Thus, precedents from other systems support our findings that spectrin, adducin, and p4.1 can act independently during bacterial pathogenesis. Conclusions Invasion of intestinal epithelial cells and comet tail-based motility in host cells are key for S. flexneri to access replicative niches and disseminate throughout host tissues . Here we have demonstrated that the actin-rich structures generated by these microbes also employ another cytoskeletal system, the spectrin cytoskeleton. Our identification of this structural network at these sites further highlights the importance of this system in bacterial pathogenesis and
indicates that these crucial segments in the pathogenesis of S. flexneri require a hybrid cytoskeletal meshwork, previously thought to be exclusive www.selleckchem.com/products/gsk2126458.html to actin. Methods Cells, bacteria and growth conditions HeLa cells (ATCC) were grown on #1 cover slips in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The bacterial strain utilized was S. flexneri (strain M90T). Bacteria were grown in standard trypticase soy. Infections HeLa cells were grown to approximately 70% confluency prior to infections. S. flexneri were grown overnight in standing culture, then diluted 80×, followed by growth in shaking
selleck culture at 37°C for 2.5 hours (OD600 nm = 0.6) after which 400 μl of the culture was added to the cells with 200 μl of growth media . Infections were initiated by centrifugation for 10 mins at 700 g and 21°C. To www.selleckchem.com/products/SB-202190.html quantify invasion events, investigate initial tail formation and study comet tails, total infection times consisted of 0.5, 2.5 and 4.5 hours respectively. For classical invasion assays, cells were washed 2× with PBS after 20 minutes of infections and incubated in 100 ug/mL of gentamycin in 10% DMEM for 1 hour. Cells were washed 3× with PBS, lysed using 1% triton and plated for CFU counts. Invasion assays examined by microscopy
To quantify S. flexneri invasion, mafosfamide similar infection parameters were followed as in the classical invasion assay, however after 1 hour of gentmycin treatment the cells were washed with PBS three times prior to fixation and quantification of bacterial invasion via microscopy. Immunofluorescence Immunofluorescence procedures were performed as described previously . Briefly, samples were fixed using 3% paraformaldehyde for 15 minutes then permeabilized using 0.1% Triton X-100 in PBS (without calcium or magnesium) (Hyclone) for 5 minutes. Prior to primary antibody treatments, samples were blocked in 5% normal goat serum in TPBS/0.1% BSA (0.05% Tween-20 and 0.1% BSA in PBS) for 20 minutes. Antibodies were then incubated on the cover slips overnight at 4°C. The next day secondary antibodies were applied for 1.5 hrs at 37°C. The cover slips were then mounted on glass slides using Prolong Gold with DAPI (Invitrogen).