Role of the S. typhimurium Actin-binding protein SipA in bacterial internalization

Science

Volumn 283, Issue 5410, 1999, Pages 2092-2095

  Zhou, Daoguo ^a   Mooseker, Mark S ^b   Galán, Jorge E ^a  

^a YALE UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b YALE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ACTIN; ACTIN BINDING PROTEIN; BACTERIAL PROTEIN; GUANINE NUCLEOTIDE BINDING PROTEIN; MAGNESIUM CHLORIDE; POTASSIUM CHLORIDE; PROTEIN CDC42; PROTEIN SIPA; PYRENE; RAC PROTEIN; UNCLASSIFIED DRUG;

ACTIN FILAMENT; ARTICLE; CELL MEMBRANE; CONTROLLED STUDY; DEPOLYMERIZATION; HELA CELL; HOST CELL; HUMAN; HUMAN CELL; INTERNALIZATION; NONHUMAN; PRIORITY JOURNAL; PROTEIN PROTEIN INTERACTION; SALMONELLA TYPHIMURIUM;

ACTINS; ANTIGENS, BACTERIAL; BACTERIAL PROTEINS; BINDING SITES; BIOPOLYMERS; CELL MEMBRANE; HELA CELLS; HUMANS; MICROFILAMENT PROTEINS; MICROSCOPY, FLUORESCENCE; MUTATION; RECOMBINANT FUSION PROTEINS; SALMONELLA TYPHIMURIUM; SIGNAL TRANSDUCTION; VINCULIN;

BACTERIA (MICROORGANISMS); SALMONELLA; SALMONELLA TYPHIMURIUM; TYPHIMURIUM;

EID: 0033605618     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5410.2092     Document Type: Article

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15. The internalization into HeLa cells of wild-type S. typhimurium strain SL1344 [S. K. Hoiseth and B. A. Stocker, Nature 291, 238 (1981)], the isogenic sipA mutant strain SB848, and its complemented derivative strain SB850 was measured by a fluorescence microscopy assay that can distinguish internalized from extracellular bacteria (5).
16. 7) were harvested in 4.5 ml of phosphate-buffered saline (PBS) containing 0.1% NP-40 and 1 mM phenylmethylsulfonyl fluoride (PMSF) and disrupted with a glass grinder, and the cell lysates were cleared by centrifugation at 16,000g for 15 min. Alternatively, E. coli cells expressing GST-SipA were harvested in PBS containing 0.1% NP-40 and 1 mM PMSF and lysed by sonication. Total protein concentrations in the bacterial lysates were adjusted to ∼2 mg/ml, and F-actin (Cytoskeleton Inc., Denver, CO) was added to a final concentration of 5 μg/mL HeLa cell lysates (500 μl) were mixed with 10 to 20 μg of purified GST or GST-SipA bound to 50 μl of Sepharose beads, and the mixtures were incubated at 4°C for 4 hours (for most experiments, a fusion between GST and the COOH-terminal 226 amino acids of SipA was used). Samples were then centrifuged for 10 min at 1000g and supernatants were saved as postcapture samples. Beads were washed four times with ice-cold PBS containing 0.1% NP-40, resuspended in Laemmli sample buffer, and proteins separated on an 8% SDS-polyacrytamide gel. Proteins were stained, excised from the gel, and their mass determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Alternatively, interacting proteins were transferred to nitrocellulose membranes and probed with a mouse monoclonal antibody (mAb) to actin or vinculin (Sigma).
17. Introduction into the yeast indicator strain Y153 of a plasmid encoding a fusion of the last 226 amino acid residues of SipA to the Gal4 DNA-binding domain, along with a plasmid encoding full-length actin fused to the Gal4 activation domain, resulted in transformants that grew robustly in the absence of histidine and exhibited significant β-galactosidase activity (25.9 Miller units vs. 0.15 Miller units of the vector control).
18. HeLa cells seeded in 150-mm dishes were infected at a multiplicity of infection of 50 for 30 min. Cells were washed and then harvested into 2.5 ml of ice-cold PBS containing 0.1% NP-40 and 1 mM PMSF and disrupted with a glass grinder. SipA was immunoprecipitated with a mouse antibody to SipA from cell lysates that had been precleared for 2 hours at 4°C with normal mouse serum and protein A - Sepharose beads or GammaBind Plus Sepharose (Pharmacia). Immunocomplexes were recovered with protein A or GammaBind Plus Sepharose beads, and the presence of actin or vinculin was probed by immunoblotting with actin mAb (Sigma).
19. 2 for 30 min at room temperature immediately before the binding assay and subsequently mixed with SipA. Mixtures (100 μl) were incubated at room temperature for 60 min and then centrifugea at 100,000g for 30 min at 4°C. Supernatants and pellets were analyzed by SDS-polyacrylamide gel electrophoresis (PACE) and the relative amounts of the proteins in each fraction were estimated by scanning densitometry of the stained gels. In the absence of actin, no SipA (at all concentrations used in the assay) was detected in pellets after centrifugation at 100,000g.
20. Samples were loaded onto carbon-coated grids, stained with 1% uranyl acetate, and visualized under the electron microscope.
21. 2, and the fluorescence intensity was measured over time.
22. 2, and the fluorescence intensity was measured over time.
23. D. Zhou and J. E. Galán, unpublished results.
24. We thank E. Taylor for providing pyrene-labeled actin, and members of the Galán laboratory for critical reading of the manuscript. Supported by NIH grants AI30492 and GM52543 (J.EG.) and DK25387 (M.S.M.). J.E.G. is an investigator of the American Heart Association.