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.

An O antigen can interfere with the function of the Yersinia pseudotuberculosis invasin protein.

Escherichia coli strains harbouring the Yersinia pseudotuberculosis inv gene are able to enter cultured mammalial cells. We show here that this property is not shared by all enteric bacteria, since Shigella flexneri 2a cured of its virulence-associated plasmid and harbouring the inv gene is unable to enter mammalian cells efficiently. Mapping studies showed that the region of the chromosome responsible for this phenotype includes rfaB, a locus involved in the production of O antigen. S. flexneri 2a strains that express O antigen were unable to enter mammalian cells, even though invasin was efficiently expressed and localized, showing that this structure interferes with invasin activity. The O antigen either masks invasin or sterically hinders the ability of the mammalian cell receptor to bind this protein.

Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells.

Bacterial strains harboring the Yersinia pseudotuberculosis inv locus were analyzed in order to investigate the mechanism of host cell penetration by an invasive pathogen. The inv locus was found to be necessary for Y. pseudotuberculosis to enter HEp-2 cells and sufficient to convert E. coli into a microorganism able to penetrate cultured cells. Both E. coli and Y. pseudotuberculosis strains harboring inv mutations were defective for entry into HEp-2 cells. Furthermore, molecular clones containing inv, and little additional DNA, converted E. coli into a microorganism that was indistinguishable from the parental Yersinia strain with regard to the entry of cultured cells. Data from in vitro protein synthesis indicated that a 103 kd protein was synthesized from inv, saturating the coding capacity of the locus. The nucleotide sequence shows an open reading frame corresponding to a protein of similar size. This protein, called invasin, is necessary for the microorganisms to penetrate HEp-2 cells, and is compartmentalized on the outer surface of the bacterium.