Name: infection of zebrafish embryos with intracellular bacterial pathogens
Uploaded: 2012-03-15T12:00:00
Duration: 11 min 18 s
Description: Watch this Scientific Journal Video about Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens at JoVE.com

The authors would like to thank Chao Cui, Floor de Kort, Esther Stoop and Ralph Carvalho for images in Figure 4, Ulrike Nehrdich and Davy de Witt for fish care, and other lab members for helpful discussions. This work is supported by the Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Ministry of Education, Culture and Science, the European Commission 7th framework project ZF-HEALTH (HEALTH-F4-2010-242048), the European Marie-Curie Initial Training Network FishForPharma (PITN-GA-2011-289209), and by the Australian NHMRC (637394). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.

<ol>
	<li>Meijer, A. H., Spaink, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+made+transparent+with+the+zebrafish+model.">Host-pathogen interactions made transparent with the zebrafish model.</a> Curr. Drug Targets. 12, 1000-1017 (2011).</li><li>Davis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+visualization+of+mycobacterium-macrophage+interactions+leading+to+initiation+of+granuloma+formation+in+zebrafish+embryos.">Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.</a> Immunity. 17, 693-702 (2002).</li><li>vander Sar, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryos+as+a+model+host+for+the+real+time+analysis+of+Salmonella+typhimurium+infections.">Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections.</a> Cell Microbiol. 5, 601-611 (2003).</li><li>Zakrzewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-specific+gene+functions+in+Spi1-directed+innate+immunity.">Macrophage-specific gene functions in Spi1-directed innate immunity.</a> Blood. 116, 1-11 (2010).</li><li>Le Guyader, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+unconventional+behavior+of+neutrophils+in+developing+zebrafish.">Origins and unconventional behavior of neutrophils in developing zebrafish.</a> Blood. 111, 132-141 (2008).</li><li>Herbomel, P., Thisse, B., Thisse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+behaviour+of+early+macrophages+in+the+zebrafish+embryo.">Ontogeny and behaviour of early macrophages in the zebrafish embryo.</a> Development. 126, 3735-3745 (1999).</li><li>Alibaud, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mycobacterium+marinum+TesA+mutant+defective+for+major+cell+wall-associated+lipids+is+highly+attenuated+in+Dictyostelium+discoideum+and+zebrafish+embryos.">A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.</a> Mol. Microbiol. 80, 919-934 (2011).</li><li>Carvalho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+screen+for+tuberculosis+progression.">A high-throughput screen for tuberculosis progression.</a> PLoS One. 6, 16779-16779 (2011).</li><li>van der Sar, A. M., Spaink, H. P., Zakrzewska, A., Bitter, W., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+the+zebrafish+host+transcriptome+response+to+acute+and+chronic+mycobacterial+infection+and+the+role+of+innate+and+adaptive+immune+components.">Specificity of the zebrafish host transcriptome response to acute and chronic mycobacterial infection and the role of innate and adaptive immune components.</a> Mol. Immunol. 46, 2317-2332 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115-e1115 (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book.">The Zebrafish Book.</a> A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). , (1993).</li><li>Gutzman, J. H., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Brain+Ventricle+Injection.">Zebrafish Brain Ventricle Injection.