Cross-talk between QseBC and PmrAB two-component systems is crucial for regulation of motility and colistin resistance in Enteropathogenic Escherichia coli.

The quorum sensing two-component system (TCS) QseBC has been linked to virulence, motility and metabolism regulation in multiple Gram-negative pathogens, including Enterohaemorrhagic Escherichia coli (EHEC), Uropathogenic E. coli (UPEC) and Salmonella enterica. In EHEC, the sensor histidine kinase (HK) QseC detects the quorum sensing signalling molecule AI-3 and also acts as an adrenergic sensor binding host epinephrine and norepinephrine. Downstream changes in gene expression are mediated by phosphorylation of its cognate response regulator (RR) QseB, and 'cross-talks' with non-cognate regulators KdpE and QseF to activate motility and virulence. In UPEC, cross-talk between QseBC and TCS PmrAB is crucial in the regulation and phosphorylation of QseB RR that acts as a repressor of multiple pathways, including motility. Here, we investigated QseBC regulation of motility in the atypical Enteropathogenic E. coli (EPEC) strain O125ac:H6, causative agent of persistent diarrhoea in children, and its possible cross-talk with the KdpDE and PmrAB TCS. We showed that in EPEC QseB acts as a repressor of genes involved in motility, virulence and stress response, and in absence of QseC HK, QseB is likely activated by the non-cognate PmrB HK, similarly to UPEC. We show that in absence of QseC, phosphorylated QseB activates its own expression, and is responsible for the low motility phenotypes seen in a QseC deletion mutant. Furthermore, we showed that KdpD HK regulates motility in an independent manner to QseBC and through a third unidentified party different to its own response regulator KdpE. We showed that PmrAB has a role in iron adaptation independent to QseBC. Finally, we showed that QseB is the responsible for activation of colistin and polymyxin B resistance genes while PmrA RR acts by preventing QseB activation of these resistance genes.

Repurposing Hsp90 inhibitors as antimicrobials targeting two-component systems identifies compounds leading to loss of bacterial membrane integrity.

The discovery of antimicrobials with novel mechanisms of action is crucial to tackle the foreseen global health crisis due to antimicrobial resistance. Bacterial two-component signaling systems (TCSs) are attractive targets for the discovery of novel antibacterial agents. TCS-encoding genes are found in all bacterial genomes and typically consist of a sensor histidine kinase (HK) and a response regulator. Due to the conserved Bergerat fold in the ATP-binding domain of the TCS HK and the human chaperone Hsp90, there has been much interest in repurposing inhibitors of Hsp90 as antibacterial compounds. In this study, we explore the chemical space of the known Hsp90 inhibitor scaffold 3,4-diphenylpyrazole (DPP), building on previous literature to further understand their potential for HK inhibition. Six DPP analogs inhibited HK autophosphorylation in vitro and had good antimicrobial activity against Gram-positive bacteria. However, mechanistic studies showed that their antimicrobial activity was related to damage of bacterial membranes. In addition, DPP analogs were cytotoxic to human embryonic kidney cell lines and induced the cell arrest phenotype shown for other Hsp90 inhibitors. We conclude that these DPP structures can be further optimized as specific disruptors of bacterial membranes providing binding to Hsp90 and cytotoxicity are lowered. Moreover, the X-ray crystal structure of resorcinol, a substructure of the DPP derivatives, bound to the HK CheA represents a promising starting point for the fragment-based design of novel HK inhibitors. IMPORTANCE: The discovery of novel antimicrobials is of paramount importance in tackling the imminent global health crisis of antimicrobial resistance. The discovery of novel antimicrobials with novel mechanisms of actions, e.g., targeting bacterial two-component signaling systems, is crucial to bypass existing resistance mechanisms and stimulate pharmaceutical innovations. Here, we explore the possible repurposing of compounds developed in cancer research as inhibitors of two-component systems and investigate their off-target effects such as bacterial membrane disruption and toxicity. These results highlight compounds that are promising for further development of novel bacterial membrane disruptors and two-component system inhibitors.

Synthesis of indazoles from 2-formylphenylboronic acids.

A method for the synthesis of indazoles was developed which involves a copper(ii) acetate catalysed reaction of 2-formylboronic acids with diazadicaboxylates followed by acid or base induced ring closure. Hydrazine dicarboxylates were also shown as competent reaction partners for the synthesis of indazoles, however, they required a stoichiometric amount of copper(ii) acetate for the C-N bond formation step. The transformation can be efficiently performed as a two step-one pot procedure to give a range of 1N-alkoxycarbonyl indazoles.