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. 2011 Jul;4(4):526-36.
doi: 10.1242/dmm.006676. Epub 2011 Mar 3.

Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component

Esther J M Stoop  1 , Tim Schipper, Sietske K Rosendahl Huber, Alexander E Nezhinsky, Fons J Verbeek, Sudagar S Gurcha, Gurdyal S Besra, Christina M J E Vandenbroucke-Grauls, Wilbert Bitter, Astrid M van der Sar
Affiliations

Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component

Esther J M Stoop et al. Dis Model Mech. 2011 Jul.

Abstract

The hallmark of tuberculosis (TB) is the formation of granulomas, which are clusters of infected macrophages surrounded by additional macrophages, neutrophils and lymphocytes. Although it has long been thought that granulomas are beneficial for the host, there is evidence that mycobacteria also promote the formation of these structures. In this study, we aimed to identify new mycobacterial factors involved in the initial stages of granuloma formation. We exploited the zebrafish embryo Mycobacterium marinum infection model to study initiation of granuloma formation and developed an in vivo screen to select for random M. marinum mutants that were unable to induce granuloma formation efficiently. Upon screening 200 mutants, three mutants repeatedly initiated reduced granuloma formation. One of the mutants was found to be defective in the espL gene, which is located in the ESX-1 cluster. The ESX-1 cluster is disrupted in the Mycobacterium bovis BCG vaccine strain and encodes a specialized secretion system known to be important for granuloma formation and virulence. Although espL has not been implicated in protein secretion before, we observed a strong effect on the secretion of the ESX-1 substrates ESAT-6 and EspE. We conclude that our zebrafish embryo M. marinum screen is a useful tool to identify mycobacterial genes involved in the initial stages of granuloma formation and that we have identified a new component of the ESX-1 secretion system. We are confident that our approach will contribute to the knowledge of mycobacterial virulence and could be helpful for the development of new TB vaccines.

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Figures

Fig. 1.
Fig. 1.
Initiation of granuloma formation in M. marinum E11 (Mma11)-infected embryos. (A) Overview of a zebrafish embryo at 28 hpf. The arrow indicates the caudal vein injection site used in this study. (B) Embryo 5 days after infection with 110 CFUs Mma11. Overlay of brightfield and fluorescent images is shown. Aggregates of red fluorescent bacteria are seen in the tail and head region. Scale bar: 500 μm. (C) Bacterial clustering in tail at 5 dpi with 92 CFUs Mma11 expressing hsp::DsRed and gap7::eGFP. Of the total amount of bacteria [constitutively expressing red fluorescence (top panel)], the majority expresses eGFP (middle panel), indicating that the bacteria reside in clusters that resemble granulomas. Overlay shows overlapping red and green fluorescence as yellow (bottom panel). Scale bar: 50 μm. (D) The Mma11 eccCb1::tn mutant is highly attenuated for initiation of granuloma formation at 5 dpi with 171 CFUs. Loose spots of red fluorescent bacteria are detected, but aggregates are not found. Scale bar: 500 μm. (E) Quantification of infection as determined with specially designed software. The amount of red (fluorescent) pixels of fluorescent images of embryos infected with the eccCb1::tn mutant is set as a percentage of the amount of red pixels of fluorescent images of embryos infected with wild-type bacteria (WT). Data shown are mean + standard deviation of three independent experiments (***P<0.001, unpaired Student’s t-test). (F) From embryos used in E, bacterial loads were determined by plating whole embryos (***P<0.001, unpaired Student’s t-test).
Fig. 2.
Fig. 2.
Three out of 200 transposon mutants were identified as early granuloma mutants. Infection of embryos with mutants FAM53, FAM58 and FAM67 reproducibly resulted in either smaller or fewer early granulomas at 5 dpi, as compared with the wild type (see Fig. 1B). (A–C) Representative overlays of brightfield and fluorescent images of embryos infected with 71 CFUs of mutant FAM53 (A), 70 CFUs of mutant FAM58 (B) and 86 CFUs of mutant FAM67 (C) are shown. Scale bars: 500 μm. (D) Relative levels of infection determined by automated quantification of fluorescent pixels. The results represent mean + standard deviation of all experiments performed (n=3 to 9) (*P<0.029, ***P<0.001, unpaired Student’s t-test).
Fig. 3.
Fig. 3.
Intracellular growth and spread of early granuloma mutants. (A) Intracellular growth of M. marinum in THP1 cells. THP1 macrophage-like cells were infected at MOI 1 with wild-type Mma11 (WT), the eccCb1::tn mutant and the three early granuloma mutants. The number of CFUs was determined by lysing infected THP1 cells and plating lysates at the indicated time points. Figure shows mean ± standard deviation for three independent experiments. (B) Growth rate of bacteria in 7H9 medium. Results represent mean ± standard deviation of two independent replicates. (C) Intercellular spread of M. marinum in THP1 cells. THP1 cells were infected at MOI 1 with wild-type Mma11 (WT), the eccCb1::tn mutant and the three early granuloma mutants. Percentage of infected cells was determined by microscopic enumeration of bacilli within THP1 cells. Mean + standard deviation of two independent replicates are shown.
Fig. 4.
Fig. 4.
Transcript levels of espLare strongly decreased in the FAM58 mutant. (A) Equivalent amounts of bacterial pellet (P) and culture supernatant (S) fractions of wild-type Mma11 and mutant FAM58 were separated on SDS-PAGE, immunoblotted and incubated with an antibody directed against EspB. As a fractionation control, the cytosolic protein GroEL2 was analyzed. (B) espL and espB transcription levels were analyzed for mutant FAM58, mutant FAM58 complemented with either espL (C58a) or the espL-espB operon (C58b) by quantitative RT-PCR and compared with wild-type Mma11 and the eccCb1::tn mutant. Additionally, transcript levels of espE and eccCb1 were measured. Transcription levels were normalized to sigA because it has previously been shown that sigA mRNA levels remain unchanged during various growth conditions. Transcript levels of mutants and complemented mutants are presented as relative values compared with wild-type levels. Data shown are mean + standard deviation for three independent replicates.
Fig. 5.
Fig. 5.
ESX-1 secretion is affected in the FAM58 mutant. Equivalent amounts of bacterial cell pellet (P) and supernatant (S) fractions of the three early granuloma mutants and mutant FAM58 complemented with either espL (C58a) or the espL-espB operon (C58b) were analyzed for the presence of the ESX-1 substrates ESAT-6 (A) and EspE (B) by SDS-PAGE separation and immunoblot, and compared with those of wild-type Mma11 and the eccCb1::tn mutant. For immunoblots with anti-EspE antibodies, in addition to pellet and supernatant samples, extracted cell wall fractions (CW) were analyzed. As a fractionation control, the cytosolic protein GroEL2 was used.
Fig. 6.
Fig. 6.
Complementation of early granuloma mutant FAM58. Embryos 5 days after infection with wild-type Mma11, mutant FAM58 and FAM58 carrying a plasmid for expression of espL (C58a) or the espL-espB operon (C58b). (A–D) Representative images of embryos for all experiments performed (n=4 to 9) are shown. Infection doses were 137 CFUs for wild-type Mma11 (A), 128 CFUs for mutant FAM58 (B), 171 CFUs for C58a (C) and 127 CFUs for C58b (D). Scale bars: 500 μm. (E) Automated quantification of relative infection level. Results represent mean + standard deviation for all experiments performed (n=4 to 9) (***P<0.001, unpaired Student’s t-test).
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