Engagement of integrins as a cellular route of invasion by bacterial pathogens.

Integrins are heterodimeric receptors that mediate important cell functions, including cell adhesion, migration and tissue organisation. These transmembrane receptors regulate the direct association of cells with each other and with extracellular matrix proteins. However, by binding their ligands, integrins provide a transmembrane link for the bidirectional transmission of mechanical forces and biochemical signals across the plasma membrane. Interestingly, several of this family of receptors are exploited by pathogens to establish contact with the host cells. Hence, microbes subvert normal eukaryotic cell processes to create a specialised niche which allows their survival. This review highlights the fundamental role of integrins in bacterial pathogenesis.

Flavoridin inhibits Yersinia enterocolitica uptake into fibronectin-adherent HeLa cells.

In this study, three structurally distinct disintegrins (flavoridin, echistatin, kistrin) were used as molecular probes to further characterize the molecular mechanisms underlying Yersinia enterocolitica infection of host cells. The activity of the three disintegrins on Y. enterocolitica uptake into fibronectin-adherent HeLa cells was evaluated at disintegrin doses which were non-cytotoxic and unable to induce cell detachment. Flavoridin resulted to be the most effective in inhibiting bacterial entry into host cells; echistatin was almost 50% less effective than flavoridin, whereas kistrin was definitely inactive. Our results suggest that alpha(5)beta(1) integrin receptor, which binds flavoridin with higher affinity than the other two disintegrins, plays a major role in Y. enterocolitica uptake into HeLa cells. Furthermore, flavoridin binding to this integrin prevented the disruption of the functional complex FAK-Cas, which occurs in the Y. enterocolitica uptake process.

Fast track selection of immunogens for novel vaccines through visualisation of the early onset of the B-cell response.

A most essential step in vaccine research and development, ie vaccine studies in animals, seriously suffer from long timespans needed to arrive at effective immunogens. In this report we show how almost immediately after vaccination the antibody inducing potential of low immunogenic 'self' antigens can be accurately assessed. (We expect that this timespan can be reduced even more when 'non self' antigens are used, since such responses should be stronger.) The method takes advantage of the immediate onset after vaccination of the immune response in the spleen. This novel method allows detection of antigen-specific B cells of the spleen as early as 7 days after immunization and at frequencies as low as 10 in 1,000,000 cells. The method depends on sequential staining with PE- and APC-conjugated tetramers, made with the same biotinylated peptide. The antigenic peptides are biotinylated and tetramerized with either PE neutravidin or APC streptavidin. We expect that this method can be generally applied to visualize B cell responses, irrespective of the way they are induced. In addition to the fast selection and development of novel immunogens, this procedure can be used to delineate the kinetics of the B cell response, to phenotypically characterize and to isolate antigen-specific B cells, and, perhaps most importantly, to count them at the clonal level before any circulating antibodies can be detected.

Pro-apoptotic signaling pathway activated by echistatin in GD25 cells.

Disintegrins, low molecular weight RGD-containing polypeptides isolated from snake venoms, have seen use as integrin antagonists in the field of tumor biology and angiogenesis. In this study, we investigated the molecular mechanism by which the disintegrin echistatin affects cell adhesion and signaling resulting in an apoptotic response in the GD25 cell system. Wild-type GD25 cells, which lack expression of the beta(1) family of integrin, and stable transfectants expressing the A isoform of beta(1) integrin subunit were used. Nanomolar concentrations of echistatin detached fibronectin- and vitronectin-adherent GD25 cells from immobilized substratum. However, prior to inducing detachment of adherent cells, echistatin caused apoptosis as measured by caspase-3 activation. Either cell detachment or apoptotic response induced by echistatin were more pronounced on fibronectin-adherent GD25 cells than on vitronectin-adherent ones. GD25 cell exposure to echistatin caused a reduction of tyrosine phosphorylation levels of pp125(FAK), whereas it didn't affect either Shc tyrosine phosphorylation levels or Shc-Grb2 functional association. The down-regulation of pp125(FAK) preceded apoptosis and cell detachment induced by echistatin. Our results indicate that pp125(FAK) and not Shc pathway is involved in echistatin-induced apoptotic response in the GD25 cell system.

Ochratoxin A affects COS cell adhesion and signaling.

Ochratoxin A (OTA), a metabolite produced by strains of Aspergillus and Penicillium, has nephritogenic, carcinogenic, and teratogenic activity in animals and humans. Nanomolar concentrations of OTA promote apoptosis in a cell-type specific fashion. In this study, we have analyzed the molecular mechanism by which OTA affects COS cell adhesion and signaling resulting in an apoptotic response. OTA, at noncytotoxic doses, was able to detach collagen- and fibronectin-adherent cells from immobilized substratum. However, prior to inducing detachment of adherent cells, OTA caused apoptosis as measured by caspase-3 activation. The treatment of adherent cells by OTA caused a reduction of tyrosine phosphorylation levels of FAK and of the adapter protein paxillin. The down-regulation of FAK preceded apoptosis and cell detachment induced by OTA. The mycotoxin was also able to cause a decrease of the phosphorylation levels of the two Shc isoforms, P66 and P52, in adherent cells. Since these Shc isoforms have been implicated in the activation of protein kinase c-Src, which is required for FAK tyrosine phosphorylation, the observed dephosphorylation of FAK and of the FAK substrate paxillin by OTA could be ascribed to the early down-regulation of Shc isoforms. However, whether FAK and Shc phosphorylation contribute both to the same pathway leading to the induction of apoptosis by OTA or are involved in two parallel signaling pathways remains to be investigated.

Expression levels of the focal adhesion-associated proteins paxillin and p130CAS in canine and feline mammary tumors.

Paxillin and p130CAS are two adaptor proteins localized at the focal adhesions which play an important role in cell signaling, cell motility and oncogenic transformation. In this study we evaluated the levels of paxillin and p130CAS in feline and canine mammary tumor tissues at different stages of malignancy. The results obtained by Western blotting analysis showed no significant differences in the amounts of paxillin and p130CAS between normal and non-invasive tumor tissues. By contrast, mammary tumor tissues with the invasive phenotype showed lower levels of paxillin P < 0.01 and higher levels of p130CAS P < 0.001 than normal tissues. The decrease P < 0.001 of the amount of paxillin and the increase P < 0.001 of p130CAS levels were correlated with the progression stage of malignancy. Since paxillin and p130CAS are involved in regulating cell migration, our results suggest that low levels of paxillin together with high levels of p130CAS expression may cause certain breast cancers to be more motile and possibly more aggressive. Thus, both paxillin and p130CAS may represent useful prognosticators of feline and canine breast cancer malignancy.