pubmed.ncbi.nlm.nih.gov

pH sensing by intracellular Salmonella induces effector translocation - PubMed

  • ️Fri Jan 01 2010

pH sensing by intracellular Salmonella induces effector translocation

Xiu-Jun Yu et al. Science. 2010.

Abstract

Salmonella enterica is an important intracellular bacterial pathogen of humans and animals. It replicates within host-cell vacuoles by delivering virulence (effector) proteins through a vacuolar membrane pore made by the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS). T3SS assembly follows vacuole acidification, but when bacteria are grown at low pH, effector secretion is negligible. We found that effector secretion was activated at low pH from mutant strains lacking a complex of SPI-2-encoded proteins SsaM, SpiC, and SsaL. Exposure of wild-type bacteria to pH 7.2 after growth at pH 5.0 caused dissociation and degradation of SsaM/SpiC/SsaL complexes and effector secretion. In infected cells, loss of the pH 7.2 signal through acidification of host-cell cytosol prevented complex degradation and effector translocation. Thus, intravacuolar Salmonella senses host cytosolic pH, resulting in the degradation of regulatory complex proteins and effector translocation.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. SsaL is required for translocon protein secretion, suppresses effector secretion, and interacts with SsaM and SpiC.

(A) Model of the SPI-2 T3SS spanning the inner and outer membranes of the bacterial cell and connected to a translocon pore formed in the vacuolar membrane. Translocon proteins must be secreted before effectors can be translocated. SsaV is thought to be located in the inner membrane and is essential for the function of the secretion system. (B) Wild-type (wt), ssaV, spiC, ssaM, or ssaL deletion mutant strains expressing 2HA-tagged SseJ from chromosome were grown in minimal medium pH 5.0, and secreted and bacterial-associated (lysate) proteins were examined by means of immunoblotting to detect the HA epitope, SseB, and DnaK. (C) A plasmid expressing SsaL-2HA was introduced into the ssaL deletion mutant, and secreted levels of SseJ-2HA, SseB, and SseC were compared with the ssaL mutant by means of immunoblotting. (D) Interaction between SpiC-2HA, T7-SsaM, and SsaL-3Flag. In the wt-3tag strain, spiC, ssaM, and ssaL are replaced with versions expressing epitope-tagged proteins. This and an isogenic strain lacking SpiC (spiC) were grown in minimal medium pH 5.0, and whole-cell lysates were immunoprecipitated with an antibody to T7. The presence of the three proteins was detected in input samples (input) and after immunoprecipitation (output) by means of immunoblotting.

Fig. 2
Fig. 2. Phenotypes of SsaL and SsaM variants that block ternary complex formation.

(A) The SsaL/SpiC/SsaM complex requires the C-terminal 18 amino acids of SsaM. A strain expressing SsaL-3Flag, glutathione S-transferase (GST)–SpiC, and either SsaM-2HA or a nonfunctional version lacking its C-terminal 18 amino acids (SsaM104-2HA) (4) were grown in minimal medium pH 5.0, and whole lysates were used for GST pull-down (GST-SpiC) or immunoprecipitation (SsaL-3Flag, SsaM-2HA, and SsaM104-2HA). (B) Plasmids encoding T7-SsaM and 2HA-tagged SsaL or mutant variants were introduced into an ssaL deletion mutant. Whole bacterial lysates were immunoprecipitated with antibody to HA. SsaL-2HA and T7-SsaM were detected in input samples (input) and after immunoprecipitation (output) by means of immunoblotting. (C) The ssaL deletion strain expressing SseB and SseJ-2HA, and SsaL or mutant variants from a plasmid, were grown in minimal medium pH 5.0 for 5 hours. Secreted fractions were analyzed by means of immunoblotting for SseB and SseJ-2HA. (D) The wild-type strain, an ssaM mutant, and the mutant with or without a plasmid expressing SsaM-2HA or SsaM104-2HA were grown at pH 5.0 and analyzed as in (C).

Fig. 3
Fig. 3. Effect of pH on effector secretion and translocation.

(A) Bacterial strains were grown in minimal medium pH 5.0 for 4 hours, then exposed to pH 5.0 or 7.2 for 90 min. Secreted and bacteria-associated (lysate) 2HA-tagged effectors and DnaK were examined by means of immunoblotting. (B) HeLa cells were infected with wild-type or ssaV mutant Salmonella for 3.5 hours in order to allow expression of the SPI-2 T3SS, then some samples were permeabilized with digitonin and exposed to pHe 6.0. Cells were fixed 2.5 hours later and immunolabeled in order to detect Salmonella and secreted SseB. (C) HeLa cells were infected for 3.5 hours with wild-type Salmonella expressing SseF-2HA, then permeabilized with digitonin and exposed to pH 6.0 or 7.2 for a further 2.5 hours. In one sample, pHe was changed from 6.0 to 7.2, 1 hour before fixation. Fixed cells were immunolabeled to detect Salmonella, LAMP-1, and SseF-2HA. In (B) and (C), values below the images represent the percentage of cells in which secreted SseB or translocated SseF-2HA was detected, ±SE of three experiments (n > 100 cells per experiment). Scale bars, 2 µm.

Fig. 4
Fig. 4. Effect of pH on the SsaL/SsaM/SpiC complex.

(A) The wt-3tag strain was grown in minimal medium at pH 5.0 for 4 hours, then exposed to pH 5.0 or 7.2 for 1 hour, then membrane-associated and cytosolic SpiC-2HA and membrane-associated T7-SsaM were immunoprecipitated (IP). Proteins were detected in input samples (input) and after immunoprecipitation (output) by means of immunoblotting. (B) The wt-3tag strain and a strain in which the chromosomal copy of ssaN is replaced with a functional version carrying a 3Flag epitope (bottom) were subjected to pH shift in the presence of tetracycline. Samples were removed at various times, and lysates were analyzed by means of immunoblot. (C) HeLa cells were infected with the wt-3tag strain (wt) or an isogenic translocon mutant (sseA-D). At 3.5 hours after invasion, some wt-infected cells were treated with digitonin (wt + dig), and exposed to pHe 6.0 for 2.5 hours (6.0) or pHe 6.0 for 1.5 hours, then changed to pHe 7.2 for another 1 hour (6.0 → 7.2). Cells were lysed at 6 hours after invasion and analyzed by means of immunoblotting.

Comment in

Similar articles

Cited by

References

    1. Galán JE, Wolf-Watz H. Nature. 2006;444:567. - PubMed
    1. Rappl C, Deiwick J, Hensel M. FEMS Microbiol Lett. 2003;226:363. - PubMed
    1. Chakravortty D, Rohde M, Jäger L, Deiwick J, Hensel M. EMBO J. 2005;24:2043. - PMC - PubMed
    1. Yu X-J, Liu M, Holden DW. Mol Microbiol. 2004;54:604. - PubMed
    1. Pallen MJ, Beatson SA, Bailey CM. BMC Microbiol. 2005;5:9. - PMC - PubMed

Publication types

MeSH terms

Substances