The gastric pathogen Helicobacter pylori uses a type IV secretion system to inject the bacterial CagA protein into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates cytoskeletal rearrangements. We demonstrate here that Src-like protein-tyrosine kinases mediate CagA phosphorylation in vitro and in vivo. First, the Src-specific tyrosine kinase inhibitor PP2 specifically blocks CagA phosphorylation and cytoskeletal rearrangements thereby inhibiting the CagA-induced hummingbird phenotype of gastric epithelial cells. Second, CagA is in vivo phosphorylated by transiently expressed c-Src. Third, recombinant c-Src and lysates derived from c-Src-expressing fibroblasts but not lysates derived from Src-, Yes-, and Fyn-deficient cells phosphorylated CagA in vitro. Fourth, a transfected CagA-GFP fusion protein is phosphorylated in vivo in Src-positive fibroblasts but not in Src-, Yes-, and Fyn-deficient cells. Because a CagA-GFP fusion protein mutated in an EPIYA motif is not efficiently phosphorylated in any of these fibroblast cells, the CagA EPIYA motif appears to constitute the major c-Src phosphorylation site conserved among CagA-positive Helicobacter strains.

The gastric pathogen Helicobacter pylori translocates the CagA protein into epithelial cells by a type IV secretion process. Translocated CagA is tyrosine phosphorylated (CagA P-Tyr ) on speci®c EPIYA sequence repeats by Src family tyrosine kinases. Phosphorylation of CagA induces the dephosphorylation of as yet unidenti®ed cellular proteins, rearrangements of the host cell actin cytoskeleton and cell scattering. We show here that CagA P-Tyr inhibits the catalytic activity of c-Src in vivo and in vitro. c-Src inactivation leads to tyrosine dephosphorylation of the actin binding protein cortactin. Concomitantly, cortactin is speci®cally redistributed to actin-rich cellular protrusions. c-Src inactivation and cortactin dephosphorylation are required for rearrangements of the actin cytoskeleton. Moreover, CagA P-Tyr -mediated c-Src inhibition downregulates further CagA phosphorylation through a negative feedback loop. This is the ®rst report of a bacterial virulence factor that inhibits signalling of a eukaryotic tyrosine kinase and on a role of c-Src inactivation in host cell cytoskeletal rearrangements.

Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H+-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor‐like (‘hummingbird’) phenotype. In this study, using a cagA mutant complemented with wild‐type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti‐phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA–green fluorescent protein (GFP) fusion proteins and two‐dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C‐terminus. Site‐directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).