Supplementary MaterialsS1 Fig: Cloning and purification of PtsA. Immunoblotting of the purified PtsA protein with anti-HAT antibodies. Lane 1: Immunoblotting with pre-immune serum. Lane 2: Immunoblotting with anti-HAT antiserum. (C) Coomassie brilliant blue staining of untagged rPtsA separated on SDS PAGE. (D) An immunoblot of untagged rPtsA probed with rabbit anti HAT tagged rPtsA antiserum. (E). MALDI TOF analysis of the untagged rPtsA.(TIF) pone.0150320.s001.tif (1.9M) GUID:?6774E52C-8307-4678-9FAE-8BC7FA5E959A S2 Fig: Identification of rPtsA binding sequences. A combinatorial peptide library was screened with rPtsA. The phages that bound rPtsA were tested for their ability to inhibit adhesion to A549 cells. These phages were incubated with strain WU2 for 1 h and added to A549 cells; excess bacteria were removed; and cells were detached with trypsin and plated onto blood agar plates for counting. (A) Phage D3 (p 0.0001, r = -1); (B) Phage E6 (p 0.0001, r = -0.6); (C) Phage D8 p 0.0001, r = -0.8); (D) Phage D10 p 0.0001, r = -0.8); (E) Phage H9 (p 0.0001, r = -0.7); (F) Phage H10 (p 0.0001, r not significant but there was a 75% reduction in adhesion); (G) The phage without Y-33075 dihydrochloride an insert did interfere with pneumococcal adhesion to A549 cells, even though it reduced adhesion by only 20% in comparison to 75% reduction in adhesion in the above active phages (p 0.001 r = -0.7). (H) An inactive phage with an insert demonstrated about 15% reduction in bacterial adhesion (p 0.0001, r = -0.7). Experiments were performed in 3C6 replicates and repeated at least 3 times. Values are meansSD. *Student’s cells (WU2 strain) were treated for 1 h with each peptide and added to Detroit 562 cells for 2 h; non-adherent bacteria were removed, and cells were detached with trypsin and plated onto blood agar plates for bacterial colony counting. (A) BMPER (p 0.0001; r = ?0.09); (B) PCDH19 (p 0.0001; r = ?0.829); (C) Int 4 (p 0.0001; r = no dose dependency but 75% inhibition of Y-33075 dihydrochloride bacterial adhesion); (D) Eps 1 (p 0.0001; r = ?0.771). Experiments were performed in 3C6 replicates and repeated at least 3 times. Values are meansSD. *Student’s strains. clinical isolates from serotypes 1, 5, 6B, 9V, 14DW, 14R, 23F and laboratory strains from serotypes 2 (D39) and 3 (WU2) were used. Cell wall fractions were isolated using mutanolysin. The cell walls proteins were isolated by 2D PAGE. Protein spots were excised from the gel and subjected to MALDI-TOF-MS Y-33075 dihydrochloride analysis(DOC) pone.0150320.s005.doc INSR (241K) GUID:?C4E40204-16E1-4331-ADC1-4283D6FD6B85 Y-33075 dihydrochloride S2 Table: Background information for the healthy children. To test whether PtsA is antigenic in children, rPtsA was immunoblotted with sera obtained from healthy children. These healthy children served as control for a Pneumovax clinical trial from 2001C2007.(DOCX) pone.0150320.s006.docx (17K) GUID:?021C3F8B-9E3D-4B4A-9862-D1613F2524D6 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract In to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human Y-33075 dihydrochloride BMPER, multimerin1, protocadherin19, integrin4, epsin1 and collagen type VII1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with (pneumococcus) colonizes the human nasopharynx asymptomatically and may therefore spread through the population. The asymptomatic colonization and the rapid spread of the bacteria are in.