Listerial colonies are regular white rounded colonies occurring singly or in short chains. F-actin and bacteria were stained using rhodamine-conjugated phalloidin and rabbit anti-LPS, respectively. Ruest P. J., Shin N. Y., Polte T. R., Zhang X., Hanks S. K. Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src, p130cas but not paxillin is essential for Caco-2 intestinal epithelial cell spreading and migration on collagen IV. 287, No. Here, we show that Salmonella recruit focal adhesion proteins including FAK, Cas, and paxillin, but not 1 integrin, to sites of invasion at the apical surface of epithelial cells and demonstrate a requirement for both FAK and p130Cas, but not paxillin, in the invasion of host cells by Salmonella. This finding is in agreement with an earlier observation that tyrosine kinase activity is not required for Salmonella entry (Rosenshine et al., 1994).
The medium (100 l) and Griess reagents (100 l) were mixed and left for 30 min at room temperature. For the adhesion assays, C. albicans cells were maintained in contact with fibroblasts for 2 h. After this, the wells were washed three times with PBS to remove unattached yeasts and the GF cells detached at 37C for 2 min using 1 ml trypsin-EDTA (Gibco) diluted in PBS. We then used the reconstitution approach to determine which domain(s) of Cas are necessary for its function in Salmonella invasion. [1] assessed the in vitro invasion of C. albicans in human tissue (fibroblasts and epithelial cells) and found that reduction in the formation of hyphae (a characteristic of the less hydrophobic cells) decreases the invasive properties and reduces the ability of the yeast to invade tissue cells. To determine if the kinase activity of FAK is required for its function in Salmonella internalization, we reconstituted FAK/ cells with either FRNK, the autonomously expressed C-terminus of FAK that lacks the catalytic domain (Figure 4B), or FAK Y397F, a mutant lacking the autophosphorylation site, and quantitated bacterial internalization using the immunofluorescence-based invasion assay.
The CytoSelect Cell Adhesion Assays developed by Cell Biolabs quantify cell adhesion using a microplate reader, no manual cell counting is necessary. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. As we observed in the FAK/ cells, bacterially induced actin focus formation was also dramatically reduced in Cas/ cells (Figure 5B). Cells were infected at 37C for 20 min, then fixed, and immunostained for both F-actin and bacteria. Accessibility Figure 8. [13]. The FAK C-terminal nonkinase domain contains two proline-rich motifs (Figure 4B), one of which (PXXP718) binds to Cas. Although Cas phosphorylation is not essential for Salmonella internalization, we found that cells lacking Cas were severely impaired in bacterial uptake. sharing sensitive information, make sure youre on a federal In contrast to FAK+/+ cells, where actin foci were robustly induced, focus formation was reduced by 80% in FAK/ cells (Figure 3A). For example: adhered cells 100/control. Focal adhesions are a complex assembly of proteins that provide a physical linkage between integrins and the actin cytoskeleton (Zamir and Geiger, 2001). Ozeri V., Rosenshine I., Mosher D. F., Fassler R., Hanski E. Roles of integrins and fibronectin in the entry of. 7, 1 January 2016 | RSC Advances, Vol. 282, No. However, Salmonella activate Rac directly through the secreted effector protein SopE (Hardt et al., 1998; Criss et al., 2001) and presumably do not require host proteins to perform this function. The role of C. albicans in periodontal disease is not clear and further studies are needed to demonstrate the clinical significance of the findings. Yersinia, Staphylococcus, and Streptococcus species have all been reported to enter host cells by integrin-dependent mechanisms, either directly (Yersinia; Isberg and Leong, 1990; Rankin et al., 1992) or indirectly through interaction with components of the extracellular matrix (Staphylococcus and Streptococcus; Ozeri et al., 1996, 1998; Okada et al., 1997; Sinha et al., 1999; Fowler et al., 2000). Adherent cells are quantified using either colorimetric or fluorometric detection. In contrast, we found that Salmonella internalization does not appear to induce significant tyrosine phosphorylation of FAK. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. However, cells deficient in paxillin were actually infected more efficiently by Salmonella, suggesting that paxillin may play a negative role in this process. (A) MDCK cells were infected apically with S. typhimurium strain SL1344 for 20 min. In agreement with these observations, Salmonella internalization in SYF (Src/Yes/Fyn)/ MEFs was found to be only slightly reduced compared with wild-type MEFs (unpublished data). Bacterial counts obtained at different time points during invasion assay give more information about the survival of listeria in the human cells and the comparison of these counts between mutants and WT strains gives information about the changes in the adaptation of listeria after mutation in human cells. Frequency of focus formation was quantified as described in Materials and Methods. Cell surface hydrophobicity plays an important role in mediating the adhesion of yeasts to epithelial, splenic, kidney, fibroblast and lymph node cells [15]. Paxillin+/+ and paxillin/ MEFs were provided by A. R. Horwitz (University of Virginia).
