Supplementary Figure 2: Deletion of PtpA in BCG increases IL-12p40 production in infected mice, reduces acid-fast bacilli accumulation in lungs, and mitigates liver pathology of the infected mice.

(a) Deletion of PtpA in BCG increases the protein levels of IL-12p40 in mice infected intratracheally with 2.0 × 10⁶ CFU of wild-type BCG, BCG△PtpA, or BCG (△PtpA + PtpA) for 0–20 days. IL-12p40 protein levels in serum from the infected mice were analyzed by ELISA assay. (b) Lung sections of mice infected with BCG△PtpA had a marked reduction of acid-fast bacilli at day 12 post-infection. Ziehl-Neelsen staining for acid-fast bacilli (arrows) was performed for lung sections of the mice infected as in (a). Scale bars, 5 μm. (c, d) Deletion of PtpA in BCG reduces total cellular and neutrophilic infiltration in the livers of the mice infected as in (a). Enlarged views of the boxed regions in (c) are shown in (d). Foci of cellular infiltration and neutrophils (with a lobulated nucleus) are indicated by arrows in (c) and (d), respectively. Liver sections from BCG-infected mice were stained with hematoxylin and eosin. Scale bars, 200 μm (c) or 20 μm (d). (e) The △PtpA mutant strain cultivated in 7H9 media under laboratory conditions exhibits no growth defect as compared to the wild-type BCG strain. A total of 1 × 10⁶ CFU of mycobacterial strains were inoculated into Middlebrook 7H9 broth supplemented with 10% oleic acid-albumin-dexrose-catalase (OADC) and 0.05% Tween-80 and cultivated at 37 °C, and then three sets of serial 10-fold dilutions of the culture from each time point were plated on 7H10 agar for bacterial colony forming unit (CFU) counting after 3–4 weeks of incubation at 37 °C. *P < 0.05 and **P < 0.01 (two-tailed unpaired t-test). Data are representative of one experiment with two independent biological replicates (a – d; mean and s.e.m., n = 6) or are representative of one experiment with at least three independent biological replicates (e; mean and s.e.m., n = 3).