High-efficiency entry of the enteropathogenic bacterium Yersinia pseudotuberculosis into nonphagocytic cells is mediated by the bacterial outer membrane protein invasin. Invasin-mediated uptake requires high affinity binding of invasin to multiple ␤1 chain integrin receptors on the host eukaryotic cell. Previous studies using inhibitors have indicated that high-efficiency uptake requires tyrosine kinase activity. In this paper we demonstrate a requirement for focal adhesion kinase (FAK) for invasin-mediated uptake. Overexpression of a dominant interfering form of FAK reduced the amount of bacterial entry. Specifically, the autophosphorylation site of FAK, which is a reported site of c-Src kinase binding, is required for bacterial internalization, as overexpression of a derivative lacking the autophosphorylation site had a dominant interfering effect as well. Cultured cells expressing interfering variants of Src kinase also showed reduced bacterial uptake, demonstrating the involvement of a Src-family kinase in invasin-promoted uptake.

Efficient uptake of Yersinia pseudotuberculosis into cultured mammalian cells is the result of high‐affinity binding of invasin to β1 chain integrins. We demonstrate here that uptake requires Rac1 and Arp 2/3 function. Bacterial uptake was stimulated by GTPγS, but was inhibited in mammalian cells transfected with the interfering Rac1‐N17 derivative. Rac1 was found to be activated in response to integrin engagement by invasin, whereas Rac1 and Arp 2/3 were found to be intensely localized around phagosomes bearing bacteria, indicating a specific role for Rac1 signalling from the nascent phagosome to downstream effectors. To determine whether the Arp 2/3 complex was a component of this proposed pathway, cells overproducing various derivatives of Scar1/WAVE1, an Arp 2/3‐binding protein, were analysed. Sequestration of Arp 2/3 away from the phagocytic cup as a result of Scar1/WAVE1 overproduction dramatically inhibited uptake. To determine whether signalling from Rac1 to Arp 2/3 occurred via N‐WASP, uptake was analysed in a cell line lacking expression of WASP and N‐WASP. Uptake was unaffected by the absence of these proteins, indicating that β1 integrin signalling from Rac1 to Arp 2/3 can occur in the absence of N‐WASP function.

The FasD protein is essential for the biogenesis of 987P fimbriae of Escherichia coli. In this study, subcellular fractionation was used to demonstrate that FasD is an outer membrane protein. In addition, the accessibility of FasD to proteases established the presence of surface-exposed FasD domains on both sides of the outer membrane. The fasD gene was sequenced, and the deduced amino acid sequence was shown to share homologous domains with a family of outer membrane proteins from various fimbrial systems. Similar to porins, fimbrial outer membrane proteins are relatively polar, lack typical hydrophobic membrane-spanning domains, and possess secondary structures predicted to be rich in turns and amphipathic n-sheets. On the basis of the experimental data and structural predictions, FasD is postulated to consist essentially of surface-exposed turns and loops and membrane-spanning interacting amphipathic ,-strands. In an attempt to test this prediction, thefasD gene was submitted to random in-frame linker insertion mutagenesis. Preliminary experiments demonstrated that it was possible to producefasD mutants, whose products remain functional for fimbrial export and assembly. Subsequently, 11 fasD alleles, containing linker inserts encoding P-turninducing residues, were shown to express functional proteins. The insertion sites were designated permissive sites. The inserts used are expected to be least detrimental to the function of FasD when they are inserted into surface-exposed domains not directly involved in fimbrial export. In contrast, FasD is not expected to accommodate such residues in its amphipathic ,-strands without being destabilized in the membrane and losing function. All permissive sites were sequenced and shown to be located in or one residue away from predicted turns. In contrast, 5 of 10 sequenced nonpermissive sites were mapped to predicted amphipathic P-strands. These results are consistent with the structural predictions for FasD.Many gram-negative bacteria express adhesive fimbriae (39). These organelles consist of helical arrangements of protein subunits (4, 13). Fimbrial biogenesis on the bacterial surface requires subunit export across two membrane barriers. Typically, genes encoding the proteins required for fimbrial biogenesis are clustered together with fimbrial structural genes on a single replicon. Many of the accessory proteins have aminoterminal export signal sequences which are cleaved off by a host-or fimbrial type-specific signal peptidase (25, 46), indicating that they cross the cytoplasmic membrane. Moreover, since the export of the well-studied type 1 fimbriae has been shown to require SecA (10), it is commonly assumed that all fimbria-specific proteins which translocate through the cytoplasmic membrane utilize host components of the general export system (46).After crossing the cytoplasmic membrane of Escherichia coli, the fimbria-specific accessory proteins form a second transport system translocating fimbrial subunits across the outer membrane (9). Genetic studies have indic...