Peptides were tested for their ability to bind to the A549 alveolar cell line (ATCC CLL-185) and to macrophages derived from U937 monocytes (ATCC CRL-2367).

Briefly, 1.5 × 106 cells cultured in Roux flasks were dislodged using 1× Non-enzymatic Cell Dissociation Solution (Sigma) and incubated with increasing concentrations of 125I-labeled peptide (0-950 nM) in the presence or absence of unlabeled peptide (40 μM). Unbound peptide was removed using a dioctylphthalate-dibutylphthalate cushion, before measuring cell-associated radioactivity in a gamma counter (Gamma Counter Cobra II, Packard Instrument Co., Meriden, CT, USA). Total binding minus nonspecific binding yielded the specific binding curve, whose slope corresponded selleck to the binding activity of the peptide. Any peptide displaying a specific binding activity of ≥1% was considered a HABP [23–25, 37]. Binding constants were determined by performing a saturation assay using U937 cells and peptide concentrations larger than the ones used for binding assays (0-4500 nM). Circular dichroism analyses of Rv0679c peptides The secondary find more structure elements of the peptides spanning the entire length of Rv0679c were studied by circular dichroism. CD spectra of peptides (5 μM) dissolved in 30% trifluoroethanol

click here (TFE) were acquired at 20°C by averaging three scans taken in a Jasco J-810 spectropolarimeter (wavelength range: 260-190 nm, scan rate: 20 nm/min, bandwidth: 1 nm), using a 1.00-cm pathway cuvette (Jasco Inc, Easton, MD). Data were corrected for baseline deviation [38]. The results were expressed as mean residue ellipticity [θ], the units being degrees × cm2 × dmol-1 according to the [Θ] = Θλ/(100lcn) function, where θλ is the measured ellipticity, l is the optical path length, c is the peptide concentration, and n is the number of residues in the amino acid sequence. Invasion inhibition assays Rv0679c HABPs were assessed for their

ability to inhibit mycobacterial invasion using a flow-cytometry-based assay developed by Bermúdez and Goodman [39] and later modified by us [26]. In brief, A549 and U937 cells (1 × 106) seeded overnight on 6-well Cobimetinib plates were incubated for 1 h with different peptide concentrations. SYBR-safe stained mycobacteria (10 × 106) suspended in RPMI medium were added to each well (MOI: 1:10) and incubated overnight at 37°C. Inhibition controls consisted of Cytochalasin D (3 μM) or colchicine (50 μM). Extracellular bacilli were first inactivated by incubation with Amikacin (200 μg/mL) for 1 h and then removed by successive washes with Hanks Balanced Salt Solution (HBSS). Cells were dislodged from monolayers and stained with methylene blue for FACscan flow cytometry analysis (Becton Dickinson).