Drosophila melanogaster is a genetically tractable model host for Mycobacterium marinum

Abstract

Mycobacterium marinum is a pathogenic mycobacterial species that is closely related to Mycobacterium tuberculosis and causes tuberculosis-like disease in fish and frogs. We infected the fruit fly Drosophila melanogaster with M. marinum. This bacterium caused a lethal infection in the fly, with a 50% lethal dose (LD(50)) of 5 CFU. Death was accompanied by widespread tissue damage. M. marinum initially proliferated inside the phagocytes of the fly; later in infection, bacteria were found both inside and outside host cells. Intracellular M. marinum blocked vacuolar acidification and failed to colocalize with dead Escherichia coli, similar to infections of mouse macrophages. M. marinum lacking the mag24 gene were less virulent, as determined both by LD(50) and by death kinetics. Finally, in contrast to all other bacteria examined, mycobacteria failed to elicit the production of antimicrobial peptides in Drosophila.We believe that this system should be a useful genetically tractable model for mycobacterial infection.

FIG. 1.

Survival of wild-type (Oregon R) flies infected either with M. marinum strain M carrying the msp12::GFP plasmid or with M. smegmatis strain JCM89. The error bars indicate 95% confidence intervals. Groups of 30 to 50 flies were injected with different quantities of M. marinum or M. smegmatis freshly diluted into sterile 7H9 medium containing OADC, 0.2% Tween 80, and 30 μg of apramycin per ml (or 30 μg of kanamycin per ml for M. smegmatis). Uninfected flies were injected under the same conditions with the same volume of sterile medium. Dead flies were counted at 24-h intervals. A total of 428 animals were infected with 500 CFU of M. marinum, 340 animals were infected with 50 CFU of M. marinum, 139 animals were infected with 5 CFU of M. marinum, 50 animals were infected with 3,000 CFU M. smegmatis, and 288 animals were not infected. (A) Survival after infection with 5 to 500 CFU of M. marinum. (B) Survival after infection with 500 CFU of M. marinum (Mm) compared with survival after infection with 1,000 CFU of M. smegmatis (Ms).

FIG. 2.

y w flies, with the wings removed, infected with 500 CFU of msp12::GFP M. marinum and examined by GFP fluorescence at different times after infection. Panels A to D are photographs of one infected fly, panels E to J are photographs of a second fly, and panels K to M are photographs of a third fly. (A to D) Dorsal view of a whole fly. (A) Bright-field view before infection. The dashed rectangle indicates the approximate area seen more closely in panels E to M. (B) GFP fluorescence visible before infection. (C) The infection first became visible at 96 h at this magnification; fluorescence was initially visible near the dorsal midline at the anterior end of the abdomen (arrowhead). (D) The first visible focus of fluorescence expanded steadily through the rest of the infection. More fluorescent foci were visible at 144 h in the thorax (arrowhead), as well as in the legs and head (visible but out of the plane of focus). By 168 h, this fly was dead. (E to J) Close views of a second fly, showing the punctate fluorescence characteristic of the first stage of infection. (E) Bright-field view before infection. (F) GFP fluorescence visible before infection. (G to J) Foci of infection were visible as early as 8 h after bacterial injection (arrowhead in panel G). The number of foci steadily increased during the first 72 to 96 h of infection, and the foci were a different color than the autofluorescence visible in panel F. (K to M) Close views of a third fly, showing the spreading patches of fluorescence characteristic of the second stage of infection. Panel K is significantly dimmer than panel J because the brightness scale was changed for panels K to M in order to make the brighter fluorescence in panel M more interpretable. The dashed line in panel L shows the plane of section used for Fig. 3. Scale bar in panel A (for panels A to D), 1 mm. Scale bar in panel E (for panels E to M), 0.1 mm.

FIG. 3.

