adhesion invasion assay

(B) FAK+/+ and FAK/ cells were infected with SL1344 (lane 3 and 7) or the entry-deficient strain VV341 (lane 4 and 8), or maintained in HBSS+ (lane 12 and 56). Sonic dismembrator settings: setting 3 (operation frequency: 20 kHz and power rating: 120 watts), 3 pulses, 5 sec each helps in further lysis of Caco-2 cells but does not harm. Using a type III secretion system encoded by the Salmonella pathogenicity island-1 (SPI-1) chromosomal locus (Collazo and Galan, 1997; Darwin and Miller, 1999), a set of bacterial effector proteins are translocated into host cells, where they manipulate host actin dynamics and signaling pathways to promote extensive reorganization of the actin cytoskeleton that culminates in bacterial entry (Patel and Galan, 2005). Note: Data can be analyzed by performing Students t-test using Microsoft Excel. All assays were performed in duplicate, in two independent experiments. GST alone was used as negative control (lane 1 and 5). These tests were carried out according to the methods, with modifications, previously described by Cogo et al. For example: adhered cells 100/control. 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. Received 2016 Dec 13; Revised 2017 Mar 26; Accepted 2017 Mar 29. Salmonella were labeled with polyclonal anti-LPS antibody followed by a TR-conjugated anti-rabbit IgG (red). Criss A. K., Ahlgren D. M., Jou T. S., McCormick B. We also found that ectopic expression of FAK was able to rescue Salmonella invasion in Cas/ cells (Figure 9). The glycemic control was performed by each patient using insulin supplementation. 6, No. Hydrophobicity assays were conducted according to those as described by Rodrigues et al. 7, American Journal of Physiology-Cell Physiology, Vol.

Two strains (one HH and one LH) were obtained from each patient. B., Bouton A. H. Yano H., Uchida H., Iwasaki T., Mukai M., Akedo H., Nakamura K., Hashimoto S., Sabe H. Paxillin alpha and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation, This is the final version - click for previous version, http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-06-0492, Temporal genome-wide DNA methylation signature of post-smolt Pacific salmon challenged with Instead, Cas appears to be necessary for the proper assembly of actin into a productive phagocytic apparatus. For example, the invasin protein of Yersinia binds 1 integrin, whereas the internalin proteins of Listeria bind to both E-cadherin and the receptor tyrosine kinase c-Met. Each bar represents the mean of six replicates; standard error is indicated by error bars. However, the bacterial staining is uniformly brighter and more focal than the transfected protein, making it possible to clearly identify both. 284, No.