The production of NO was higher for the most hydrophobic strains, but did not reach statistical difference with the weakly hydrophobic isolates. Candida albicans is a regular inhabitant of the human mucosal surfaces and part of its microbiota, but can also cause infections ranging from those of the superficial mucosal to haematogenously disseminated disease [1]. ). (B) Means and standard deviations of NO (M) produced in each assay. Alternatively, FAK and Cas may function in distinct pathways, and the FAK-dependent pathway may be sufficient to compensate for the lack of Cas when FAK is overexpressed. (B) Transfected cells were labeled with an anti-myc antibody 9E10 followed by a Cy2-conjugated donkey anti-mouse secondary antibody. To determine if Cas is required for bacterial entry, we compared Salmonella invasion in Cas+/+ and Cas/ MEFs. Experiments have suggested that high CSH impairs phagocytosis, increasing resistance to blood clearance and as a consequence, the virulence of C. albicans [12]. Once lysed cells float in the solution, using a pipette carefully mix the solution (without splashing) to remove adherent cells if any. An aliquot of 20 l was removed, serially diluted, inoculated onto SDA and incubated for 48 h. The number of adhered yeasts in the fibroblasts was assessed by counting the colony-forming units/ml (CFU/ml). Therefore, future experiments to examine the mechanisms by which paxillin may attenuate Salmonella invasion will provide more insight into the molecular regulation of this process. Bacteria of the genus Salmonella are the causative agents of diseases ranging from gastroenteritis to typhoid fever. Additionally, it is possible that the PXXP718 motif on FAK binds proteins other than Cas that are necessary for bacterial entry. The invasion defect in Cas/ cells can be suppressed by overexpression of FAK, suggesting a functional link between FAK and Cas in the regulation of Salmonella invasion. Because it has been shown that c-Src is the major tyrosine kinase that phosphorylates Cas (Astier et al., 1997; Ruest et al., 2001; Agerer et al., 2005), we assayed Salmonella internalization in cells treated cells with a Src inhibitor, PP2. Focal adhesion-like complexes form at sites of Salmonella invasion. It has been shown that tyrosine phosphorylation of paxillin and Cas play opposing roles in cell migration and contact inhibition of cell growth, where phosphorylated paxillin reduces cell migration and Cas has the opposite effect (Yano et al., 2000). Bourdet-Sicard R., Rudiger M., Jockusch B. M., Gounon P., Sansonetti P. J., Nhieu G. T. Bouton A. H., Riggins R. B., Bruce-Staskal P. J. 2B). HeLa cells were grown in DMEM with 4.5 g/l glucose, 10% FBS, 1 mM sodium pyruvate, 2 mM l-glutamine, and antibiotics. Cell invasion is related to, and encompasses, cell migration, except that cells do more than migrate. Wash pellet and resuspend it in 1 ml of fresh warm (37 C) cEMEM. However, we did not observe a corresponding accumulation of 1 integrin at Salmonella invasion sites (Figure 1A), suggesting that 1 integrins are not clustered at sites of Salmonella invasion. An alternative interpretation is that FAK and Cas function independently of one another, but that FAK-dependent processes are sufficient to support bacterial internalization in the absence of Cas. C. albicans has been isolated from periodontal pockets in different forms of periodontitis, especially in HIV-positive and diabetic patients [35]. Although only a small enhancement of coprecipitation was observed in cells infected with the noninvasive VV341 strain, infection with SL1344 robustly enhanced the interaction of p130Cas with both FAK and paxillin.
