The authors are grateful to K. Kissa for helpful discussions and for providing lipo-clodronate and L. Ramakrishnan for the generous gift of pTEC27 and pTEC15 that allow expression of tdTomato and Wasabi, respectively. This work forms part of the projects of the French National Research Agency (ZebraFlam ANR-10-MIDI-009 and DIMYVIR ANR-13-BSV3-007-01) and the European Community&#8217;s Seventh Framework Program (FP7-PEOPLE-2011-ITN) under grant agreement no. PITN-GA-2011-289209 for the Marie-Curie Initial Training Network FishForPharma. We wish also to thank the Association Gregory Lemarchal and Vaincre La Mucoviscidose (RF20130500835) for funding C.M. Dupont.

Zebrafish experimental procedures must comply with the relevant institutional and governmental regulations. For the present study, zebrafish experiments were done at the University Montpellier, according to European Union guidelines for handling of laboratory animals (http://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm) and approved&#160;under the reference CEEA-LR-13007.
1. Preparation of Reagents and Microinjection Equipment
<ol>
	<li>Prepare fish water by dissolving 0.06 g I...

The zebrafish has recently emerged as an excellent vertebrate model system for studying the dynamics of bacterial infection using wide field and confocal imaging in real-time36. The combination of dispersed mycobacterial suspensions (protocol 2.2) together with micro-injection methods (protocol 4) allows reproducible systemic infections, and subsequent monitoring and visualization of the progression of the infection with a special focus on the bacterial interactions with host macrophages. Virulence of M. a...

Mycobacterium abscessus is an emerging pathogen that causes a wide spectrum of clinical syndromes in humans. These include cutaneous infections as well as severe chronic pulmonary infections, mostly encountered in immunocompromised and in cystic fibrosis patients1,2,3,4. M. abscessus is also regarded as a major rapidly-growing mycobacterial species responsible for nosocomial and iatrogenic infections in humans. Moreover, several recent reports highlighted the possibility that M. abscessus could cross the blood-brain barrier and induce important lesions in the central nervous system (CNS)5,6. Despite being a rapid grower, M. abscessus exhibits also several pathogenic features that are related to those of Mycobacterium tuberculosis, including the capacity to remain silent for years within granulomatous structures and to generate caseous lesions in the lungs7. More alarming is the low sensitivity of M. abscessus to antibiotics, rendering these&#160;infections extremely difficult to treat leading to a significant therapeutic failure rate8,9. The important threat of this species is mainly its intrinsic resistance to antibiotics, which is of major concern in public health institutions10 and a contraindication to lung transplantation11.
M. abscessus displays smooth (S) or rough (R) colony morphotypes that lead to different clinical outcomes. In contrast to the S strain, R bacteria have a tendency to grow end to end, leading to a rope or cord-like structure12,13. Several independent studies based on either cellular or animal models revealed the hyper-virulence phenotype of the R morphotype14,15. From epidemiological studies, the most severe cases of M. abscessus pulmonary infections appear to be associated with R variants16 which are the only variant that has been seen to persist for years in an infected host3. The morphotype difference relies on the presence (in S) or loss (in R) of surface-associated glycopeptidolipids (GPL)12. However, due to the inherent limitations of the currently available cellular/animal models used to study M. abscessus infection, our knowledge regarding the pathophysiological events of the R or S variants remains obscure. Infection of immuno-competent mice via intravenous or aerosol routes leads to transient colonization, impeding the&#160;use of mice&#160;to study persistent infections and for in vivo drug susceptibility testing17. Therefore, developing animal models amenable to the manipulation of the host response is a major challenge. In this context, non-mammalian models of infection have been developed recently, including Drosophila melanogaster18 that offers several advantages such as cost, speed and ethical acceptability over the mouse model. The zebrafish (Danio rerio) model of infection has also been explored to visualize, by non-invasive imaging, the progression and chronology of M. abscessus infection in a live animal19. Importantly, a proof of concept was also established to demonstrate its suitability for in vivo antibiotic assessments against M. abscessus17,20.
The zebrafish have been widely used during the last two decades to study the interactions between various pathogens and the host immune system21. The increasing success of this alternative vertebrate model relies on major and unique opportunities that motivated and validated its use for a better understanding of numerous viral and bacterial infections19,22,23,24,25,26,27,28,29. As opposed to most other animal models, zebrafish embryos are optically transparent, allowing non-invasive fluorescence imaging30. This has led to study M. abscessus infected zebrafish embryos with unprecedented details, culminating with the description of extracellular cording, that represent an example of bacterial morphological plasticity. Cording represents a new mechanism of subversion of the immune system and a key mechanism promoting pathogenesis of acute M. abscessus infection19.
This report describes new tools and methods using the zebrafish embryo to decipher the pathophysiological traits of M. abscessus infection and to study the intimate interactions between the bacilli and the innate immune system. First, a detailed microinjection protocol that includes processing of the bacterial inoculum, embryo preparation, and infection per se, is presented. Methods specifically adapted to assess M. abscessus virulence by measuring various parameters, such as host survival and bacterial burden, are presented. Special focus is given on how to monitor, at a spatiotemporal level, the fate and progression of the infection and the host immune response to M. abscessus using video microscopy. Moreover, to investigate the contribution and role of macrophages during M. abscessus infection, methods to generate macrophages-depleted embryos (using either genetically- or chemically-based approaches) are described. Finally, protocols to visualize the specific interactions with macrophages or neutrophils using either fixed or living embryos are documented.
The aim of this report is to stimulate further studies to shed new light into M. abscessus virulence mechanisms and particularly the role of cording in the establishment of an acute and uncontrolled infection process.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BBL MGIT PANTA</td>
			<td>BD Biosciences</td>
			<td>245114</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>Euromedex</td>
			<td>04-100-811-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase from Bovine Liver&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H10 Agar</td>
			<td>BD Biosciences</td>
			<td>262710</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>BD Biosciences</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Delta Microscopie</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma-Aldrich</td>
			<td>319244</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich</td>
			<td>P4780</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Gibco Life Technologie</td>
			<td>30391-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting agarose</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean Sea Salts&#160;</td>
			<td>Aquarium Systems Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries&#160;</td>
			<td>Sutter instrument Inc</td>
			<td>BF100-78-10</td>
			<td>1mm O.D. X 0.78 mm I.D.</td>
		</tr>
		<tr>
			<td>Micropipette puller device&#160;</td>
			<td>Sutter Instrument Inc</td>
			<td></td>
			<td>Flamming/Brown Micropipette Puller p-87</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Tritech Research&#160;</td>
			<td></td>
			<td>Digital microINJECTOR, MINJ-D</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Sciences Tools inc</td>
			<td></td>
			<td>Dumont # M5S&#160;</td>
		</tr>
		<tr>
			<td>Microloader Tips</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

deciphering and imaging pathogenesis and cording of mycobacterium abscessus in zebrafish embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Although various anatomical sites can be injected32, caudal vein injections are often used to generate systemic infection for subsequent analyses including survival experiments, bacterial burden determination, phagocytosis activity or cord formation. Injections in the tail muscles are used to assess the recruitment of macrophages at the site of injection (Figure 3A). To investigate and compare the virulence of R and S variants of M. abscessus, fluorescent bacterial suspensions are inj...

Watch this Scientific Journal Video about Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos at JoVE.com

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

deciphering-imaging-pathogenesis-cording-mycobacterium-abscessus

Research

JoVE Journal

Immunology and Infection

10.1K ビュー.  Université Montpellier. Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos. 