Tuberculous Granuloma Formation Is Enhanced by a Mycobacterium Virulence Determinant
Figure 3
ΔRD1 Is Attenuated in Zebrafish Larvae
(A) Diagram of the zebrafish embryo/larva. Arrows indicate the two injection sites used in this study.
(B) Survival of embryos infected with ΔRD1 (410 CFU) or WT bacteria (250 CFU) and null-injected embryos.
(C) Whole embryo bacterial counts of WT- and ΔRD1-infected embryos. Infecting doses: 32 CFU for WT, 36 CFU for ΔRD1. Error bars are ± SEM (** p = 0.0075 comparing 7-d postinfection WT to 7-d postinfection ΔRD1; * p = 0.05 comparing 9-d postinfection WT to 9-d postinfection ΔRD1, unpaired Student's t-test).
(D) Time of aggregate formation, showing delayed aggregation in the ΔRD1-infected embryos (n = 13) as compared to WT-infected embryos (n = 15). Infecting doses: 131 CFU for WT, 301 CFU for ΔRD1.
(E) Whole embryo bacterial counts of WT- and ΔRD1-infected embryos on day of aggregate formation. Infecting doses: 36 CFU for WT, 78 CFU for ΔRD1. Error bars are ± SEM (*** p = 0.0008, unpaired Student's t-test; ΔRD1 n = 28, WT n = 29).
(F) Fluorescent image of WT-infected embryo at 6 d postinfection with two aggregates (arrows). Scale bar, 200 μm.
(G) WT-infected embryos with higher magnification overlay of fluorescent and DIC images showing an aggregate (arrow) with individual infected macrophages that are migrating toward aggregate (arrowheads). Scale bar, 50 μm.
(H) Fluorescent image of ΔRD1-infected embryo at 6 d postinfection that has not formed any aggregates. Note the numerous infected macrophages throughout the head, body, and tail. Arrowhead and close-up insert (scale bar, 50 μm) show infected macrophages close to each other, but not aggregating. Scale bar, 200 μm.
(I) ΔRD1-infected embryo under higher-magnification overlay of DIC and fluorescent images showing three individual infected macrophages (arrowheads). Scale bar, 50 μm.