Residues added to the carboxyl terminus of the Yersinia pseudotuberculosis invasin protein interfere with recognition by integrin receptors.
Escherichia coli strains encoding the Yersinia pseudotuberculosis invasin protein are efficiently internalized by mammalian cells. Bacterial uptake into cultured cell lines became defective, however, if invasin was altered by fusion of its carboxyl terminus to E. coli alkaline phosphatase or by the addition of two hydrophobic amino acids to its carboxyl-terminal end. Probing with anti-invasin monoclonal antibodies revealed that the amino-terminal end of invasin was properly localized on the bacterial cell surface in strains encoding invasin with 2 additional amino acids, whereas the carboxyl terminus was not accessible to the monoclonal antibody. Therefore, the 2 additional amino acids interfered with the folding or orientation of the carboxyl terminus in the outer membrane. Alkylation experiments in the absence of reduction indicated that this defect was not caused by a gross inability to form a critical disulfide bond. Revertants were selected from a strain encoding this mutant protein by enriching for organisms able to enter cultured mammalian cells. The vast majority of revertants that were isolated following this enrichment contained a stop codon at the usual position found in the wild type inv gene. The most efficient of the remaining revertants resulted in the introduction of a glycine residue at the site of the wild type stop codon, presumably restoring proper conformation of the carboxyl-terminal region.
Isberg
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RR
Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, Massachusetts 02111.
Yang
Y
Y
Voorhis
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DL
D018528:ATP-Binding Cassette Transporters; D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D004269:DNA, Bacterial; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D016023:Integrins / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D015011:Yersinia pseudotuberculosis / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D018829:Adhesins, Bacterial; D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004220:Disulfides; D000939:Epitopes; D029968:Escherichia coli Proteins; D016023:Integrins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; C053476:invasin, Yersinia; D008320:Maltose