Salmonella inhibits retrograde trafficking of mannose-6-phosphate receptors and lysosome function

Abstract

Salmonella enterica is an intracellular bacterial pathogen that replicates within membrane-bound vacuoles through the action of effector proteins translocated into host cells. Salmonella vacuoles have characteristics of lysosomes but are reduced in hydrolytic enzymes transported by mannose-6-phosphate receptors (MPRs). We found that the effector SifA subverted Rab9-dependent retrograde trafficking of MPRs, thereby attenuating lysosome function. This required binding of SifA to its host cell target SKIP/PLEKHM2. Furthermore, SKIP regulated retrograde trafficking of MPRs in noninfected cells. Translocated SifA formed a stable complex with SKIP and Rab9 in infected cells. Sequestration of Rab9 by SifA-SKIP accounted for the effect of SifA on MPR transport and lysosome function. Growth of Salmonella increased in cells with reduced lysosomal activity and decreased in cells with higher lysosomal activity. These results suggest that Salmonella vacuoles undergo fusion with lysosomes whose potency has been reduced by SifA.

Fig 1. The SPI-2 T3SS interferes with Syn10-dependent retrograde trafficking of MPRs and reduces lysosome function.

(A and B) HeLa cells were infected for 14 hours with green fluorescent protein (GFP)–expressing wild-type (wt) (Aa and c) or ssaV mutant (Ab and d) Salmonella (blue) or (B) a replication-deficient aroC,purD mutant (23) that hasa functional SPI-2 T3SS (fig. S1C). Cells were immunolabeled for TGN46 (green) and CD-MPR (red) (a and b) or CI-MPR (red) (c and d). Scale bars, 10 μm. (B) Quantitation of mean fluorescent signals of TGN-associated TGN46 (24), CD-MPR, and CI-MPR, analyzed by three-dimensional (3D) confocal microscopy and normalized to signals in uninfected cells from the same sample. Error bars in all figures indicate SEM. (C to E) Plasma membrane–TGN transport assays. HeLa cells either were subjected to siRNA to deplete Syn6 or infected with wt or ssaV mutant Salmonella (blue in E) for 14 hours, then pulse-chased with either fluorescent CTxB [red in (E)] or CI-MPR antibody [red in (E)]. Fixed cells were immunolabeled for CI-MPR and TGN46 [green in (E)] or Rab6 (to identify the TGN in si Syn6– and si Scramble–treated cells), and TGN-associated CI-MPR or CTxB was quantified. Scale bars, 10 μm. (F and G) Rerouting of lysosomal enzymes. (F) HeLa cells were exposed to dimethyl sulfoxide (DMSO) or amiodarone (ami) [which redistributes MPRs (13)] and were not infected (ni) or infected with wt or ssaV mutant Salmonella for 14 hours. Secreted levels of cathepsin D (CatD) or β-hexosaminadase (β-hexo) are a percentage of total levels. (G) Intra- and extracellular proteins were analyzed by immunoblotting, using actin as a loading control. (H and I) Cathepsin B (CatB) activity was quantified by measuring fluorescent cleavage product of Magic Red-RR in living cells. (H) HeLa cells were infected for 14 hours with wt or ssaV mutant Salmonella and exposed to Magic Red-RR, and steady-state fluorescent product intensity was measured by flow cytometry. Bars represent the percentage of fluorescence of infected compared with uninfected cells in the same sample. (I) Cells as in (H) were photobleached and analyzed for new fluorescent product. Data were normalized to the mean fluorescence of uninfected cells. Vertical lines represent SEM for each of 100 time points. Statistically significant relationships are denoted (*P < 0.05; **P < 0.01; ***P < 0.005).

Fig 2. SifA inhibits lysosome function.

