Fig. 3

Lysosomal Trafficking Is a Host-Beneficial Process, which Is Counteracted by Bacterial MarP

(A) Confocal images of 3 dpf larvae that were infected with Mm in the CV at 2 dpf and then treated for 24 hr with 50 nM Bafilomycin (Baf) or DMSO control. Arrowheads denote bacteria labeled with pHrodo, while arrows show pHrodo-negative bacteria.

(B) Percent of Mm colocalizing with pHrodo at 24 hpi as shown in (A), representative of two experiments.

(C) Confocal images of 3 dpf larvae that were infected in the CV with blue fluorescent Mm at 2 dpf and stained with red LT at 3 dpf following treatment with DMSO, 50 nM Baf, or morpholino targeting atp6v1a at 0 dpf. Arrowheads denote bacteria that colocalize with LT, while arrows denote LT-negative bacteria.

(D) Bacterial burden (FPC) measured at 24 hpi with 300 Mm; fish were randomly assigned to DMSO or 50 nM Baf treatment immediately after infection, representative of three experiments.

(E) Bacterial burden measured at 24 hpi with 300 Mm in larvae injected at 0 dpf with a morpholino targeting atp6v1a or control, representative of two experiments.

(F) Average intramacrophage burden at 40 hpi with 60 Mm into the CV comparing larvae treated with DMSO or 50 nM Baf following infection, or injected with a morpholino targeting tnfr1 at 0 dpf, which was used as a positive control, representative of three experiments.

(G) Bacterial burden measured at 3 dpi following infection with wild-type Mm, marP::Tn, marP::Tn transformed with a plasmid containing Mtb marP, or marP::Tn transformed with a plasmid containing Mtb marP with a mutation in the active site serine (S343A), representative of two experiments.

(H) Bacterial burden at 1 dpi following infection with wild-type or marP::Tn Mm and treatment with either DMSO or 50 nM Baf, representative of three experiments. The fold increase in burden following treatment with Baf is shown for wild-type and marP::Tn.

(I) Fold increase in bacterial burden during infection with wild-type or marP::Tn Mm following treatment with 50nM Baf. Shown is the average of four experiments as in (H) ± SEM.

(J) Bacterial burden measured at 0, 3, and 5 dpi following infection with 250 wild-type or ptpA::Tn Mm with mean and 95% CI shown, representative of three experiments.

(K) Bacterial burden at 0, 3, and 5 dpi following infection with wild-type or marP::Tn Mm with mean and 95% CI shown, representative of three experiments. Significance tested using two-tailed unpaired t test (B, D, E, and I-K) or one-way ANOVA with Tukey’s post-test (F-H). Each point in (B) and (D)-(H) represents one larva. See also Figure S3.