Histological examination of M. marinum-induced pathology. (A and B) Sections through uninfected (A) and infected (B) animals, roughly in the plane shown by the dashed line in Fig. 2L. The infected animal was examined 96 h after injection of 5,000 CFU of M. marinum. The cuticle (open arrowhead), dorsal vessel (solid arrowhead), muscle (solid arrow), and fat body (open arrow) are indicated. (C and D) Close-ups of the areas indicated by the rectangles in panels A and B. Extracellular (solid arrow) and intracellular (solid arrowhead) bacteria are evident, as is a small abscess of the fat body (open arrowhead).

FIG. 4.

Failure of internalized M. marinum to colocalize with phagocytosed E. coli or with acidified vesicles. (A, D, G, and J) Hemocyte from a larva injected with both mag24::GFP M. marinum and TRITC-labeled dead E. coli, bled and examined 48 h after injection. (B, E, H, and K) Hemocyte from an animal injected with both mag85::GFP M. marinum and TRITC-labeled dead E. coli, bled and examined 48 h after injection. (C, F, I, and L) Hemocyte from an animal injected with msp12::GFP, incubated for 24 h, injected with Lysotracker Red DND-99, bled, and examined. In each case, the red fluorescence (dead E. coli or Lysotracker) failed to colocalize with the internalized mycobacteria.

FIG. 5.

Effect of blockage of phagocytosis on mag24 activation and progression of disease. The animals shown were all infected in parallel, as part of a single experiment. (A) Typical fly shown under bright-field illumination in order to orient the views in panels B to F. (B and C) Flies injected with water and then 3 days later with M. marinum carrying the msp12::GFP (B) or mag24::GFP (C) plasmid, examined for green fluorescence 5 days after infection. The patterns of infection in the two animals are apparently identical. (D to F) Flies injected with polystyrene beads to block phagocytosis and then 3 days later with sterile medium (D) or M. marinum carrying the msp12::GFP (E) or mag24::GFP (F) plasmid, examined for fluorescence 5 days after infection. In animals injected with beads first, the anterior dorsal abdomen was no longer the predominant focus of infection (E). Moreover, the mag24 promoter was not activated in these animals (F).

FIG. 6.

Survival of Oregon R flies infected with wild-type M. marinum (WT) or the L1D mutant of M. marinum. The error bars indicate 95% confidence intervals. Groups of 30 to 50 animals were injected with different quantities of M. marinum freshly diluted into sterile 7H9 medium containing OADC and 0.2% Tween 80. Uninfected flies were injected under the same conditions with the same volume of sterile medium. Dead flies were counted at 24-h intervals (i.e., 2 days postinfection [48 h postinfection]).

FIG. 7.

Antimicrobial peptide transcripts are not upregulated in response to M. marinum. (A to H) Animals injected with sterile Luria broth and 7H9 (A and E), L. monocytogenes (B and F), M. marinum (C and G), or M. smegmatis (D and H) and carrying GFP reporters for drosocin (A to D) and metchnikowin (E to H). M. marinum did not induce expression of these antimicrobial agents at levels above the levels induced by the process of injection (compare panels A to D with panels E to H). (I) Animals were injected with sterile medium (mock), S. enterica serovar Typhimurium (S.t.), L. monocytogenes (L.m.), or M. marinum (M.m.). The levels of mRNA encoding attacin A, cecropin A1, and diptericin were determined 6, 30, or 50 h after infection by quantitative real-time RT-PCR. The error bars indicate standard deviations. The experiment was repeated three times. In each case, antimicrobial agents were induced by L. monocytogenes and S. enterica serovar Typhimurium but not by M. marinum. The transcript levels were normalized to the expression level of Drosophila ribosomal protein 15A.

FIG. 8.

Survival of Oregon R, spaetzle, and kenny flies infected with M. marinum. The error bars indicate 95% confidence intervals. Groups of 30 to 50 animals were injected with different quantities of M. marinum freshly diluted into sterile 7H9 medium containing OADC and 0.2% Tween 80. Uninfected flies were injected under the same conditions with the same volume of sterile medium. Dead flies were counted at 24-h intervals (i.e., 2 days postinfection [48 h postinfection]). spz²/spz^rm7 flies were used because the chromosomes carrying each of the two spaetzle alleles also carry different recessive lethal mutations.