Killing of Δpgm Y. pestis in postactivated macrophages depends on iNOS expression. BMM from iNOS^+/+ (a- d) or iNOS^-/- (e- h) mice were infected with KIM10+/GFP (a, b, e, and f) or KIM10/GFP (c, d, g, and h), exposed to IFN-γ, and processed for phase and fluorescence microscopy as described in the legend of Fig. 1.

Role of the pgm locus in the replication of Y. pestis in naïve macrophages. Unactivated BMM were infected with KIM10+/GFP or KIM10/GFP for 20 min before gentamicin addition to the medium (0 h postinfection). At the indicated times postinfection, intracellular bacteria were recovered and enumerated by cfu assay. Fold increase values are normalized relative to the 1-h value and represent averages of triplicate samples from one representative experiment. Error bars show the standard deviations.

Δpgm Y. pestis does not survive in postactivated macrophages. BMM were infected with KIM10+/GFP (pgm⁺)(a, b, e, and f) or KIM10/GFP (Δpgm) (c, d, g, and h) for 20 min. Gentamicin was then added to prevent survival of extracellular bacteria. The BMM were then left untreated (a- d) or exposed to IFN-γ (e- h; postactivated). After 24 h of infection, isopropyl β-d-thiogalactoside was added to the infected cells to induce GFP expression. One hour later, the samples were fixed and examined by fluorescence or phase microscopy. Representative images were captured by digital photography. Overlays of GFP expression and phase-contrast images are shown (a, c, e, and g).

Reduction of NO levels in postactivated macrophages infected with pgm⁺ Y. pestis is not controlled at the level of iNOS expression. (A) BMM infected with KIM10+/GFP or KIM10/GFP were exposed to IFN-γ. Uninfected BMM were exposed to LPS and IFN-γ. Twenty-five hours after infection or LPS/IFN-γ exposure, the nitrite concentration in the supernatant of the macrophages was measured. Nitrite levels in infected cells were normalized relative to the nitrite levels in uninfected macrophages exposed to LPS/IFN-γ. Each bar represents the average of five infections from two independent experiments. (B) Uninfected BMM exposed to LPS, or BMM infected with KIM10+/GFP or KIM10/GFP, were incubated for 25 h in the presence or absence of IFN-γ. Lysates of the infected macrophages were analyzed by immunoblotting with an anti-iNOS antibody or with an anti-SKAP-HOM antiserum to control for loading.

Identification of rip genes in the pigmentation segment. (A) Map of the pgm locus. The pgm locus contains the pigmentation segment that encodes the hemin storage locus (hms) and the HPI that encodes the yersiniabactin locus (ybt) and carries a P4-like integrase gene (int). Positions of inserts in recombinant plasmids are indicated below the map. (B) BMM were infected with the indicated strains, treated or not with IFN-γ, and processed for microscopy, as in the legend to Fig. 1. (C) ORF map of the insert in pSDR973 and comparison with SPI6. The insert in pSDR973 corresponds to nucleotides 2624985-2634761 of the KIM chromosome (48). ORFs shown as arrows are named according to the nomenclature of Deng et al. (48) or according to our nomenclature for ripA and ripB. In the right end of the insert, black parallel lines indicate a truncation of Y2387 in the insert. Positions and identification numbers of transposon insertions in the insert are indicated as arrowheads below the ORF map. Filled arrowheads represent insertions that prevented Y. pestis replication in postactivated macrophages. The SPI6 (S.e.) is compared with the rip region (percent identities in parentheses). (D) The concentration of nitrite in the supernatant of postactivated macrophages infected with the indicated strains was assessed 25 h after infection as described in the legend to Fig. 3A. Each bar represents the average of values obtained in single infections from three independent experiments. Error bars show the standard deviations.