NH125 Sensitizes Staphylococcus aureus to Cell Wall-Targeting Antibiotics through the Inhibition of the VraS Sensor Histidine Kinase.

Staphylococcus aureus utilizes the two-component regulatory system VraSR to receive and relay environmental stress signals, and it is implicated in the development of bacterial resistance to several antibiotics through the upregulation of cell wall synthesis. VraS inhibition was shown to extend or restore the efficacy of several clinically used antibiotics. In this work, we study the enzymatic activity of the VraS intracellular domain (GST-VraS) to determine the kinetic parameters of the ATPase reaction and characterize the inhibition of NH125 under in vitro and microbiological settings. The rate of the autophosphorylation reaction was determined at different GST-VraS concentrations (0.95 to 9.49 µM) and temperatures (22 to 40°C) as well as in the presence of different divalent cations. The activity and inhibition by NH125, which is a known kinase inhibitor, were assessed in the presence and absence of the binding partner, VraR. The effects of inhibition on the bacterial growth kinetics and gene expression levels were determined. The GST-VraS rate of autophosphorylation increases with temperature and with the addition of VraR, with magnesium being the preferred divalent cation for the metal-ATP substrate complex. The mechanism of inhibition of NH125 was noncompetitive in nature and was attenuated in the presence of VraR. The addition of NH125 in the presence of sublethal doses of the cell wall-targeting antibiotics carbenicillin and vancomycin led to the complete abrogation of Staphylococcus aureus Newman strain growth and significantly decreased the gene expression levels of pbpB, blaZ, and vraSR in the presence of the antibiotics. IMPORTANCE This work characterizes the activity and inhibition of VraS, which is a key histidine kinase in a bacterial two-component system that is involved in Staphylococcus aureus antibiotic resistance. The results show the effect of temperature, divalent ions, and VraR on the activity and the kinetic parameters of ATP binding. The value of the KM of ATP is vital in designing screening assays to discover potent and effective VraS inhibitors with high translational potential. We report the ability of NH125 to inhibit VraS in vitro in a noncompetitive manner and investigate its effect on gene expression and bacterial growth kinetics in the presence and absence of cell wall-targeting antibiotics. NH125 effectively potentiated the effects of the antibiotics on bacterial growth and altered the expression of the genes that are regulated by VraS and are involved in mounting a resistance to antibiotics.

Comparative expression of soluble, active human kinases in specialized bacterial strains.

Kinases act as molecular switches for cellular functions and are involved in multiple human pathogeneses, most notably cancer. There is a continuous need for soluble and active kinases for in-vitro drug discovery and structural biology purposes. Kinases remain challenging to express using Escherichia coli, the most widely utilized host for heterologous expression. In this work, four bacterial strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta, and Arctic Express, were chosen for parallel expression trials along with BL21 (DE3) complemented with folding chaperones DnaJ/K and GroEL/ES to compare their performance in producing soluble and active human kinases. Three representative diverse kinases were studied, Epidermal Growth Factor Receptor kinase domain, Aurora Kinase A kinase domain, and Mitogen-activated protein Kinase Kinase. The genes encoding the kinases were subcloned into pET15b bacterial plasmid and transformed into the bacterial strains. Soluble kinase expression was tested using different IPTG concentrations (1-0.05 mM) at varying temperatures (37°C- 10°C) and induction times (3-24 hours). The optimum conditions for each kinase in all strains were then used for 1L large scale cultures from which each kinase was purified to compare yield, purity, oligomerization status, and activity. Although using specialized strains achieved improvements in yield and/or activity for the three kinases, none of the tested strains was universally superior, highlighting the individuality in kinase expression.

Engineering soluble artificial epidermal growth factor receptor mimics capable of spontaneous in vitro dimerization.

Epidermal growth factor receptor (EGFR) is a clinically validated target for a multitude of human cancers. The receptor is activated upon ligand binding through a critical dimerization step. Dimerization can be replicated in vitro by locally concentrating the receptor kinase domains on the surface of lipid-based vesicles. In this study we investigated the use of coiled coils to induce spontaneous receptor kinase domain dimerization in vitro to form non-membrane-bound artificial receptor mimics in solution. Two engineered forms of EGFR kinase domain fused to coiled coil complementary peptides were designed to self-associate upon mixing. Two fusion protein species (P3-EGFR and P4-EGFR) independently showed the same activity and polymerization profile known to exist with EGFR kinase domains. Upon mixing the two species, coiled coil heterodimers were formed that induced EGFR association to form dimers of the kinase domains. This was accompanied by 11.5-fold increase in the phosphorylation rate indicative of kinase domain activation equivalent to the levels achieved using vesicle localization and mimicking in vivo ligand-induced activation. This study presents a soluble tyrosine kinase receptor mimic capable of spontaneous in vitro activation that can facilitate functional and drug discovery studies for this clinically important receptor class.