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Volume 5, Issue 3 (Suppl)
J Infect Dis Ther, an open access journal
ISSN:2332-0877
Infectious Diseases 2017
August 21-23, 2017
3
rd
Annual Congress on
Infectious Diseases
August 21-23, 2017 San Francisco, USA
Fluorescence high-throughput screening for inhibitors of TonB action
Phillip E Klebba, Olivia S Eliasson, Dallas R Hyder, Noah J Long, Aritri Majumdar, Somnath, Chakravorty, Peter McDonald, Anuradha Roy, Salete M
Newton
and
Brittany L Nairn
Kansas State University, USA
G
ram (-) bacteria acquire ferric siderophores through TonB-dependent transporters (TBDT) in their outer membrane.
By fluorescence spectroscopic high-throughput screening (FLHTS) we identified inhibitors of TonB-dependent ferric
enterobactin (FeEnt) uptake through
E. coli
FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441
compounds, we evaluated 20 candidates in secondary tests of TonB activity, including ferric siderophore uptake and colicin
killing. 6 of the 20 primary hits inhibited TonB action in all the tests. Further analysis of the inhibitors in [
59
Fe] Ent and [
14
C]-
lactose accumulation experiments suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, did
not behave like proton ionophores and may inhibit TonB-ExbBD. The success of FLHTS against
E. coli
led us to adapt it to the
ESKAPE pathogen
Acinetobacter baumannii
. We identified its FepA ortholog (
AbaFepA
), confirmed its involvement in FeEnt
uptake by deleting the structural gene, cloned
AbafepA
, genetically engineered 8 Cys substitutions in its surface loops, modified
them with fluorescein and made fluorescence spectroscopic observations of FeEnt uptake in
A. baumannii
. Among the Cys
substitutions in
AbaFepA
, several (S279C, T562C, S665C) were well labeled by fluorescein and suitable for measurements of
FeEnt transport. As in
E. coli
, the test monitored TonB-dependent FeEnt uptake by
AbaFepA
. In micro titer format FLHTS with
A. baumannii
produced Z’ factors from 0.6-0.8. Overall these experiments both identified agents that block TonB action, and
revealed the potential of FLHTS for larger screens of bigger libraries to find novel antimicrobial compounds against Gram (-)
bacteria, including the CRE/ESKAPE pathogens.
peklebba@ksu.eduJ Infect Dis Ther 2017, 5:3 (Suppl)
DOI: 10.4172/2332-0877-C1-027