(A) Quantitation from 3D confocal microscopy of the mean fluorescence signals of TGN-associated CD-MPR in HeLa cells infected with the indicated strains. (B) HeLa cells infected for 14 hours with sifA, sseJ double mutant Salmonella expressing SifA-HA or SifA_L130D-HA were immunolabeled for HA (blue), TGN46 (green), and CD-MPR (red). (C) Magic Red-RR recovery assay of CatB activity in HeLa cells infected for 14 hours with the indicated strains. (D) HeLa cells were infected for 14 hours with the indicated Salmonella strains and exposed to Magic Red-RR, and the percentage of fluorescent product intensity of infected cells compared with uninfected cells from the same sample was measured at steady state by flow cytometry. (E) HeLa cells were infected as above and pulse chased with DQ-bovine serum albumin (DQ-BSA, Life Technologies, Paisley, UK), and the percentage of fluorescence between infected and uninfected cells from the same sample was measured by flow cytometry. (F) BALB/c mice were inoculated orally with the indicated Salmonella strains. After 48 hours, CD11b(+) splenocytes were harvested and subjected to Magic Red-RR assay. Bars represent the percentage of steady-state fluorescence of infected cells compared with uninfected cells from the same sample or of cells from uninfected mice compared with uninfected cells from infected animals. (G) HeLa cells were transfected with vectors encoding GFP-SifB, GFP-SifA, or GFP-SifA_L130D, and 24 hours later cells were subjected to Magic Red-RR assay. In each case, fluorescence intensity in cells expressing similar levels of the indicated protein was normalized to that of untransfected control cells within the same sample and then to fluorescence caused by expression of SifB. Statistically significant relationships are denoted (*P < 0.05; **P < 0.01; ***P < 0.005).

Fig 3. SKIP negatively regulates lysosome function.

(A and B) HeLa cells depleted of SKIP by siRNA or exposed to scrambled siRNA were analyzed for TGN-associated CD-MPR (A) and by Magic Red-RR assay of recovery of CatB activity after photobleaching (B). (C) Steady-state levels of CatB activity and DQ-BSA hydrolysis analysed by flow cytometry. For each substrate, the data were normalized to that obtained by using scrambled siRNA. (D) Skip^−/− MEFs expressing either an N-terminal domain of SKIP (N550) or full-length protein were analyzed for steady-state levels of CatB by Magic Red-RR assay. Data are normalized to SKIP (N550) samples. (E) HeLa cells were depleted of SKIP by siRNA (si SKIP) or exposed to scrambled siRNA and subjected to plasma membrane–TGN transport assays using fluorescent CTxB or CI-MPR antibody. Data were analyzed by 3D confocal microscopy and were normalized to that obtained by using scrambled siRNA. (F) Wild-type (Skip^+/+) or Skip^−/− MEFs were transfected with vectors encoding either GFP-SifA or GFP-SifB and assayed for CatB activity by Magic Red-RR. Bars represent the percentage of fluorescence of transfected cells compared with untransfected cells from the same samples, normalized to GFP-SifB in either Skip^+/+ or Skip^−/− cells. Statistically significant relationships are denoted (*P < 0.05; **P < 0.01; ***P < 0.005).

Fig 4. Interactions between SifA, SKIP, and Rab9.

(A) HeLa cells depleted of SKIP (si SKIP) or infected for 14 hours with wt or sifA,sseJ double mutant Salmonella were fixed, labeled, and analyzed by quantitative confocal immunofluorescence microscopy. Pearson’s correlation coefficient was calculated between Rab9 and CD-MPR and compared to noninfected or si Scramble–treated cells. (B) HeLa cells stably expressing Flag-SKIP and GFP-Rab9 were infected for 14 hours with sifA,sseJ mutant Salmonella expressing either SifA_wt-HA or SifA_L130D-HA. Cells were fixed, labeled, and analyzed by confocal immunofluorescence microscopy. Arrows indicate co-localization of the three proteins. (C) Cells infected as in (B) were lysed and proteins were immuno-precipitated with GRP antibody–conjugated beads, or protein G (prG)–beads as a control. GFP-Rab9, SifA-HA, SifA_L130D-HA, and Flag-SKIP were detected in input samples (input) and after immunoprecipitation (output) by means of SDS–polyacrylamide gel electrophoresis and immunoblotting. (D) Model depicting interference of Rab9-mediated retrograde transport of MPR to the TGN by sequestration of Rab9 by SifA and SKIP. (E) HeLa cells treated with scramble, Syn6, or Syn10 oligos or with lysosome inhibitor were infected with GFP-expressing wild-type Salmonella. Bacterial load, measured as GFP fluorescence per HeLa cell, was measured by flow cytometry (n > 10,000). The % fluorescence/cell represents the ratio between the median GFP fluorescence at 2 and 8 hours after infection, normalized to mock-transfected or DMSO-treated cells. Statistically significant relationships are denoted (*P < 0.05).