Finally, we show that overexpression of FAK in Cas/ cells completely restores Salmonella internalization, suggesting that FAK and Cas may act in concert to promote bacterial invasion. However, when controls were compared to the adhesion and invasion groups, this difference was remarkable (p < 0.05). Interestingly, the few actin-rich foci that did form in FAK/ cells appeared phenotypically normal. For each time point use one plate. Invasive bacteria were generated by growth under O2-limited conditions as originally described (Lee and Falkow, 1990). Eight subjects, ranging in age from 3168 years, from the Graduate Clinic of the Piracicaba Dental School, State University of Campinas (UNICAMP) who had generalized chronic periodontitis and diabetes mellitus type 2 were included in the studies. B., Fry J., Rock E. P., Falkow S. Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors. (A) Paxillin+/+, paxillin/ (mock transfected), or paxillin/ MEFs transfected with GFP-paxillin were infected with SL1344 at a MOI = 30 for 1 h and were subjected to the two-color immunofluorescence assay. When phosphorylated, these pYDxP motifs bind two adaptor proteins, CrkII and Nck, which can link Cas to other regulators of the actin cytoskeleton. 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. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Plates are precoated with a uniform substrate layer of a single ECM protein: Collagen I, Collagen IV, Fibrinogen, Fibronectin, or Laminin. Thus human intestinal cells are used as an in vitro medium to study adhesion and intracellular survival of Listeria monocytogenes. MF-Millipore filters (EMD Millipore, catalog number: SCGPU05RE), Eppendorf tubes (Eppendorf, catalog number: 022363204), Cell culture flasks (Corning, catalog number: 3275), Cell culture plates (12-well cell culture receiver plate, sterile) (EMD Millipore, catalog number: PIMWS1250), Cell scrapers (Corning, catalog number: 3010), ZapCap bottle-top filters, pore size 0.2 m (Maine Manufacturing, catalog number: 10443421), Serological pipettes (1, 5, 10, 25, 50 ml) (Corning, catalog numbers: 4010, 4050, 4100, 4250, 4501), Culture tubes (Sigma-Aldrich, catalog number: T1661), Caco-2 cell line (ATCC, catalog number: HTB-37), 70% ethanol (Fisher Scientific, catalog number: BP82014), Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T3924), Brain Heart Infusion (BHI) agar (Sigma-Aldrich, catalog number: 70138), BHI broth (Sigma-Aldrich, catalog number: 53286), Gentamicin (Sigma-Aldrich, catalog number: G1397), Eagles minimum Essential medium (EMEM) (ATCC, catalog number: 30-2003), Fetal bovine serum, certified, heat inactivated (FBS) (Thermo Fisher Scientific, Gibco, Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco, Dulbeccos phosphate buffered saline (Sigma-Aldrich, catalog number: D8537), Triton X-100 (Sigma-Aldrich, catalog number: 234729), Phosphate-buffered saline, 10x (PBS) (Sigma-Aldrich, catalog number: P5493), Water bath, 37 C (Thermo Fisher Scientific), Biological safety cabinet (Thermo Fisher Scientific, Thermo Scientific, Inverted microscope(Nikon Instruments, model: Eclipse Ti-S), Pipette (Thermo Fisher Scientific, Thermo Scientific, Pipettor (Daigger Scientific, model: Portable Pipet-Aid XP Pipette Controller), Hemacytometer (Fisher Scientific, catalog number: S17040), 37 C incubator (Thermo Fisher Scientific), Fisher Scientific Sonic dismembrator (Fisher Scientific, model: 100), Vortex (Thermo Fisher Scientific, Thermo Scientific, Growing and maintaining Caco-2 cell lines, Using MF-Millipore filters prepare media, Caco-2, human colon adenocarcinoma cells are maintained in cEMEM (complete EMEM, see Recipes), Thaw a vial of frozen Caco-2 cells from ATCC (or previously frozen cells in the lab) by gentle agitation in a 37 C water bath. The resulting suspensions are diluted (1/100, 1/1,000 and 1/10,000) using 1xPBS. One possible explanation for this observation is that Cas can localize to focal adhesions independently of FAK, as has been previously shown (Harte et al., 2000) and that direct interaction between FAK and Cas is not absolutely required. The new PMC design is here! [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. Briefly, cells were infected at an MOI of 30 for 1 h at 37C. Briefly, biopsies of healthy gingiva were obtained from the retromolar area of a single individual, i.e., a 23-year-old, non-smoker, without systemic alterations, who was undergoing third molar extractions for orthodontic reasons. In contrast to the integrin-mediated internalization of Staphylococcus, which requires FAK kinase activity, the catalytic domain of FAK is not necessary for invasion. 516, No. However, whether focal adhesion proteins play a role in Salmonella invasion remains largely unknown. In addition to the presence of several cytokines, NO production in the microenvironment of periodontal disease, has been observed and acts directly on the maintenance or progression of inflammation and tissue destruction [10]. To test this hypothesis, we examined the effects of Salmonella infection on the association of endogenous p130Cas, FAK, and paxillin in a coprecipitation assay. HHS Vulnerability Disclosure, Help The green colonies grown on the chromogenic agar plates were randomly selected and cultures stored in glycerol stock at 20C for laboratory analysis. The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). Cell adhesion is a complex mechanism involved in a variety of processes including cell migration & invasion, embryogenesis, wound healing and tissue remodeling. A key feature of Salmonella pathogenesis in humans and other animals is the ability of the bacteria to enter and penetrate the intestinal epithelium. [19] quantitatively evaluated the adhesion of oral strains of C. albicans, C. tropicalis, and C. glabrata to human gingival epithelial cells, gingival fibroblasts and pulmonary fibroblasts. Note: This product has been discontinued. Therefore all unattached and attached yeasts were collected to obtain the total number of CFU/ml. assay adhesion This finding is in agreement with an earlier observation that tyrosine kinase activity is not required for Salmonella entry (Rosenshine et al., 1994). Reddy S., Akgul A., Karsi A., Abdelhamed H., Wills R. W. and Lawrence M. L.(2016). (C) Cas+/+, Cas/ (mock transfected), and Cas/ cells transfected with full-length Cas, CasSH3, or CasYXXP mutant were subjected to the two-color immunofluorescence invasion assay. Care was taken to ensure that cells used for quantitation expressed similar levels of exogenous protein. In contrast, expression of FAK in Cas/ cells completely restored Salmonella internalization. adhesion migration cell vitro invasion imaging assays The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton, The integrin-binding domain of invasin is sufficient to allow bacterial entry into mammalian cells. NO produced by fibroblasts in contact Candida albicans strains during adhesion and invasion assays. At least 40 foci were measured. Cells from these culture flasks, at approximately 1 105 cells/well, were used to inoculate 24-well tissue culture plates containing supplemented RPMI and incubated at 37C in 5% CO2 for 24 h to reach a confluent monolayer. Wash pellet and resuspend it in 1 ml of fresh warm (37 C) cEMEM. Mitra S. K., Hanson D. A., Schlaepfer D. D. Focal adhesion kinase: in command and control of cell motility, Role of tyrosine kinases and the tyrosine phosphatase SptP in the interaction of. Focal adhesions are a complex assembly of proteins that provide a physical linkage between integrins and the actin cytoskeleton (Zamir and Geiger, 2001). 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). Each point represents the mean of six replicates; standard error is indicated by error bars. Several other enteric pathogens, such as Yersinia and Listeria, utilize a zipper mechanism to enter host cells, in which a bacterial surface ligand is used to engage host cell surface receptors. (B) MDCK cells were treated with either DMSO (control) or 10 M PP2 for 30 min before performing a gentamicin resistance invasion assay. Rac1 activation is not impaired in FAK/ MEFs. The C terminus also contains a paxillin-binding site (Parsons, 2003; Mitra et al., 2005). (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. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. Blots were then stripped and reprobed for Cas.

