The adapter protein Myd88 plays an important role in limiting mycobacterial growth in a zebrafish model for tuberculosis

Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (www.​zfin.​org). Wild-type and the myd88^hu3568 zebrafish strains were used for this study, and culturing the myd88^hu3568 strain was approved by the local animal welfare committee (DEC) of the University of Leiden (protocol 12232). All fish were raised and grown at 28.5 °C on a 14-h light:10-h dark cycle. Embryos were obtained from natural spawning at the beginning of the light period and kept in egg water (60 μg/ml Instant Ocean sea salts).

The M. marinum M strain fluorescently labelled with E2-crimson was used and prepared at ~ 50 colony-forming units per 1 nl as previously described [32]. Borosilicate glass micro-capillaries (Harvard Apparatus, USA) were used with a micropipette puller device (Sutter Instruments Inc., USA) for preparing microinjection needles. Zebrafish larvae were injected in the tail fin at 3 dpf using the Eppendorf microinjection system with a fine (~ 5 to 10 micron) needle tip broken off with tweezers and mounted at a 30-degree angle. Larvae were anesthetized in egg water with 200 μg/mL 3-aminobenzoic acid (tricaine; Sigma-Aldrich, USA) and injected between the 2 epidermal layers at the ventral part of the tail fin (Fig. 1), as previously described [31]. Larvae were fixed at desired time points after infection with 4% paraformaldehyde in PBS-T (phosphate-buffered saline; NaCl 150 mM, K₂HPO₄ 15 mM, KH₂PO₄ 5 mM) with 0.05% Tween 20 (Merck Millipore, Germany) with gentle agitation for 18 h at 4 °C. The larvae were washed the next day with PBS-T and stored at 4 °C for further staining or until imaging.

For Lcp1 immunostaining, larvae were anesthetized with 200 μg/ml tricaine and then immediately fixated in 4% paraformaldehyde in PBS (phosphate-buffered saline, pH 7.2) for 16 h at 4 °C. After fixation, the larvae were rinsed in PBS-DTx (phosphate-buffered saline with 0.5% DMSO and 0.3% Triton X-100) and treated with proteinase K (10 μg/ml in PBS-DTx; Roche) for 10 min at 37 °C. The larvae were blocked in 5% normal sheep serum (Sigma-Aldrich) in PBS-DTx for 2 h at room temperature, incubated with Lcp1/L-Plastin antibody (a gift from Dr. Anna Huttenlocher, University of Wisconsin, USA) in 1:1000 dilution at 4 °C overnight and subsequently incubated with Alexa-488 conjugated secondary antibody (1:200; Invitrogen) for 2 h at room temperature. The larvae were washed with PBS-DTx and stored at 4 °C until imaging.

The TUNEL experiments were performed after fixation (anesthetized with 200 μg/ml tricaine and afterwards immediately fixated in 4% paraformaldehyde in PBS for 16 h at 4 °C) of larvae using Millipore ApopTag Peroxidase In Situ Apoptosis detection kit and Roche Anti-Digoxigenin-POD Fab fragments using a protocol adapted for use in zebrafish larvae [33]. TSA Fluorescence kits (Perkin Elmer, USA) Fluorescein was used for fluorescence detection.

Larvae were mounted in 1% low melting agarose (Sigma-Aldrich, USA) and imaged with a Leica TCS SPE (Wetzlar, Germany) confocal laser scanning microscope (CLSM) using 488 and the 633 laser lines and a 20X (NA 0.7) objective (for the images used in Fig. 1b–e) or a Nikon A1 CLSM (Tokyo, Japan) using 488 and the 641 laser lines and a 20X (NA 0.75) objective (for the images used in Figs. 1f, 2 and 3). Images were analysed using Fiji [34] or NIS-elements 4 (Nikon) software. The images of larvae used in Fig. 1b–e were visually inspected using maximum z-projections, and cropped regions of infection were generated using Fiji software. The bacterial burden was measured as the total volume (in μm³) of the fluorescent signal of M. marinum in the 3D images (12 bit) in Nis-elements 4, i.e. the total volume of the voxels with a signal above a certain fluorescence intensity threshold (using the same threshold for all larvae). The numbers of TUNEL- and Lcp1-positive cells were counted manually for each image.