</a> J. Vis. Exp. (26), e1218-e1218 (2009).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish.">mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.</a> Blood. 117, 49-56 (2011).</li><li>Stockhammer, O. W., Zakrzewska, A., Hegedus, Z., Spaink, H. P., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+and+functional+analyses+of+the+zebrafish+embryonic+innate+immune+response+to+Salmonella+infection.">Transcriptome profiling and functional analyses of the zebrafish embryonic innate immune response to Salmonella infection.</a> J. Immunol. 182, 5641-5653 (2009).</li><li>Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108, 3976-3978 (2006).</li><li>Stoop, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryo+screen+for+mycobacterial+genes+involved+in+the+initiation+of+granuloma+formation+reveals+a+newly+identified+ESX-1+component.">Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component.</a> Dis. Model Mech. 4, 526-536 (2011).</li><li>Levraud, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+zebrafish+IFN+receptor:+implications+for+the+origin+of+the+vertebrate+IFN+system.">Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system.</a> Journal of Immunology. 178, 4385-4394 (2007).</li><li>Levraud, J. P., Colucci-Guyon, E., Redd, M. J., Lutfalla, G., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+zebrafish+innate+immunity.">In vivo analysis of zebrafish innate immunity.</a> Methods Mol. Biol. 415, 337-363 (2008).</li><li>Brothers, K. M., Newman, Z. R., Wheeler, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+disseminated+candidiasis+in+zebrafish+reveals+role+of+phagocyte+oxidase+in+limiting+filamentous+growth.">Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth.</a> Eukaryot. Cell. 10, 932-944 (2011).</li><li>Colucci-Guyon, E., Tinevez, J. Y., Renshaw, S. A., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+of+professional+phagocytes+in+vivo:+unlike+macrophages,+neutrophils+engulf+only+surface-associated+microbes.">Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils engulf only surface-associated microbes.</a> J. Cell Sci. 124, 3053-3059 (2011).</li><li>van Soest, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+static+immersion+and+intravenous+injection+systems+for+exposure+of+zebrafish+embryos+to+the+natural+pathogen+Edwardsiella+tarda.">Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda.</a> BMC Immunology. 12, 58-58 (2011).</li><li>Adams, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+tolerance+in+replicating+mycobacteria+mediated+by+a+macrophage-induced+efflux+mechanism.">Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism.</a> Cell. 145, 39-53 (2011).</li><li>Ellett, F. L., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+quantification+of+fluorescent+leukocyte+numbers+in+zebrafish+embryos.">Computational quantification of fluorescent leukocyte numbers in zebrafish embryos.</a> Methods in Enzymology. , (2011).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113-e1113 (2009).</li><li>Mathias, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation+by+retrograde+chemotaxis+of+neutrophils+in+transgenic+zebrafish.">Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish.</a> J. Leukoc. Biol. 80, 1281-1288 (2006).</li><li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Dev. Biol. 7, 42-42 (2007).</li><li>Gray, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+intravital+imaging+of+macrophage+and+neutrophil+behaviour+during+inflammation+using+a+novel+transgenic+zebrafish.">Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish.</a> Thromb. Haemost. 105, 811-819 (2011).</li></ol>