The samples were read at 550nm [14], with the assay performed in duplicate. Figure 3. Rac1 and Cdc42 were detected using monoclonal antibodies (BD Transduction Laboratories, Lexington, KY; Rac1, 1:1000; Cdc42, 1:250). (A) MDCK cells were infected with wild-type S. typhimurium (SL1344) or the invasion-defective mutant VV341 (HilA) at a MOI = 20 for 20, 40, or 60 min. Both the gentamicin-resistance assay (Figure 5A) and the fluorescence-invasion assay (Figure 5, C and D) revealed that invasion efficiency was reduced by 80% (5%; p < 0.001) in Cas/ cells compared with Cas+/+ controls. In contrast to FAK+/+ cells, where actin foci were robustly induced, focus formation was reduced by 80% in FAK/ cells (Figure 3A).

To determine if any of these binding partners is necessary for Salmonella invasion, we analyzed bacterial internalization in FAK/ cells transfected with a panel of FAK mutants: FAK P715A, which largely attenuates binding to Cas (Polte and Hanks, 1995; Harte et al., 1996); FAK P878A, which impairs binding to ASAP1 and GRAF1 (Taylor et al., 1998; Randazzo et al., 2000; Liu et al., 2002); or FAK I937/999E, which reduces binding to paxillin (K. H. Martin and J. T Parsons, unpublished observation). To test this possibility, we examined the activation of endogenous Rac1 and Cdc42 during Salmonella infection using a pulldown assay as previously described (Criss et al., 2001). Being a foodborne pathogen, it enters the blood stream in humans via intestines by crossing intestinal barriers. The For adhesion and invasion, the percentage was calculated based on each control group, e.g., adhered cells 100/control adhesion. Emerging studies have shown that FAK/Cas interaction is essential for mediating signaling to downstream effectors and that a FAK mutant lacking the Cas binding site compromises cell migration (Parsons, 2003). 67, No. All strains were able to adhere and invade gingival fibroblasts but the HH strains had a greater capacity to adhere and invade cells (Table 1) than LH isolates. Focal adhesion-like complexes form at sites of Salmonella invasion.

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