Then, cells were fixed in 2% paraformaldehyde, and extracellular Salmonella were stained using a rabbit polyclonal antibody against Salmonella LPS (1:500), followed by a Cy2-conjugated goat anti-rabbit antibody (1:400). Tran Van Nhieu G., Ben-Ze'ev A., Sansonetti P. J. Modulation of bacterial entry into epithelial cells by association between vinculin and the. Cells were then permeablized by incubation with PBS-NGS (10% normal goat serum) containing 0.2% saponin. Centrifuge the plates briefly for 45 sec and incubate them at 37 C for 2 h. After incubation wash twice with 1x PBS, and add fresh media containing gentamycin (100 g/ml) to kill extracellular bacteria. To detect cells expressing myc-tagged FAK or Cas constructs, a mouse anti-myc antibody 9E10 (1:1000) was included in the second incubation with anti-LPS antibody, followed by a Cy2-conjugated goat anti-mouse antibody (1:500). Re-feed cells every 48-72 h or when the color of the media changes. Moreover, reconstitution of Cas/ cells with wild-type p130Cas was sufficient to restore invasiveness (Figure 5, C and D), indicating that Cas is also required for Salmonella entry. Include negative controls as shown and described in. *p < 0.05 compared with mock-transfected Cas/ cells (Student's t test). Bars, 10 m. As observed in apically infected MDCK cells, Cas was strongly concentrated at sites of bacterial internalization in mouse fibroblasts (see Supplementary Material, Figure S1B). 71, No. Nitric oxide: cytotoxicity versus cytoprotection how, why, when, and where? Liu Y., Loijens J. C., Martin K. H., Karginov A. V., Parsons J. T. The association of ASAP1, an ADP ribosylation factor-GTPase activating protein, with focal adhesion kinase contributes to the process of focal adhesion assembly. Salmonella typhimurium colonizes the intestinal epithelium by injecting an array of effector proteins into host cells that induces phagocytic uptake of attached bacteria. In addition to the presence of microbial adhesins and receptors on host cells, microbial surface hydrophobicity has been described as an important factor that influences adhesion of microorganisms to biological or inert surfaces [12]. All statistical analyses were conducted at a significance level of 5% (SPSS, version 17.0). Bacterial internalization was normalized to invasion efficiency in Cas+/+ cells. The number of adhered cells was always higher than those that invaded with both HH and LH isolates, except for strains 5LH and 8HH (Fig.
Expression levels of FAK in these cells were detected using the FAK mAb 4.47. Cells were infected with wild-type S. typhimurium SL1344 for 20 min, washed with PBS, and fixed in 4% paraformaldehyde. Cell migration can be monitored in real time by microscopy. The other (PXXP881) is a binding site for two different GTPase regulators, the Arf GAP ASAP1 and the Rho GAP GRAF. receptor and dysregulation of p130cas phosphorylation, The Leishmania Surface Protease GP63 Cleaves Multiple Intracellular Proteins and Actively Participates in p38 Mitogen-activated Protein Kinase Inactivation, Abelson Tyrosine Kinase Facilitates
(A) FAK+/+ and FAK/ MEFs were infected with wild-type S. typhimurium (SL1344) at a MOI = 30 for 1 h. Salmonella internalization was assayed by a standard gentamicin resistance assay as described in Materials and Methods. MDCK cells infected apically with either the wild-type Salmonella strain SL1344 or the syngeneic entry-deficient mutant VV341(hilA) were lysed and immunoprecipitated with antibody to p130Cas, and the levels of associated FAK or paxillin were determined by immunoblotting. 24 h after seeding, check the cells under microscope for attachment, if attached to flask surface, slowly aspirate media to remove dead cells and add pre-warmed (37 C) cEMEM.
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