TY - JOUR
T1 - Stress-based high-throughput screening assays to identify inhibitors of cell envelope biogenesis
AU - Steenhuis, Maurice
AU - Ten Hagen-Jongman, Corinne M.
AU - van Ulsen, Peter
AU - Luirink, Joen
PY - 2020/11
Y1 - 2020/11
N2 - The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.
AB - The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.
KW - Antibiotics
KW - Cpx
KW - Escherichia coli
KW - Heat-shock
KW - High-throughput screening
KW - Potentiators
KW - Rcs
KW - SigmaE
UR - http://www.scopus.com/inward/record.url?scp=85096071019&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85096071019&partnerID=8YFLogxK
U2 - 10.3390/antibiotics9110808
DO - 10.3390/antibiotics9110808
M3 - Article
AN - SCOPUS:85096071019
SN - 2079-6382
VL - 9
SP - 1
EP - 15
JO - Antibiotics
JF - Antibiotics
IS - 11
M1 - 0808
ER -