Cell Host Microbe 14:683C695. PhoP R112 could be dimethylated at high Mg2+. Download FIG?S1, TIF file, 2.0 MB. Copyright ? 2021 Su et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S2. Growth curves of eWT and mutants in LB and high-magnesium medium. The overnight cultures of eWT, eE8A, eD9A, eE107A, eE108A, and eR112A strains were diluted to an OD600 of 0.01 in fresh LB medium (A) or M9CA medium supplemented with 10 mM Mg2+ (B). Cultures were grown at 37C with shaking, and OD600 was measured each hour. Download FIG?S2, TIF file, 0.5 MB. Copyright ? 2021 Su et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S3. E8, D9, E107, E108, and R112 are important for the activation of PhoP and contribute to mutants. HeLa cells were infected at an MOI of 100 by exponential-phase bacterial cultures. At the same GSK-5498A time, the number of total alive bacteria was determined by plating an aliquot of culture on LB plates. At 2?h postinfection, cells were lysed, and released intracellular bacteria were enumerated on LB agar plates. Invasion efficiency was calculated by dividing the number of intracellular bacteria with the input alive bacteria and expressed as a percentage. Results are shown as mean SD. ***, test. Statistical difference was calculated between eWT and individual mutant. (B) The proliferation of the wild-type strain and mutant strains in macrophages. At 2?h or 24?h postinfection, cells were lysed and plated on LB agar plates, and bacterial colonies were counted. Bacterial replication folds GSK-5498A between 2?h and 24?h were calculated. Results are shown as mean SD; *, test. Statistical difference was calculated between eWT and individual mutant. (C) Survival rates of mice infected by intraperitoneal injection. BALB/c mice were injected intraperitoneally by 1.5??105 bacteria (wild type or mutants) in 100?l PBS or PBS of equal volume as control (seven mice/group). The mortality of mice was recorded twice per day. Mantel-Cox test was performed between eWT-infected and individual mutant-infected mice, ****, test. Statistical difference was calculated between eWT and individual mutant. (F) Bacterial burdens in ceca of mice. The ceca from streptomycin-pretreated mice were harvested 48?h after oral gavage infection and prepared as paraformaldehyde-fixed paraffin section. These sections were stained for lipopolysaccharide (LPS) (red), actin (green), and nuclei with DAPI (4,6-diamidino-2-phenylindole; blue). Images are pseudocolor representations at 200 magnification. (G) H&E-stained ceca of mice. The ceca of the wild type- or mutant-infected mice were fixed and embedded in paraffin, and then 5-m-thin sections were cut and stained with H&E. (H) Neutrophil infiltration in ceca. The paraffin section was stained by hematoxylin and incubated with the anti-MPO antibody and followed by immunohistochemistry. Blue indicates the nucleus, and claybank indicates polymorphonuclear neutrophils (PMN). Download FIG?S3, TIF file, 1.7 MB. Copyright ? 2021 Su et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S4. Structure analysis and dimer formation of PhoP E8, D9, E107, E108, and R112. (A) Conservation analysis of PhoP E8, D9, E107, E108, and SIRT7 R112. Asterisks denote the conserved E8, D9, E107, E108, and R112. The sequences were analyzed by BioEdit 7.0. (B) Interactions between E8, D9, E107, E108, and R112 and other residues. (B, Left) E8 and D9 are involved GSK-5498A in forming salt bridges with K102, which might regulate PhoP phosphorylation via acetylation. (B, Right) E107, E108, and R112 are located GSK-5498A within 4-5-5 motif, which might regulate PhoP dimerization. The relative distance between R112 and DNA is closer than the other residues, indicating its higher binding affinity with the promoter. (C) Phosphorylation GSK-5498A of PhoP. PhoP was incubated with 20 mM PAM for different time as indicated. The samples were resolved on 10% SDS-PAGE gel containing Phos tag followed by Western blotting using anti-His antibody. (D) Dimer formation of PhoP variants. PhoP and variants were subjected to cross-linking with 1 mM DSS. The samples were analyzed by Western blotting using anti-His antibody..
Categories