Peptide sequences identified by phage display are immunodominant functional motifs of Pet and Pic serine proteases secreted by Escherichia coli and Shigella flexneri.
Plasmid-encoded toxin (Pet) and protein involved in colonization (Pic), are serine protease autotransporters of Enterobacteriaceae (SPATEs) secreted by enteroaggregative Escherichia coli (EAEC), which display the GDSGSG sequence or the serine motif. Our research was directed to localize functional sites in both proteins using the phage display method. From a 12mer linear and a 7mer cysteine-constrained (C7C) libraries displayed on the M13 phage pIII protein we selected different mimotopes using IgG purified from sera of children naturally infected with EAEC producing Pet and Pic proteins, and anti-Pet and anti-Pic IgG purified from rabbits immunized with each one of these proteins. Children IgG selected a homologous group of sequences forming the consensus sequence, motif, PQPxK, and the motifs PGxI/LN and CxPDDSSxC were selected by the rabbit anti-Pet and anti-Pic IgGs, respectively. Analysis of the amino terminal region of a panel of SPATEs showed the presence in all of them of sequences matching the PGxI/LN or CxPDDSSxC motifs, and in a three-dimensional model (Modeller 9v2) designed for Pet, both these motifs were found in the globular portion of the protein, close to the protease active site GDSGSG. Antibodies induced in mice by mimotopes carrying the three aforementioned motifs were reactive with Pet, Pic, and with synthetic peptides carrying the immunogenic mimotope sequences TYPGYINHSKA and LLPQPPKLLLP, thus confirming that the peptide moiety of the selected phages induced the antibodies specific for the toxins. The antibodies induced in mice to the PGxI/LN and CxPDDSSxC mimotopes inhibited fodrin proteolysis and macrophage chemotaxis biological activities of Pet. Our results showed that we were able to generate, by a phage display procedure, mimotopes with sequence motifs PGxI/LN and CxPDDSSxC, and to identify them as functional motifs of the Pet, Pic and other SPATEs involved in their biological activities.
Ulises
Hernández-Chiñas
HC
Unidad de Posgrado, Departamento de Salud Pública, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México DF, Mexico.
Tatiana
Gazarian
G
Karlen
Gazarian
G
Guillermo
Mendoza-Hernández
MH
Juan
Xicohtencatl-Cortes
XC
Carlos
Eslava
E
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000276:immunology; D004768:Enterotoxins / Q000737:chemistry* / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000276:immunology; D016056:Immunodominant Epitopes / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000276:immunology; D019151:Peptide Library; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000276:immunology; D057057:Serine Proteases / Q000737:chemistry* / Q000276:immunology; D012763:Shigella flexneri / Q000201:enzymology*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D019151:Peptide Library; D057057:Serine Proteases; C530786:Pet protein, E coli; C541114:Pic protein, E coli; D012697:Serine Endopeptidases