Conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies.
Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single-chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single-chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high-throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the recipient cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr-26 and involved in the conjugative DNA-processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the recipient cell and open a new approach to identify specific inhibitors of bacterial conjugation.
Garcillán-Barcia
M Pilar
MP
Departamento de Biología Molecular (Laboratorio asociado al Centro de Investigaciones Biológicas, C.S.I.C.), Universidad de Cantabria, C/Cardenal Herrera Oria s/n, 39011 Santander, Spain.
Jurado
Paola
P
González-Pérez
Blanca
B
Moncalián
Gabriel
G
Fernández
Luis A
LA
de la Cruz
Fernando
F
D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology*; D003227:Conjugation, Genetic; D004254:DNA Nucleotidyltransferases / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000276:immunology; D008958:Models, Molecular
D000907:Antibodies, Bacterial; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D004254:DNA Nucleotidyltransferases; C510362:TrwC protein, E coli