S. flexneri growth curves The growth curves of S. flexneri 2a strains were determined by PLX3397 manufacturer measuring the optical density at 600 nm (OD600) as described previously [28]. Briefly, overnight cultures were diluted 1:200 and incubated at 37°C with shaking (220 rpm). Samples (1 mL) of the bacterial cultures were taken every 30 min over 16 h and OD measured. Growth curves were created by plotting

OD600 against incubation time (h). S. flexneri HeLa cell invasion assays S. flexneri cell invasion assays were used to test the virulence of a SF51 clinical strain without set1B, SF301-∆ pic, wild-type SF301, SF301-∆ pic/pPic and SF51/pPic. The CFTRinh-172 ability of bacteria to invade HeLa cells was determined using a gentamicin protection assays [29]. HeLa cells were grown in 6-well tissue culture plates in DMEM supplemented with

10% FCS and incubated at 37°C/5% CO2 until they formed semi-confluent monolayers. SF51, SF301-∆ pic, SF301-∆ pic /pPic, SF51 /pPic and SF301 were individually added to semi-confluent HeLa cells at an MOI of 100. Bacteria were diluted and plated on LB agar plates. Colony-forming units (CFUs) were counted and added to HeLa cells. Plates were centrifuged at 900 × g for 5 min. After incubating at 37°C for 90 min, cells were washed three times with PBS, and gentamicin added to the medium at a final concentration of 10 μg/mL. The mixture was then incubated BEZ235 in vivo for 20 min at 37°C. HeLa cells in each well were lysed with 1 mL of

PBS containing 0.1% Triton X-100 for 10 min at room temperature. Lysates were diluted and plated onto LB agar plates in triplicate. Colonies that grew on LB plates were counted. Results were expressed as the number of bacteria recovered from gentamicin-treated cells divided by the number of inoculated bacteria added to the cell. Cells inoculated with E. coli ATCC 25922, an avirulent strain, were the negative controls. Cell invasion assays were performed in triplicate for each strain, and the assay repeated twice. Sereny tests and pathohistological examination A mouse Sereny test was used Molecular motor to evaluate the virulence of all strains we examined in this study, as described by Murayama [30]. A single red colony of S. flexneri on Congo red agar [Tryptic soy broth (Oxoid), 1.5% (w/v) agar and 0.01% (w/v) Congo red] was inoculated into LB broth at 37°C for 8 h with constant shaking. Female BALB/c mice (4–5-weeks-old) were infected with 1 × 108 CFUs per eye (n = 4 eyes, two mice in each group). Symptoms and signs of keratoconjunctivitis in mice infected with bacteria were observed at 24, 48, 72, and 96 h post-inoculation [28, 30]. Eyes inoculated with E. coli ATCC 25922 and normal saline (NS) served as the negative controls. The invasiveness of bacteria was scored according to the following system: ‘−’ indicates no inflammation, and an infection level score of 0; ‘±’ is indicative of low levels of keratoconjunctivitis, and an infection level score of 0.