Regulated expression of the Escherichia coli lepB gene as a tool for cellular testing of antimicrobial compounds that inhibit signal peptidase I in vitro.
Escherichia coli under-expressing lepB was utilized to test cellular inhibition of signal peptidase I (SPase). For the construction of a lepB regulatable strain, the E. coli lepB gene was cloned into pBAD, with expression dependent on L-arabinose. The chromosomal copy of lepB was replaced with a kanamycin resistance gene, which was subsequently removed. SPase production by the lepB regulatable strain in the presence of various concentrations of L-arabinose was monitored by Western blot analysis. At lower arabinose concentrations growth proceeded more slowly, possibly due to a decrease of SPase levels in the cells. A penem SPase inhibitor with little antimicrobial activity against E. coli when tested at 100 micro M was utilized to validate the cell-based system. Under-expression of lepB sensitized the cells to penem, with complete growth inhibition observed at 10 to 30 micro M. Growth was rescued by increasing the SPase levels. The cell-based assay was used to test cellular inhibition of SPase by compounds that inhibit the enzyme in vitro. MD1, MD2, and MD3 are SPase inhibitors with antimicrobial activity against Staphylococcus aureus, although they do not inhibit growth of E. coli. MD1 presented the best spectrum of antimicrobial activity. Both MD1 and MD2 prevented growth of E. coli under-expressing lepB in the presence of polymyxin B nonapeptide, with growth rescue observed when wild-type levels of SPase were produced. MD3 and MD4, a reactive analog of MD3, inhibited growth of E. coli under-expressing lepB. However, growth rescue in the presence of these compounds following increased lepB expression was observed only after prolonged incubation.
Barbosa
Maria D F S
MD
Departments of Antimicrobial Research, Bristol-Myers Squibb Company, Wilmington, Delaware 19880, USA. mbarbosa@xencor.com
Lin
Siqi
S
Markwalder
Jay A
JA
Mills
Jonathan A
JA
DeVito
Joseph A
JA
Teleha
Christopher A
CA
Garlapati
Vasudha
V
Liu
Charles
C
Thompson
Andy
A
Trainor
George L
GL
Kurilla
Michael G
MG
Pompliano
David L
DL
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015153:Blotting, Western; D015780:Carbapenems / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D008565:Membrane Proteins; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D008565:Membrane Proteins; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; C026515:type I signal peptidase