Experimental design. Used was the strain M. tuberculosis H37Rv (Mtb Berlin) without plasmid obtained from the Kaufman lab, MPI, Berlin, Germany. In the case of the fluorescent imaging and survival curves an additional strain containing a DsRed expressing plasmid (Mtb^DsRed) was used. Numbers indicate the number of replicates followed (after symbol x) by the number of larvae per replicate. Vertical arrows indicate the time points of sampling.

Survival of zebrafish larvae at 34 °C upon M. tuberculosis infection. Zebrafish embryos between 16 and 128-cell stage were robotically infected with 500 CFU M. tuberculosis wild-type () or M. tuberculosis-DsRed ()H37Rv strains and raised at 34 °C along with control groups of PVP-injected () and uninjected () embryos. The last viable larvae were collected at 9 dpf. Percent survival for each group is shown on the survival curve. The number of injected embryos was 1000, 1200, 2000 and 400 for the M. tuberculosis wild-type strain, M. tuberculosis-H37Rv DsRed strain, PVP-injected and uninjected embryos, respectively. Raw data is presented in Table S4.

Fluorescent microscopy imaging of Mycobacterium tuberculosis infection in zebrafish larvae. Images taken with a fluorescent stereomicroscope of 0, 6, 8 and 9 dpi M. tuberculosis-DsRed infected larvae are shown together with the bright field image of the same embedded larvae. Image of 0 dpi embryo represents a successful injection of fluorescent bacteria into the yolk. At 6, 8 and 9 dpi bacteria showed dissemination throughout the body of the larvae and the formation of bacterial aggregates. Scale bar: 200 micrometer.

M. tuberculosis CFU counts per embryo based on molecular bacterial load (MBL) assay. Zebrafish embryos were infected with M. tuberculosis H37Rv at 0 dpf. Total RNA was purified at 0, 1, 2, 3, 4, 5 and 9 dpi and used as template in the MBL assay Mtb-specific 16S rRNA in a RT-qPCR. The figure shows the amount of CFU per embryo. Raw data are provided in Supplementary Table S2.

Differential gene expression of il1b (A) and mmp9 (B). Differential gene expression measured by qPCR of cDNA for RNA isolated from zebrafish larvae infected with M. tuberculosis (Mtb) at different times after injection (dpi). Strain H37Rv without a plasmid was used for these experiments. A comparison is made with the mock-injection results with the carrier PVP. The raw data and statistical analysis is presented in Supplementary Table S2.

Differentially expressed genes in the time-course infection experiment with Mycobacterium tuberculosis. (A) The total number of significantly differentially expressed genes at 3, 4, 5, 6, and 8 dpi larvae infected with Mycobacterium tuberculosis compared to the PVP-injected control group. Strain H37Rv without a plasmid was used for these experiments. Significance was set to a fold change larger than 2 for up-regulated genes (green bars) or smaller than −2 for down-regulated genes (red bars), with a p value smaller than 0.05. (B,C) Venn diagrams show the overlap of the significantly up-regulated (B) or down-regulated (C) genes between the five time points of mycobacterium infection.

Comparison of differentially expressed host genes in M. tuberculosis and M. marinum infection experiments. The expression profiles are compared after aligning the developmental stages of larvae grown at an increased temperature (34 °C) in the M. tuberculosis (Mtb) infection experiment with the ones developed under normal condition (28 °C) in the M. marinum (Mm) infection. Area-proportional Venn diagrams visualize the number of the significantly up- and down-regulated genes in the stage-matched larvae of M. tuberculosis (white) and M. marinum (gray) infection. The 3 dpi Venn diagrams represent 3 dpi (3 dpf) Mtb vs. 3 dpi (4 dpf) Mm data set, the 4 dpi Venn diagrams represent 4 dpi (4 dpf) Mtb vs. 4 dpi (5 dpf) Mm data set, and the 5 dpi Venn diagrams show 5 dpi (5 dpf) Mtb vs. 5 dpi (6 dpf) Mm data set.