Before being used for electron microscopy, the zebrafish larvae were anesthetized with 200 μg/ml tricaine and afterwards immediately fixated in 2% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2) for 3 h at room temperature followed by fixation for 16 h at 4 °C. Postfixation was performed in 1% osmium tetroxide in sodium cacodylate buffer for 1 h at room temperature. After dehydration through a graded series of ethanol, all specimens were kept in epoxy resin (Agar Scientific, UK) for 16 h before embedding. Ultra-thin sections were collected on Formvar coated 200 mesh or one-hole copper grids (Agar Scientific, UK) stained with 2% uranyl acetate in 50% ethanol and lead citrate for 10 min each. Electron microscopy images were obtained with a JEM-1010 transmission electron microscope (JEOL, Japan) equipped with an Olympus Megaview camera (Tokyo, Japan). A 200 kV Tecnai 20 FEG transmission electron microscope (FEI Company, the Netherlands) equipped with a 4 k × 4 K CCD camera (Gatan, USA) was used to image large specimen tissues with the automatic montaging of individual images into large overviews with high resolution [35].

For TEM imaging and a quantitative analysis of the images, three larvae for each group (wild type and mutant) were used, and a section in the middle of the initial granulomas was imaged. Quantification was done by manually counting and classifying individual bacteria. The total number of bacteria counted for this analysis was 2178 and 1655, for wild type and mutant, respectively.

We have previously shown that TLR/IL1R-Myd88 signalling is required for defence of zebrafish larvae systemically infected with M. marinum (Mm). In the present study, we used a previously established localized infection in the tail fin [31], to investigate the initial granuloma formation and subcellular localization of Mm at the site of infection. Homozygous myd88 mutant (myd88^-/-) and wild-type larvae (myd88^+/+) were infected with ~ 50 colony-forming units (cfu) of fluorescently labelled Mm in the tail fin at 3 days post fertilization (dpf). The infection in these larvae develops into a granuloma-like structure within 3 to 5 days post infection (dpi).

To provide a detailed description of the infection process and the development of the granuloma structure, confocal laser scanning microscopy (CLSM) was performed on the tail fin of infected myd88^-/- and wild-type (myd88^+/+) larvae. The bacterial burden in representative myd88^+/+ and myd88^-/- larvae is shown in Fig. 1. The infection in each of these larvae was imaged at 4 h post infection (hpi) and at 1, 2, 3, 4 and 5 dpi. From 2dpi onward we observed compacted bacterial aggregates in myd88^-/- larvae (Fig. 1e), while in the myd88^+/+ larvae, these aggregates were smaller and more widely distributed over the infected tissue (Fig. 1d).

In a separate experiment, the bacterial burden was quantified in the myd88^+/+ and myd88^-/- larvae, based on fixed samples at 0 to 5 dpi (Fig. 1F). The bacterial burden was measured as the total volume of the fluorescent signal of Mm. The burden increased significantly between 0 and 3 dpi in the myd88^+/+ and the myd88^-/- larvae but increased at a lower rate in the myd88^+/+ larvae, compared to the rate in the myd88^-/- larvae, resulting in larger burdens at later time points in the mutant. At 4dpi, the difference in infection level between myd88^+/+ and myd88^-/- larvae was maximal with a bacterial volume of 69·10³ (± 1.6·10³) μm³ and 328·10³ (± 52·10³) μm³, respectively. In line with previous results obtained using a systemic infection model [28], localized Mm infection in the tail fin of myd88^-/- larvae results in an increased bacterial burden.

In order to investigate the presence of leukocytes at the site of infection in myd88^-/- zebrafish, we performed Lcp1/L-plastin immunostaining at 4 dpi for visualization of all leukocytes (Fig. 2). The larvae were imaged using CLSM, and representative images are shown for myd88^+/+ and myd88^-/- larvae (Fig. 2 a and b, respectively). At 4 dpi, the infection in the tail fin has resulted in the formation of an initial stage granuloma, which in myd88^+/+ larvae was observed as a large local accumulation of L-plastin-positive cells (35.4 ± 4.2) at the site of the infection in the tail fin (Fig. 2a and c). In the myd88^-/- larvae, the number of L-plastin-positive cells at the site of infection was significantly lower (10.2 ± 1.8).

