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. Author manuscript; available in PMC: 2006 Apr 10.
Published in final edited form as: Cell Microbiol. 2006 Jan;8(1):107–119. doi: 10.1111/j.1462-5822.2005.00604.x

Fig. 9.

Fig. 9

Perforation of LAMP1-positive compartments by LLO was inefficient.

A. Macrophages expressing YFP-CBD were infected with wild-type or iLLO Lm to produce <1 bacterium per macrophage and were fixed at the indicated time points. Infected cells were identified by DAPI staining and cytosolic bacteria were identified by YFP-CBD labelling. YFP-CBD labelled wild-type Lm (grey circles) by 60 min. Escape of iLLO Lm treated with 10 mM IPTG before infection (IPTG 0′, black circles) was reduced relative to wild type. iLLO Lm treated with IPTG at 10 min after infection (IPTG 10′, triangles) were present in the cytosol at 60 min. iLLO Lm treated with IPTG at 30 min (IPTG 30′, diamonds) did not become CBD-labelled until 90 min, at which point ~0.5% were YFP-CBD-positive. In the absence of IPTG (open squares, partially obscured by IPTG 30′ data), no Lm were CBD-labelled. At each time point >100 infected cells were scored for the presence of YFP-CBD. Each data point represents the mean ± SEM of two experiments.

B. Overnight cultures of iGFP Lm or macrophages infected with iGFP Lm were fixed at the indicated time points. Intracellular bacteria were identified by DAPI staining and GFP-producing bacteria were identified by indirect immunofluorescence. iGFP Lm treated with IPTG in broth (black circles) stained positive for GFP by 10 min. iGFP Lm treated with IPTG at 10 min after infection stained positive for GFP from 30 to 120 min (black triangles), whereas iGFP Lm treated with IPTG at 30 min stained positive for GFP from 60 to 120 min (black diamonds). iGFP Lm did not stain positive for GFP in the absence of IPTG treatment (open squares). At each time point 100 bacteria were scored for Alexa Fluor 594 staining. Each data point represents the mean ± SEM of two experiments.

C. Uninduced iLLO Lm acquired LAMP1-CFP by 20 min after infection, but did not decorate with YFP-CBD at any time (open squares).

D. After treatment with IPTG at 10 min after infection, iLLO Lm escaped from LAMP1-CFP-negative compartments at 60–90 min (closed triangles). Many CBD-negative vacuoles acquired LAMP1-CFP as early as 20 min (open triangles).

E. In cells treated with IPTG at 30 min after infection, iLLO Lm perforated LAMP1-CFP-positive vacuoles at 90–100 min (closed diamonds).