The invasion of colonic epithelial cells by Shigella, an early essential step for causing bacillary dysentery, is mediated by the IpaB, IpaC and IpaD proteins. Secretion of the Ipa proteins from Shigella requires functions encoded by the mxi and spa loci. In this study, we show that contact between the bacteria and epithelial cell triggers release of the Ipa proteins into the external medium, which results in a rapid decrease in levels of Ipa proteins presented on the cell surface. When the bacteria were used to infect polarized Caco-2 cells, release of Ipa proteins occurred efficiently from bacteria interacting with the basolateral surface rather than with the apical surface. Moreover, the interaction of bacteria with components of the extracellular matrix, such as fibronectin, laminin or collagen type IV, also stimulates the release of Ipa proteins. The release of Ipa proteins from Shigella required the surface-located Spa32 protein encoded by one of the spa genes on the large plasmid.

SummaryShigella is a genus of highly adapted bacterial pathogens that cause bacillary dysentery in humans.Bacteria reaching the colon invade intestinal epithehal cells by a process of bacterial-directed endocytosis mediated by the Ipa proteins: IpaB, IpaC, and IpaD of Shigella. The invasion of epithelial cells is thought to be a receptor-mediated phenomenon, although the cellular components of the host that interact with the Ipa proteins have not yet been identified. We report here that in a Shigellaflexneri invasive system and Chinese hamster ovary (CHO) cell monolayers, the Ipa proteins were capable of interacting directly with oL5131 integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of cx5131 integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both ofpp 125 F~, an integrin-regulated 125 K focal adhesion kinase, and of paxillin was stimulated. In contrast, an isogenic strain of S. flexneri that was defective in invasion owing to a mutation in its spa32 gene failed to induce such phosphorylation. Under in vitro and in vivo conditions, the released IpaB, IpaC, and IpaD proteins bound to ot5[~ 1 integrin in a manner different from that of soluble fibronectin but similar to that of the tissue form of fibronectin. At the site of attachment of S. flexneri to CHO cells, ot5131 integrin converged with polymerization of actin. These data thus suggest that the capacity oflpa proteins to interact with o~5151 integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.

Summary Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes. Lipid raft‐associated molecules such as glycosylphosphatidylinositol (GPI)‐anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella. In contrast, lysosomal glycoprotein LAMP‐1 and host cell transmembrane protein CD44 were excluded from the macropinosomes. Removing GPI‐anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB‐dependent macropinocytosis and intracellular replication. Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.

The products of the Legionella pneumophila dot/icm genes enable the bacterium to replicate within a macrophage vacuole. This study demonstrates that the Dot/Icm machinery promotes macropinocytotic uptake of L. pneumophila into mouse macrophages. In mouse strains harboring a permissive Lgn1 allele, L. pneumophila promoted formation of vacuoles that were morphologically similar to macropinosomes and dependent on the presence of an intact Dot/Icm system. Macropinosome formation appeared to occur during, rather than after, the closure of the plasma membrane about the bacterium, since a fluid-phase marker preloaded into the macrophage endocytic path failed to label the bacterium-laden macropinosome. The resulting macropinosomes were rich in GM1 gangliosides and glycosylphosphatidylinositol-linked proteins. The Lgn1 allele restrictive for L. pneumophila intracellular replication prevented dot/icm-dependent macropinocytosis, with the result that phagosomes bearing the microorganism were targeted into the endocytic network. Analysis of macrophages from recombinant inbred mouse strains support the model that macropinocytotic uptake is controlled by the Lgn1 locus. These results indicate that the products of the dot/icm genes and Lgn1 are involved in controlling an internalization route initiated at the time of bacterial contact with the plasma membrane.