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">11140-035</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjector&#160;</td>
			<td>Narishige</td>
			<td>IM-300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette puller device&#160;</td>
			<td>Sutter Instrument Co., Novato,</td>
			<td>P-87&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Mineral oil</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">P35G-1.0-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Monoclonal anti-tubulin, acetylated antibody&#160;</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">T6793</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nocodazole</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1404</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-phenylthiourea&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Paraformaldehyde</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">158127</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phalloidin&#160;</td>
			<td style="width:240px;">Molecular Probes</td>
			<td>A12379</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Phenol red solution</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0290</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Protease inhibitor cocktail</td>
			<td style="width:240px;">Roche</td>
			<td style="width:119px;">4693116001</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:412px;">p62/SQSTM1 antibody</td>
			<td style="width:240px;">Cliniscience</td>
			<td style="width:119px;">PM045</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapamycin</td>
			<td style="width:240px;">Sigma-Aldrich</td>
			<td style="width:119px;">R8781</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Sodium pyruvate</td>
			<td style="width:240px;">GIBCO</td>
			<td style="width:119px;">11360039</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:412px;">Transfection reagent</td>
			<td style="width:240px;">Life Technologies</td>
			<td style="width:119px;">12252-011</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield hard set mounting medium containing DAPI&#160;</td>
			<td style="width:240px;">Vector Laboratories</td>
			<td style="width:119px;">H-1500&#160;</td>
		</tr>
	</tbody>
</table>

