Evaluation of small molecule kinase inhibitors as novel antimicrobial and antibiofilm agents.

Antibiotic resistance is a global and pressing concern. Our current therapeutic arsenal is increasingly limited as bacteria are developing resistance at a rate that far outpaces our ability to create new treatments. Novel approaches to treating and curing bacterial infections are urgently needed. Bacterial kinases have been increasingly explored as novel drug targets and are poised for development into novel therapeutic agents to combat bacterial infections. This review describes several general classes of bacterial kinases that play important roles in bacterial growth, antibiotic resistance, and biofilm formation. General features of these kinase classes are discussed and areas of particular interest for the development of inhibitors will be highlighted. Small molecule kinase inhibitors are described and organized by phenotypic effect, spotlighting particularly interesting inhibitors with novel functions and potential therapeutic benefit. Finally, we provide our perspective on the future of bacterial kinase inhibition as a viable strategy to combat bacterial infections and overcome the pressures of increasing antibiotic resistance.

The bacterial volatile N,N-dimethyl-hexadecylamine promotes Arabidopsis primary root elongation through cytokinin signaling and the AHK2 receptor.

N,N-dimethyl-hexadecylamine (DMHDA) is a volatile organic compound (VOC) produced by some plant growth-promoting rhizobacteria (PGPR), which inhibits the growth of pathogenic fungi and induces iron uptake by roots. In this report, through the application of a wide range of concentrations, we found that DMHDA affects Arabidopsis primary root growth and lateral root formation in a dose-dependent manner where 1 and 2 µM promoted root growth and higher (4-32 µM) concentrations repressed growth. Cytokinin-inducible TCS::GFP and ARR5::uidA gene constructs showed an increased expression in columella cells and root meristem, respectively, at 2 µM DMHDA, but their expression domains strongly diminished at growth repressing treatments. To test if either primary root growth promotion or repression could involve members of the cytokinin receptor family, the growth of WT and double mutant combinations cre1-12 ahk2-2, cre1-12 ahk3-3, and ahk2-2 ahk3-3 was tested in control conditions or supplemented with 2 µM or 16 µM DMHDA. Noteworthy, the root growth promotion disappeared in cre1-12 ahk2-2 and ahk2-2 ahk3-3 combinations, whereas all double mutants had higher repression than the WT at high doses. We further show that DMHDA fails to mimic the effects of ethylene in Arabidopsis seedlings grown in darkness that include an exaggerated apical hook, stem and root shortening, and root hair elongation. Our data help unravel how Arabidopsis senses a growth-modulating bacterial volatile through changes in cytokinin responsiveness.

Effect of Phosphatase Activity of the Control of Virulence Sensor (CovS) on Clindamycin-Mediated Streptolysin O Production in Group A Streptococcus.

Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.

Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346.

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light-sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

Cold interferes with male meiotic cytokinesis in Arabidopsis thaliana independently of the AHK2/3-AHP2/3/5 cytokinin signaling module.

Previously we have shown that low temperature stress in Arabidopsis causes defects in microtubule organization and cytokinesis in male meiocytes, which leads to the formation of diploid pollen. Because cytokinin (CK) mediates multiple physiological responses to cold stress, we investigated whether CK signaling is involved in cold-induced diploid pollen formation. To this end, we monitored male sporogenesis in a series of mutants defective in CK metabolism and signalling. Arabidopsis plants with altered CK homeostasis, that is, the ahk2-2 ahk3-3 double and the ahp2-1 ahp3 ahp5-2 triple mutant, were cold sensitive and displayed similar defective male meiotic cytokinesis as wild type plants upon cold stress. These findings demonstrate that the AHK2/3-AHP2/3/5 CK-signaling module is not required for cold-induced ploidy stability of male gamete in Arabidopsis. Cytological analysis further revealed that the cold-induced cytokinesis defects in the ahk2-2 ahk3-3 mutant correlated with irregular organization of the radial microtubule array (RMA) in tetrad microspores at the end of male meiosis. Contrary to the ahk and ahp mutants, Arabidopsis plants defective for ARR1, a downstream target of ahk and ahp mediated CK signalling, displayed higher cold-tolerance of male meiotic cytokinesis program. We here suggest that the transcription regulator ARR1 may act independently from the CK AHK2/3-AHP2/3/5 signaling module in conveying the cold response to male meiocytes.

A Novel Two-Component System, GluR-GluK, Involved in Glutamate Sensing and Uptake in Streptomyces coelicolor.

Two-component systems (TCSs), the predominant signal transduction pathways employed by bacteria, play important roles in physiological metabolism in Streptomyces Here, a novel TCS, GluR-GluK (encoded by SCO5778-SCO5779), which is located divergently from the gluABCD operon encoding a glutamate uptake system, was identified as being involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor Under the condition of minimal medium (MM) supplemented with different concentrations of glutamate, deletion of the gluR-gluK operon (gluR-K) resulted in enhanced actinorhodin (ACT) but reduced undecylprodigiosin (RED) and yellow type I polyketide (yCPK) production, suggesting that GluR-GluK plays a differential role in antibiotic biosynthesis. Furthermore, we found that the response regulator GluR directly promotes the expression of gluABCD under the culture condition of MM with a high concentration of glutamate (75 mM). Using the biolayer interferometry assay, we demonstrated that glutamate acts as the direct signal of the histidine kinase GluK. It was therefore suggested that upon sensing high concentrations of glutamate, GluR-GluK would be activated and thereby facilitate glutamate uptake by increasing gluABCD expression. Finally, we demonstrated that the role of GluR-GluK in antibiotic biosynthesis is independent of its function in glutamate uptake. Considering the wide distribution of the glutamate-sensing (GluR-GluK) and uptake (GluABCD) module in actinobacteria, it could be concluded that the GluR-GluK signal transduction pathway involved in secondary metabolism and glutamate uptake should be highly conserved in this bacterial phylum.IMPORTANCE In this study, a novel two-component system (TCS), GluR-GluK, was identified to be involved in glutamate sensing and uptake as well as antibiotic biosynthesis in Streptomyces coelicolor A possible GluR-GluK working model was proposed. Upon sensing high glutamate concentrations (such as 75 mM), activated GluR-GluK could regulate both glutamate uptake and antibiotic biosynthesis. However, under a culture condition of MM supplemented with low concentrations of glutamate (such as 10 mM), although GluR-GluK is activated, its activity is sufficient only for the regulation of antibiotic biosynthesis. To the best of our knowledge, this is the first report describing a TCS signal transduction pathway for glutamate sensing and uptake in actinobacteria.