Exploration of the Effects of γ-Phosphate-Modified ATP Analogues on Histidine Kinase Autophosphorylation.

While two-component systems (TCSs), composed of a sensor histidine kinase (HK) and a response regulator, are the main signaling pathways in bacteria, global TCS activity remains poorly described. Here, we report the kinetic parameters of the HK autophosphorylation reaction using previously uncharacterized γ-phosphate-modified ATP analogues to further elucidate their utility as activity-based probes for global TCS analysis. Given the increased stability of thiophosphorylated histidine in comparison to that of the native phosphoryl modification, which is attributed to the decreased electrophilicity of this moiety, we anticipated that ATPγS may be turned over much more slowly by the HKs. Surprisingly, we found this not to be the case, with the turnover numbers decreasing <1 order of magnitude. Instead, we found that alkylation of the thiophosphate had a much more dramatic effect on turnover and, in one case, the binding affinity of this substrate analogue (BODIPY-FL-ATPγS).

Targeting multidrug resistant Staphylococcus infections with bacterial histidine kinase inhibitors.

The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), which are not susceptible to current antibiotics has necessitated the development of novel approaches and targets to tackle this growing challenge. Bacterial two-component systems (TCSs) play a central role in the adaptative response of bacteria to their ever-changing environment. They are linked to antibiotic resistance and bacterial virulence making the proteins of the TCSs, histidine kinases and response regulators, attractive for the development of novel antibacterial drugs. Here, we developed a suite of maleimide-based compounds that we evaluated against a model histidine kinase, HK853, in vitro and in silico. The most potent leads were then assessed for their ability to decrease the pathogenicity and virulence of MRSA, resulting in the identification of a molecule that decreased the lesion size caused by a methicillin-resistant S. aureus skin infection by 65% in a murine model.

Cationic Polymers Enable Internalization of Negatively Charged Chemical Probes into Bacteria.

The bacterial cell envelope provides a protective barrier that is challenging for small molecules and biomolecules to cross. Given the anionic nature of both Gram-positive and Gram-negative bacterial cell envelopes, negatively charged molecules are particularly difficult to deliver into these organisms. Many strategies have been employed to penetrate bacteria, ranging from reagents such as cell-penetrating peptides, enzymes, and metal-chelating compounds to physical perturbations. While cationic polymers are known antimicrobial agents, polymers that promote the permeabilization of bacterial cells without causing high levels of toxicity and cell lysis have not yet been described. Here, we investigate four polymers that display a cationic poly(2-(dimethylamino)ethyl methacrylate (D) block for the internalization of an anionic adenosine triphosphate (ATP)-based chemical probe into Escherichia coli and Bacillus subtilis. We evaluated two polymer architectures, linear and micellar, to determine how shape and hydrophobicity affect internalization efficiency. We found that, in addition to these reagents successfully promoting probe internalization, the probe-labeled cells were able to continue to grow and divide. The micellar structures in particular were highly effective for the delivery of the negatively charged chemical probe. Finally, we demonstrated that these cationic polymers could act as general permeabilization reagents, promoting the entry of other molecules, such as antibiotics.

Mechanistic Studies of Bioorthogonal ATP Analogues for Assessment of Histidine Kinase Autophosphorylation.

Phosphorylation is an essential protein modification and is most commonly associated with hydroxyl-containing amino acids via an adenosine triphosphate (ATP) substrate. The last decades have brought greater appreciation to the roles that phosphorylation of myriad amino acids plays in biological signaling, metabolism, and gene transcription. Histidine phosphorylation occurs in both eukaryotes and prokaryotes but has been shown to dominate signaling networks in the latter due to its role in microbial two-component systems. Methods to investigate histidine phosphorylation have lagged behind those to study serine, threonine, and tyrosine modifications due to its inherent instability and the historical view that this protein modification was rare. An important strategy to overcome the reactivity of phosphohistidine is the development of substrate-based probes with altered chemical properties that improve modification longevity but that do not suffer from poor recognition or transfer by the protein. Here, we present combined experimental and computational studies to better understand the molecular requirements for efficient histidine phosphorylation by comparison of the native kinase substrate, ATP, and alkylated ATP derivatives. While recognition of the substrates by the histidine kinases is an important parameter for the formation of phosphohistidine derivatives, reaction sterics also affect the outcome. In addition, we found that stability of the resulting phosphohistidine moieties correlates with the stability of their hydrolysis products, specifically with their free energy in solution. Interestingly, alkylation dramatically affects the stability of the phosphohistidine derivatives at very acidic pH values. These results provide critical mechanistic insights into histidine phosphorylation and will facilitate the design of future probes to study enzymatic histidine phosphorylation.

Modified nucleoside triphosphates in bacterial research for in vitro and live-cell applications.

Modified nucleoside triphosphates (NTPs) are invaluable tools to probe bacterial enzymatic mechanisms, develop novel genetic material, and engineer drugs and proteins with new functionalities. Although the impact of nucleobase alterations has predominantly been studied due to their importance for protein recognition, sugar and phosphate modifications have also been investigated. However, NTPs are cell impermeable due to their negatively charged phosphate tail, a major hurdle to achieving live bacterial studies. Herein, we review the recent advances made to investigate and evolve bacteria and their processes with the use of modified NTPs by exploring alterations in one of the three moieties: the nucleobase, the sugar and the phosphate tail. We also present the innovative methods that have been devised to internalize NTPs into bacteria for in vivo applications.

Disarming the virulence arsenal of Pseudomonas aeruginosa by blocking two-component system signaling.

Pseudomonas aeruginosa infections have reached a "critical" threat status making novel therapeutic approaches required. Inhibiting key signaling enzymes known as the histidine kinases (HKs), which are heavily involved with its pathogenicity, has been postulated to be an effective new strategy for treatment. Herein, we demonstrate the potential of this approach with benzothiazole-based HK inhibitors that perturb multiple virulence pathways in the burn wound P. aeruginosa isolate, PA14. Specifically, our compounds significantly reduce the level of toxic metabolites generated by this organism that are involved in quorum-sensing and redox-balancing mechanisms. They also decrease the ability of this organism to swarm and attach to surfaces, likely by influencing their motility appendages. Quantitative transcription analysis of inhibitor-treated cultures showed substantial perturbations to multiple pathways including expression of response regulator GacA, the cognate partner of the "super regulator" of virulence, HK GacS, as well as flagella and pili formation. These promising results establish that blocking of bacterial signaling in P. aeruginosa has dramatic consequences on virulence behaviours, especially in the context of surface-associated infections.