A Modular High-Throughput In Vivo Screening Platform Based on Chimeric Bacterial Receptors

Abstract

Multidrug resistance (MDR) is a globally relevant problem that requires novel approaches. Two-component systems are a promising, yet untapped target for novel antibacterials. They are prevalent in bacteria and absent in mammals, and their activity can be modulated upon perception of various stimuli. Screening pre-existing compound libraries could reveal small molecules that inhibit stimulus-perception by virulence-modulating receptors, reduce signal output from essential receptors or identify artificial stimulatory ligands for novel SHKs that are involved in virulence. Those small molecules could possess desirable therapeutic properties to combat MDR. We propose that a modular screening platform in which the periplasmic domain of the targeted receptors are fused to the cytoplasmic domain of a well-characterized receptor that governs fluorescence reporter genes could be employed to rapidly screen currently existing small molecule libraries. Here, we have examined two previously created Tar-EnvZ chimeras and a novel NarX-EnvZ chimera. We demonstrate that it is possible to couple periplasmic stimulus-perceiving domains to an invariable cytoplasmic domain that governs transcription of a dynamic fluorescent reporter system. Furthermore, we show that aromatic tuning, or repositioning the aromatic residues at the end of the second transmembrane helix (TM2), modulates baseline signal output from the tested chimeras and even restores output from a nonfunctional NarX-EnvZ chimera. Finally, we observe an inverse correlation between baseline signal output and the degree of response to cognate stimuli. In summary, we propose that the platform described here, a fluorescent Escherichia coli reporter strain with plasmid-based expression of the aromatically tuned chimeric receptors, represents a synthetic biology approach to rapidly screen pre-existing compound libraries for receptor-modulating activities.

Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ

“…Ala and Phe substitutions were evaluated at sites expected to be on the interior of the protein; these mutants were expected to alter the relative energetics of the kinase- versus phosphatase-promoting states by altering core packing geometry ( Airola et al, 2013 ; Ferris et al, 2011 ; Hulko et al, 2006 ; Bi et al, 2018 ). We also examined the effects of Trp substitutions in the TM helix at positions expected to map to the headgroup region of the bilayer, as similar substitutions often induce changes in signaling ( Draheim et al, 2005 ; Lehning et al, 2017 ; Inda et al, 2016 ; Monzel and Unden, 2015 ; Yusuf and Draheim, 2015 ; Yusuf et al, 2018 ; Adase et al, 2013 ). We also included several mutations, particularly in the C-terminal half of the HAMP domain, that show altered autokinase activity as compared to WT PhoQ.…”

Multiple functions of flagellar motility and chemotaxis in bacterial physiology

“… 2021 ; Lehning et al . 2017 ; Luu et al . 2019 ; Matilla, Martin-Mora and Krell 2020 ), only a tiny fraction of chemoreceptor ligands are currently known and even fewer have an established mode of binding (Ortega, Zhulin and Krell 2017 ).…”

Nitrate- and Nitrite-Sensing Histidine Kinases: Function, Structure, and Natural Diversity

“…Sensory domains are predominantly either fully α-helical (such as 4-helical bundle, 4HB fold) or mixed α/β (such as single and double Cache) [ 42 , 43 ]. Conformational changes in different sensory domains are likely to be similar, because they are often interchangeable, and functional chimeric sensor proteins are often easily constructed [ 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ]. Sometimes, more than one signal may be recognized, either by the same sensory domain [ 53 , 54 ] or by the canonical sensory domain and additional cytoplasmic modules [ 55 , 56 ].…”