The authors have nothing to disclose.

The infection methods described in this article are frequently used to study the function of host innate immunity genes or bacterial virulence genes1. The intravenous micro-injection methods (protocols 5 and 6.1) are used most often for such studies. The caudal vein at the posterior blood island is the most convenient location for intravenous injection of 1-day-old embryos. As the caudal vein becomes more difficult to penetrate at later stages, we prefer the Duct of Cuvier as intravenous injection site for embryos at 2-3 dpf. In all cases it is critical to inject the embryos with consistent numbers of bacteria, which should be checked by plating injection inocula for cfu counting. The following aspects must be considered to achieve reproducible injections. Firstly, it is essential to have high-quality glass microcapillary injection needles. If the needle tip is too large, the injection will create a puncture hole large enough for bleeding to occur and the injected bacteria will flow out of the blood circulation. Secondly, bacteria must be injected directly into the blood circulation. If the needle tip is not inside the vein or if injection fluid spreads into the yolk sac extension, the embryo must be discarded from the experiment. Thirdly, using PVP40 as a carrier for injecting M. marinum will assist in preventing the bacteria from sinking in the glass needle. PVP40 improves the homogeneity of the suspension, resulting in more reproducible inocula throughout the duration of injections. PVP40 may also be used for S. typhimurium injections, but here it is less important because the larger size and bright fluorescence of the bacteria allows a visual control over the injection inoculum during experiments. For practising the injections we recommend the use of phenol red dye (1% v/v) or 1 &mu;m fluorescent spheres available in different colors (Invitrogen). Once the technique is mastered, it takes approximately 30 minutes to inject 50-100 embryos, including the time required for aligning the embryos on agarose plates and adjusting the bacterial injection volume.
While intravenous injections into the blood island (protocol 5) or into the duct of Cuvier (protocol 6.1) are most commonly used, the alternative routes of infection described in this video article have proved useful to study macrophage and neutrophil chemotaxis (protocols 6.2, 6.3, and 6.4), to study growth of attenuated bacterial strains (protocol 6.5), and to adapt the zebrafish infection model for high-throughput applications (protocol 6.6) 4-8. The described micro-injection methods can also be applied for injections of viruses 18, 19, fungal spores 14, 20 or protozoan parasites (Maria Forlenza, personal communication). A useful addition to the injection procedures described in this video article is a recently described procedure for subcutaneous injection of bacteria into zebrafish embryos 21. This procedure was applied to study phagocytosis of bacteria by neutrophils, which were found to efficiently engulf bacteria on tissue surfaces but, in contrast to macrophages, were virtually unable to phagocytose bacteria upon injection into the blood or into fluid-filled body cavities. For subcutaneous injections embryos are positioned similar as for the tail muscle injections described in this video article, but the needle is inserted just under the skin to inject the bacteria over a somite.
It is important to consider that micro-injection is essentially an artificial route of infection. However, early embryos are highly resistant to external exposure to bacterial pathogens. In fact, immersion assays, where embryos are bathed in a bacterial suspension, are not reproducible in our hands 22. While some embryos may become infected upon immersion, we have observed a large variation in mortality rates and in the expression of inflammatory marker genes between individual embryos in such assays. The micro-injection methods described in this article achieve reproducible infections and are particularly useful to study bacterial interactions with host innate immune cells. An efficient approach to quantify bacterial infection in individual embryos is to analyze the fluorescent images of infected embryos with custom-made, dedicated pixel quantification software 17, 23. This approach has been shown to correlate well with results of cfu counts after plating of infected embryos. Similarly, relative changes in leukocyte numbers per embryo have been quantified by digital image analysis in transgenic leukocyte fluorophore reporter lines; this approach would be applicable to quantifying the host leukopoietic response to infection24.
The function of host innate immune genes can be investigated efficiently in vivo by combining the described infection models with morpholino knockdown. Morpholinos are synthetic antisense oligonucleotides that target specific RNAs and reduce the expression of the gene products when injected into 1-cell stage zebrafish embryos as shown in other video articles in this journal 10, 25 . In morpholino knockdown studies, it is of great importance that all embryo groups to be compared are staged correctly prior to bacterial injections. This is especially important because embryos have a developing immune system that becomes increasingly competent over time.
There are several transgenic reporter lines currently available to distinguish the major innate immune subsets, macrophages and neutrophils, in zebrafish embryos. The mpx and lyz promoters have been used to generate neutrophil reporter lines 16, 26, 27, and the csf1r (fms) and mpeg1 promoters were used to produce macrophage reporters 14, 28 . While csf1r (fms) is additionally expressed in xanthophores, mpeg1 expression is exclusively in macrophages. As shown in this video article, the recently developed mpeg1 reporter lines are highly useful for imaging bacterial phagocytosis by macrophages.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma</td>
			<td>P0290</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>228210</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H10 agar</td>
			<td>Becton, Dickenson and Company</td>
			<td>262710</td>
		</tr>
		<tr>
			<td>BBL Middlebrook OADC Enrichment</td>
			<td>Becton, Dickenson and Company</td>
			<td>211886</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>Becton, Dickenson and Company</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>BBL Middlebrook ADC Enrichment</td>
			<td>Becton, Dickenson and Company</td>
			<td>211887</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich</td>
			<td>P1754</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone (PVP40)</td>
			<td>Calbiochem, Merck KGaA</td>
			<td>529504</td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0387</td>
		</tr>
		<tr>
			<td>SeaPlaque Agarose (low melting point agarose)</td>
			<td>Lonza Inc.</td>
			<td>50100</td>
		</tr>
		<tr>
			<td>FluoSpheres (1.0 &mu;m, red fluorescent (580/605))</td>
			<td>Invitrogen</td>
			<td>F8821</td>
		</tr>
		<tr>
			<td>FluoSpheres (1.0 &mu;m, yellow-green fluorescent (505/515))</td>
			<td>Invitrogen</td>
			<td>F8823</td>
		</tr>
	</tbody>
</table>

infection of zebrafish embryos with intracellular bacterial pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Watch this Scientific Journal Video about Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens at JoVE.com

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

infection-of-zebrafish-embryos-with-intracellular-bacterial-pathogens

Research

JoVE Journal

Immunology and Infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...