The lower number of leukocytes observed at the site of infection in myd88^-/- larvae could either be due to a lower number of leukocytes recruited to the infected area or to a higher rate of cell death of these cells. To address this issue, we performed a fluorescent terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL) assay, which visualizes double-stranded DNA breaks, thereby labelling apoptotic as well as necrotic cells (Fig. 3). At 4 dpi, myd88^+/+ larvae show a high number of TUNEL positive cells (48.7 ± 3.6) throughout the site of infection (Fig. 3a). The myd88^-/- larvae show a lower number of TUNEL positive cells (31.6 ± 1.8; Fig. 3b and c). Although alternative explanations such as infection-induced Myd88-dependent changes in haematopoiesis, or leukocyte cell death that is occurring systemically at earlier time points, the observations suggest that the lower number of leukocytes at the infection sites in myd88^-/- larvae is a consequence of reduced recruitment rather than increased cell death.

In order to analyse effects of Myd88 deficiency at an ultrastructural level, transmission electron microscopy (TEM) was performed on the tail fin granulomas of Mm infected myd88^+/+ and myd88^-/- larvae (Fig. 4). At 5 dpi, the myd88^+/+ larvae show a necrotic centre in the infected area, visible as a hole in the tail fin, surrounded by bacteria-containing cells (Fig. 4a and b). In the myd88^-/- larvae, the majority of bacteria were found to reside extracellularly (Fig. 4c and d), and the area containing extracellular bacteria is surrounded by infected cells (Fig. 4d). Such areas with extracellular bacteria are absent in wild-type larvae.

To determine the nature and frequency of different cell-bacterium interactions in myd88^+/+ and myd88^-/- larvae, we used a previously described procedure [31], in which we quantified the occurrence of intracellular Mm in the cytoplasm without any membrane structures surrounding it, or in different types of intracellular compartments, as individual bacteria or as aggregates. The results of this quantification and representative images of each type of interaction are shown in Fig. 5. A large fraction of intracellular bacteria occurred in compartments containing more than 5 bacteria (hereafter referred to as aggregates), both in myd88^+/+ (~ 64%) and in myd88^-/- larvae (~ 50%) (Fig. 5a and b). However, electron-dense compartments containing these aggregates (hereafter referred to as electron-dense aggregates) were much less abundant in mutant larvae (~ 8%) compared to wild type (~ 39%, Fig. 5b). These compartments are characterized by a uniform electron-dense content and probably reflect lysosomes or phagosomes that have been fused with a lysosome. Individual bacteria were present in membrane-engulfed compartments, characterized by a single membrane without any cytoplasmic material, probably reflecting phagosomal structures (Fig. 5c). In mutant larvae, those membrane-engulfed compartments were found more often (~ 27%) than in the wild types (~ 13%). Bacteria in the cytoplasm, not enclosed by a membrane, were observed in the type and mutant larvae at a similar frequency, (~ 16% and ~ 17% respectively; Fig. 5d). In addition, bacteria were occasionally found in membrane-engulfed compartments containing cytoplasmic material, in the myd88^+/+ (~ 2%) and myd88^-/- (~ 1%) larvae. These compartments are characterized by partially degraded cytoplasmic content and organelles, probably as a result of engulfment by or fusion with autophagosomes. This morphology was previously shown to be associated with the localization of the Lc3 protein, using correlative light and electron microscopy, which also suggests an autophagic origin [31] (Fig. 5e). Finally, sometimes bacteria were located inside electron-dense, membrane-engulfed compartments with a regular electron-dense content in the wild-type and myd88^-/- larvae (~ 5% and ~ 4% respectively; Fig. 5f). These electron-dense compartments probably reflect lysosomes with acidic content. In conclusion, the most notable difference between myd88^-/- and wild-type larvae was the reduced presence of electron-dense aggregates in the mutant.