use of shigella flexneri to study autophagy-cytoskeleton interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Upon infection of tissue culture cells in vitro, S. flexneri can escape from the phagosome and invade the cytosol. In the cytosol, host cells can prevent the actin-based motility of Shigella by compartmentalizing bacteria inside septin cages (Figure 1A). Bacteria entrapped by septin cages can also be labeled by autophagy markers p62 (Figure 1B) and LC3 (Figure 1C). These observations highlight a novel mechanism of host defense that restricts dissemination of invasive pathogens, and also reveal new links between autophagy and the cytoskeleton. Strikingly, the depletion of autophagy markers significantly reduces septin caging of bacteria (Figure 2A), and work has also shown that the depletion of septin caging significantly reduces recruitment of autophagy markers8. Thus, at least in the case of Shigella, septin cage assembly and autophagosome formation can be viewed as interdependent processes. Other cellular requirements for compartmentalization of Shigella by septin cages include actin polymerization and actomyosin activity (Figure 2B).
There is no natural mouse model of shigellosis, and investigation of Shigella pathogenesis, septin biology and bacterial autophagy in vivo can benefit from a new animal model of infection, the zebrafish larvae19. It is possible to infect zebrafish larvae by injecting bacteria in various anatomical sites such as caudal intravenous injections for survival experiments, and tail muscle injections for in vivo microscopy (Figure 3A). Depending on the dose, S. flexneri injected in zebrafish larvae can either be cleared within 48 hr post-infection, or may result in a progressive and ultimately fatal infection (Figures 3B-3D). Shigella virulence factors are expressed at 28 &#176; C, the optimal growth temperature of zebrafish, and zebrafish infection by Shigella is strictly dependent upon its type III secretion system (T3SS)19, an essential virulence determinant in human disease. Taken together, these observations indicate that the zebrafish larva represents a valuable new host for in vivo analysis of Shigella infection.
The optical accessibility of zebrafish larvae enables visualization of septin caging in vivo (Figure 4A), an achievement that has never before been accomplished using mammalian host models. To complement evidence that septin cages entrap bacteria targeted to autophagy in vivo, one can infect transgenic zebrafish larvae expressing GFP-Lc3 and observe autophagy marker recruitment to Shigella (Figure 4B). For ultrastrucutral analysis of Shigella autophagosomes in vivo, electron microscopy can be used to clearly show the cytosolic sequestration of bacteria by double membrane vacuoles19. Autophagy is viewed as a key component of cell-autonomous immunity and a crucial defense mechanism against intracytosolic bacteria14-16. To characterize autophagy function in vivo, p62 morpholino-treated zebrafish larvae can be used. Unlike the core autophagy machinery [e.g., the 36 autophagy related proteins (ATGs)26], p62 is not essential for vertebrate development27 and thus zebrafish larvae can develop normally prior to infection. Strikingly, p62-depleted larvae inoculated with S. flexneri result in significantly increased mortality and increased bacterial burden19. In agreement with in vitro work showing that septin cage assembly is interdependent with autophagosome formation7,8, septin recruitment to Shigella is clearly reduced in p62-depleted larvae (Figure 4C). These data demonstrate that zebrafish survival depends on p62-mediated autophagy to control intracellular bacterial infection.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig1highres.jpg" width="500" /> 
Figure 1.&#160;The septin cage in vitro. (A) HeLa cells were infected with S. flexneri for 4 hr 40 min, fixed, labeled with antibodies to SEPT9 and phalloidin, and imaged by confocal microcopy. Scale bar, 1 &#181;m. (B) HeLa cells were infected with S. flexneri for 4 hr 40 min, fixed, labeled with antibodies to p62 and SEPT2, and imaged by fluorescent light microscopy. Scale bar, 1 &#181;m. (C) HeLa cells were transfected with GFP-ATG8/LC3, infected with S. flexneri for 4 hr 40 min, fixed, labeled with antibodies to SEPT2, and imaged by fluorescent light microscopy. Scale bar, 1 &#181;m. These figures have been modified from Mostowy&#160;et al7.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig2highres.jpg" width="500" /> 
Figure 2.&#160;Cellular requirements for Shigella-septin cage formation. (A) HeLa cells were treated with control (CTRL), p62, ATG5, ATG6 or ATG7 siRNA. Whole-cell lysates of siRNA-treated cells were immunoblotted for GAPDH, p62, ATG5, ATG6, or ATG7 to show the efficiency of siRNA depletion (top). siRNA-treated cells were infected with S. flexneri for 4 hr 40 min, fixed, and labeled for quantitative microscopy. Graphs (bottom) represent the mean % &#177; SEM of Shigella inside SEPT2 cages from n &#8805;3 experiments per treatment. (B) HeLa cells were infected with S. flexneri, treated with DMSO, cytochalasin D (CytD), latrunculin B (LatB), nocodazole (Noco), or blebbistatin (Bleb) and after 4 hr 40 min were fixed and labeled for quantitative microscopy. Graphs represent the mean % &#177; SEM of Shigella inside SEPT2 cages from two independent experiments per treatment. These figures have been modified from Mostowy&#160;et al7.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig3highres.jpg" width="500" /> 
Figure 3.&#160;The zebrafish model of Shigella infection. (A) Images to illustrate orientation of the zebrafish larva under the stereomicroscope. (Left panel) Zebrafish larvae 72 hr post fertilization were positioned laterally in the injection plate with their dorsal side facing the injection needle. (Middle panel) Bloodstream infection was performed by injecting the bacteria (red solution) in the caudal vein, posterior to the urogenital opening. (Right panel) Infection in the tail muscle was performed by injecting the bacteria (red solution) over a somite. (B) Survival curves of 72 hr post fertilization larvae injected with various doses of S. flexneri and incubated at 28 &#176;C for 48 hr post infection. The effective inoculum was classified as low (&#60;103 CFU, open circles), medium (~4 x 103 CFU, open triangles) or high (~104 CFU, open squares). Mean % &#177; SEM (horizontal bars) from n &#8805;3 experiments per inoculum class. (C) Enumeration of live bacteria in homogenates from individual larvae at various times post infection measured by plating onto LB. Note, only larvae having survived the infection are included in enumeration analysis. Mean &#177; SEM (horizontal bars) also shown. (D) Distribution of GFP-Shigella determined by live imaging using a fluorescent stereomicroscope at various times post infection using a low, medium, or high dose inoculum (caudal intravenous injections). Overlay of transmission image (grey) and GFP fluorescence (green). (B)-(D)These figures have been modified from Mostowy&#160;et al19.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51601/51601fig4highres.jpg" width="500" /> 
Figure 4.&#160;The cell biology of Shigella infection in vivo. (A) Zebrafish larvae were infected in the tail muscle with GFP-Shigella (low dose) for 24 hr, fixed, labeled with antibodies against SEPT7 (red) and to GFP (green), and imaged by confocal microscopy. Scale bar, 5 &#181;m. (B) GFP-Lc3 zebrafish larvae were infected with mCherry-Shigella (medium dose) for 4 hr, fixed, labeled with antibodies against mCherry (red) and to GFP (green), and imaged by confocal microscopy. Scale bar, 1.5 &#181;m. (C) Zebrafish larvae treated with either control (CTRL; left image) or p62 (right image) morpholinos were infected with GFP-Shigella for 4 hr (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 hr post infection. Scale bar, 5 &#181;m. These figures have been modified from Mostowy&#160;et al19.

Watch this Scientific Journal Video about Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions at JoVE.com

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

use-of-shigella-flexneri-to-study-autophagy-cytoskeleton-interactions

Research

JoVE Journal

Immunology and Infection

15.7K Views.  Imperial College London. To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo. 

Video: Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

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Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions