Investigation of the in vivo interaction between β-lactamase and its inhibitor protein

Abstract

The afnity of β-lactamase inhibitory protein (BLIP) for TEM-1 β-lactamase has raised hopes in the challenge of protein- based inhibitor discovery for β-lactamase-mediated antibiotic resistance. Currently, the efect of the formation of the β-lactamase:BLIPcomplex in vivo in β-lactam resistant bacteria is an open question. Te scarcity of information to the extent to which BLIP can impairβ-lactamase activity inside cells has urged us to assess the in vivo efcacy of BLIP as a potent β-lactamase inhibitor. To this end,β-lactamase and BLIP were coexpressed in Escherichia coli. Simultaneous expression of β-lactamase and BLIP and the formation ofthe TEM-1 β-lactamase:BLIP complex in the periplasmic space of E. coli were verifed by electrophoretic and Western blot techniques.Growth profles of the cells expressing both β-lactamase and its protein inhibitor, complemented with β-lactamase activity measurements,suggested that BLIP synthesis retarded cell growth and reduced β-lactamase activity. Although co-expression of β-lactamase and itsprotein inhibitor did not completely impair cell growth, the specifcity of BLIP enabled it to bind β-lactamase in the bacterial periplasm,regardless of the crowding components.