Biomedical subjects

Vassilis Koronakis

Publications and source records attributed to Vassilis Koronakis.

24 records · Page 2Linked to original sources

PubMed2002

Processing of viable Salmonella typhimurium for presentation of a CD4 T cell epitope from the Salmonella invasion protein C (SipC).

We have identified Salmonella invasion protein C (SipC) as a target antigen for CD4 T cell recognition in mice infected with Salmonella typhimurium. SipC is a product of the type III secretion system encoded by S. typhimurium pathogenicity island 1. A SipC-specific T cell response was induced by infection with either the C5 wild type or attenuated SL3261 vaccine strain of S. typhimurium. We localized the response of T cell lines from infected mice to an epitope near the carboxyl terminus of SipC (SipC(381-394)) and studied the way it was processed from viable S. typhimurium. We demonstrated that CD4 T cell recognition of this epitope required actin-dependent uptake of S. typhimurium. Presentation also occurred when transport of newly synthesized MHC class II from the endoplasmic reticulum was disrupted and when the pH of intracellular compartments was raised, suggesting presentation by mature MHC class II recycled from the macrophage surface into neutral intracellular compartments. Salmonellae are known to colonize macrophages by localizing to compartments that do not make contact with the bactericidal environment of late endosomes or lysosomes, and thus might avoid lysosomal antigen processing. However, we demonstrate that a CD4 T cell response to S. typhimurium-secreted proteins may be induced by an alternative pathway capable of antigen presentation in conditions similar to those in the compartments where Salmonella localize.

Animals↗

PubMed2002

Direct modulation of the host cell cytoskeleton by Salmonella actin-binding proteins.

Invasive Salmonella trigger their own uptake into non-phagocytic eukaryotic cells by delivering virulence proteins that stimulate signaling pathways and remodel the actin cytoskeleton. It has recently emerged that Salmonella encodes two actin-binding proteins, SipC and SipA, which together efficiently nucleate actin polymerization and stabilize the resulting supramolecular filament architecture. Therefore, Salmonella might directly initiate actin polymerization independently of the cellular Arp2/3 complex early in the cell entry process. This is an unprecedented example of a direct intervention strategy to facilitate entry of a pathogen into a target cell. Here, we discuss the Salmonella actin-binding proteins and how they might function in combination with entry effectors that stimulate Rho GTPases. We propose that membrane-targeted bacterial effector proteins might trigger actin polymerization through diverse mechanisms during cell entry by bacterial pathogens.

Actin Cytoskeleton↗

PubMed2002

An aspartate ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations.

The TolC protein of Escherichia coli comprises an outer membrane beta-barrel channel and a contiguous alpha-helical tunnel spanning the periplasm, providing an exit duct for protein export and multidrug efflux. It forms a single transmembrane pore that is open to the outside of the cell but constricted at the peri-plasmic tunnel entrance. This sole constriction is lined by a ring of six aspartate residues, two in each of the three identical monomers. When these were replaced by alanines, the resulting TolC(DADA) protein reconstituted normally in black lipid membranes but showed altered electrophysiological characteristics. In particular, it had lost the strong pH dependence of the wild type and had switched ion selectivity from cations to anions. The function of wild-type TolC as a membrane pore was severely inhibited by divalent and trivalent cations entering the channel tunnel from the channel ("extracurricular") side. Divalent cations bound reversibly to effect complete blocking of the transmembrane ion flux. Trivalent cations were more potent. Hexamminecobalt bound at nanomolar concentrations allowed visualization of single blocking events, whereas the smaller Cr(3+) cation bound irreversibly and could also access the cation binding site via the tunnel entrance. The inhibitory cations had no effect on the mutant TolC(DADA), supporting the view that the aspartate ring is the cation binding site. The electronegative entrance is widely conserved throughout the TolC family, which is essential for efflux and export my Gram-negative bacteria, suggesting that it could present a general target for drugs.

Aspartic Acid↗

PubMed2002

Topology of the Salmonella invasion protein SipB in a model bilayer.

A critical early event in Salmonella infection is entry into intestinal epithelial cells. The Salmonella invasion protein SipB is required for the delivery of bacterial effector proteins into target eukaryotic cells, which subvert signal transduction pathways and cytoskeletal dynamics. SipB inserts into the host plasma membrane during infection, and the purified protein has membrane affinity and heterotypic membrane fusion activity in vitro. We used complementary biochemical and biophysical techniques to investigate the topology of purified SipB in a model membrane. We show that the 593 residue SipB is predominantly alpha-helical in aqueous solution, and that no significant change in secondary structural content accompanies lipid interaction. SipB contains two -helical transmembrane domains (residues 320-353 and 409-427), which insert deeply into the bilayer. Their integration allowed the hydrophilic region between the hydrophobic domains (354-408) to cross the bilayer. SipB membrane integration required both the hydrophobic domains and an additional helical C-terminal region (428-593). Further spectroscopic analysis of these domains in isolation showed that the hydrophobic regions insert obliquely into the bilayer, whereas the C-terminal domain associates with the bilayer surface, tilted parallel to the membrane. The combined data suggest a topological model for membrane-inserted SipB.

Amino Acid Sequence↗

PubMed2002

A Salmonella SipB-derived polypeptide blocks the 'trigger' mechanism of bacterial entry into eukaryotic cells.

Entry into non-phagocytic mammalian cells by the invasive pathogens Salmonella and Shigella is triggered by the delivery of bacterial virulence effector proteins into the host cell. This is dependent upon Salmonella SipB or its Shigella homologue IpaB, which insert into the eukaryotic cell plasma membrane. Here we show that a SipB-derived 166 residue alpha-helical polypeptide is a potent inhibitor of SipB-directed liposome fusion in vitro, preventing the membrane-associated form of SipB from inserting deeply into the bilayer. This polypeptide blocks Salmonella entry into cultured mammalian cells at 10(-10) M, and is a heterologous inhibitor of analogous IpaB activity and Shigella cell entry. These findings reveal a potential strategy to identify inhibitors of the 'trigger' mechanism underlying cell entry by these major invasive pathogens.

Bacterial Proteins↗

PubMed2002

The Salmonella pathogenicity island 1 secretion system directs cellular cholesterol redistribution during mammalian cell entry and intracellular trafficking.

The bacterial pathogen Salmonella triggers its own uptake into non-phagocytic mammalian cells. Entry is induced by the delivery of bacterial effector pro-teins that subvert signalling and promote cytoskeletal rearrangement, although the molecular mechanisms that co-ordinate initial pathogen-host cell recognition remain poorly characterized. Here we show that cholesterol is essential for Salmonella uptake. Depletion and chelation of plasma membrane cholesterol specifically inhibited bacterial internalization but not adherence. Cholesterol accumulated at bacterial entry sites in cultured cells, and was retained by Salmonella-containing vacuoles following pathogen internalization. Cellular cholesterol redistribution required bacterial effector protein delivery mediated by the Salmonella pathogenicity island (SPI) 1 type III secretion system, but was independent of the SPI2-encoded system.

3T3 Cells↗