(A and B) The whiB4::Tn mutant is attenuated in zebrafish. (A) Adult zebrafish (n = 20) were infected with the indicated strains at 10⁴ CFU bacteria per fish and were monitored for mortalities over an 18-week period. The survival curves were plotted using the Kaplan-Meier method and differences between curves were analyzed using the log-rank test. **, P < 0.01; ****, P < 0.0001. (B) In parallel experiment, zebrafish (n = 20) were infected with the same strains at 10⁴ CFU bacteria per fish, and at indicated time points (2, 6, and 12 weeks) postinfection, 3 live fish per group were sacrificed and bacterial burdens were determined. Data are plotted as mean ± standard error of the mean (SEM). Unpaired Student’s t test: *, P < 0.05; **, P < 0.01. (C and D) whiB4::Tn in infected zebrafish is more tolerant to antibiotic killing than WT. In a separate experiment, zebrafish were infected with the indicated strains at a dose of 10⁴ CFU/fish. At 2 weeks postinfection, animals (n = 5) were treated with ethambutol (1 mM), rifampicin (0.4 mM), or moxifloxacin (0.3 mM) for 1 or 2 weeks, and they were then sacrificed and bacterial burdens were determined. Data are plotted as mean ± standard deviation (SD). Unpaired Student’s t test: *, P < 0.05; **, P < 0.01. COM, the complemented strain.

The whiB4::Tn mutant induces partially necrotic and stable granulomas. Zebrafish were infected with the indicated strains as described in Fig. 1A and B. At different time points (2, 6, and 12 weeks) postinfection, animals (n = 3) were sacrificed and subjected to histological analysis. Ziehl-Neelsen acid fast staining: panels A to F and J to L. Hematoxylin and eosin (H&E) staining: panel B (inset) and panels G to I and M to O. COM, the complemented strain.

The whiB4::Tn mutant induces lower levels of host immune response than the WT strain. RNA-seq analysis of kidneys isolated from zebrafish infected with whiB4::Tn or WT at 2 weeks postinfection was performed. The identified DEGs were subjected to GO analysis (A) and KEGG pathway analysis (B). Each circle represents a biological process or pathway, and the size of the circle is proportional to the number of genes detected. (C) Map DEGs involved in the cytokine-cytokine receptor interaction pathway. Green, downregulated; red, upregulated. (D) RT-qPCR validation of selected DEGs. Heatmap of 18 DEGs analyzed by RT-qPCR. The mRNA levels were normalized against WT and 18S rRNA was used as an internal control. COM, the complemented strain.

The whiB4::Tn mutant is defective in resuscitation from hypoxia-induced dormancy. (A) Growth of WT, whiB4::Tn, and the complemented strain (COM) under hypoxia. Data (mean ± SD) are from three biological replicates. There was no significant difference on the growth curves as analyzed by two-way analysis of variance (ANOVA). (B) Growth of hypoxic cultures of WT, whiB4::Tn, and the complemented strain (COM) after reaeration. Data (mean ± SD) are from three biological replicates. The CFU of whiB4::Tn was significantly lower than that of WT or the complemented strain at 48 and 72 h postaeration. *, P < 0.05; ****, P < 0.0001, two-way ANOVA. (C) Overlaps of DEGs in whiB4::Tn and the complemented strain (COM). Genes that were downregulated in whiB4::Tn relative to WT overlapped significantly with genes that were upregulated in the complemented strain relative to WT at 24 and 48 h postaeration. (D) Heatmap of the mRNA levels of the DosR regulon and sigE. The mRNA level of each gene was normalized against the value of the same gene at 0 h time point (7-day hypoxic cultures) and plotted as log₂.

WhiB4 binds dosS in hypoxic culture upon aeration. (A) Overlap of the WhiB4 binding sites in bacteria grown under different conditions. (B) WhiB4 binding sites at the dosR-dosS locus. Binding sites 1 and 2 were detected in cultures at 0.5 h postaeration. Binding sites 1 and 3 were detected in hypoxic cultures without aeration. The numbers in black are the genome coordinates of the indicated genes, and the numbers in red indicate the binding site of WhiB4. (C) Model for the WhiB4-mediated regulation of the DosR regulon. Upon aeration of hypoxic cultures, WhiB4 binds directly to dosS and inhibits its expression, and consequently the expression of the DosR regulon is repressed.