Pujol et al. 10.1073/pnas.0502849102. |
Supporting Table 1
Supporting Table 2
Supporting Figure 6
Supporting Figure 7
Supporting Figure 6
Fig. 6.
Analysis of intracellular replication of Yersinia pestis in IFN-g-postactivated macrophages. Bone marrow macrophages (BMM) were infected with KIM10+/GFP, and 20 min after infection, IFN-g (100 units/ml) was added (a, b, e, and f) or not (c, d) to the cells and maintained throughout the assay. After killing of the extracellular bacteria with gentamicin (8 mg/ml) for 1 h, the infected cells were either fixed (a, b) or maintained in medium containing a reduced concentration of gentamicin (2 mg/ml) during 25 h. One hour before the indicated time, isopropyl b-D-thiogalactoside (IPTG) was added to the supernatant to induce GFP expression in bacteria. Samples were analyzed by phase contrast or fluorescence microscopy. Images of GFP expression are shown (Right, b, d, f), and overlays of phase-contrast and GFP (Left, a, c, and e). (Insets) Enlargements of IFN-g -activated macrophages containing aggregates of Y. pestis in big vacuoles. Note that bacteria growing inside IFN-g-activated macrophages have the tendency to filament (arrowheads).Fig. 7.
The effect of the ripA mutation and its complementation on Y. pestis survival in postactivated macrophages. Macrophages infected with KIM6+ (a, d, g, and j), KIM6ripA (b, e, h, and k) or KIM6ripA/pripA+ (c, f, i, and l) and treated or not with IFN-g, as indicated, were fixed two (A) or 25 h postinfection (B) and labeled with an anti-Yersinia antibody (d-f; j-l). The samples were observed by phase contrast or fluorescence microscopy and overlays of phase contrast and anti-Yersinia labeling images are shown (a-c and g-i). Representative photographs are shown. Note the delay in degradation of ripA mutant in activated macrophages after 25 h of infection (k).Table 1. Y. pestis strains and plasmids used in this study
Strain names or plasmids | Relevant characteristics | Reference |
Strains | ||
KIM6+ | Biovar Medievalis, pCD1- | 1 |
KIM6+/GFP | KIM6+ containing p67GFP3.1, ApR | This work |
KIM6ripA | KIM6 + y2385::tetR, TcR | This work |
KIM6ripA/GFP | KIM6ripA containing p67GFP3.1, TcR, ApR | This work |
KIM6ripA/pripA+ | KIM6ripA containing pripA+, TcR, ApR | This work |
KIM10+ | KIM6+, pPCP1- | 2 |
KIM10+/GFP | KIM10+ containing p67GFP3.1, ApR | 3 |
KIM10 | KIM6+, pPCP1-, Dpgm | 2 |
KIM10/GFP | KIM10 containing p67GFP3.1, ApR | 3 |
KIM10/pSDR13 | KIM10 containing pSDR13, ApR | This work |
KIM10/GFP/pSDR15 | KIM10/GFP containing pSDR15, ApR, KmR | This work |
KIM10/GFP/pSDR973 | KIM10/GFP containing pSDR973, ApR, KmR | This work |
KIM10/GFP/pSDR1016 | KIM10/GFP containing pSDR1016, ApR, KmR | This work |
KIM10/GFP/pSDR973.1 | KIM10/GFP containing pSDR973.1 ApR, KmR, TcR | This work |
KIM10/GFP/pSDR973.34 | KIM10/GFP containing pSDR973.34, ApR, KmR, TcR | This work |
Plasmids | ||
pETBlue-2 | Cloning vector | Novagen |
pMMB67EH | Cloning vector, IPTG-inducible tac promoter | 4 |
pKOBEG-sacB | sacB cloned into NdeI site of pKOBEG | 5 |
p67GFP3.1 | Gfpmut3.1 cloned into XmaI-HindIII pMMB67EH | 3 |
pripA+ | Y2385 cloned into EcoRI-XbaI pMMB67EH | This work |
pSDR13 | 23.5 kb BamH1 insert from pgm locus in pUC19, ApR | 6 |
pSDR15 | 20.4 kb Sau3A insert from pgm locus in pLG338, KmR | 6 |
pSDR973 | 10.6 kb Sau3A insert from pgm locus in pLG338, KmR | 6 |
pSDR973.1 | pSDR973 with a transposon <TET-1> insertion in Y2384 | This work |
pSDR973.34 | pSDR973 with a transposon <TET-1> insertion in Y2385 | This work |
pSDR1016 | 11.9 kb Sau3A insert in pLG338, KmR | 6 |
1. Une, T. & Brubaker, R. R. (1984) Infect. Immun. 43, 895-900.
2. Perry, R. D., Pendrak, M. L. & Schuetze, P. (1990) J. Bacteriol. 172, 5929-5937.
3. Pujol, C. & Bliska, J. B. (2003) Infect. Immun. 71, 5892-5899.
4. Furste, J. P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M. & Lanka, E. (1986) Gene 48, 119-131.
5. Derbise, A., Lesic, B., Dacheux, D., Ghigo, J. M. & Carniel, E. (2003) FEMS Immunol. Med. Microbiol. 38, 113-116.
6. Fetherston, J. D., Schuetze, P. & Perry, R. D. (1992) Mol. Microbiol. 6, 2693-2704.
Table 2. Oligonucleotides used for PCR
Primer name | Sequence | Source |
TET-1 FP-1 | GGGTGCGCATGATCCTCTAGAGT | Epicentre Technologies (Madison, WI) |
TET-1 RP-1 | TAAATTGCACTGAAATCTAGAAATA | Epicentre |
973-F1 | CGCGTGTGAAATGTCGTTCT | This study |
973-R1 | CTCATGGCCATAAATCGTCAC | This study |
973-R2 | TGGCTGTTGCTGAAGAACGAC | This study |
Y2386-EcoRI | GAATTCATGACAAAATCAGTGATGATAGTTGATG | This study |
Y2386-R1 | TTACACCAAATTATGGGTGTGTGC | This study |
ripA-F1 | TATGCCGAAAGTACGGTACTTCATAAA | This study |
ripA-R1 | TTAAGGTATTCGTACATTTCTTGTTGC | This study |
ripA-EcoRI-F2 | CGGAATTCATGGATATACGTGCATTGTATG | This study |
ripA-XbaI-R2 | GCTCTAGATCAGATTAGATGGCATTTTTTTGGC | This study |
ripB-EcoR1-F2 | CGGAATTCATGAGGCTGTTGCAGGTCAGG | This study |
ripB-XbaI-R2 | GCTCTAGATCAGTATCCTGCCATTTCTTC | This study |