Evaluation of serine protein kinase HPrK as an antibacterial target in gram-positive bacteria and mycoplasmas

Antimicrobial resistance (AMR) poses a serious threat to global public health. To tackle this challenge, identifying novel effective Antibacterial targets and Antibiotics is crucial. The serine kinase HPrK, widely present in gram-positive bacteria and mycoplasmas, primarily regulates carbon metabolism and virulence factor expression. In a previous study, we failed to knockout the hprK gene using the conventional homologous recombination method in the zoonotic Streptococcus suis. Here, we employed an anhydrotetracycline (ATc)-inducible promoter (AiP) and hprK fusion to replace its original expression cassette in the S. suis genome. However, in the absence of ATc, mutations in the AiP elements resulted in ATc-independent expression of HPrK, suggesting that HPrK is essential for S. suis and a potential antimicrobial target. Accordingly, a high-throughput inhibitor screening assay based on the kinase activity of HPrK was designed, and an inhibitor compound CDK9-IN-2 was identified. Molecular docking and bio-layer interferometry revealed that CDK9-IN-2 targets highly conserved residues D242, D249, and N281 on HPrK protein in several species of gram-positive bacteria. CDK9-IN-2 exhibited significant Antibacterial effects against multidrug-resistant S. suis clinical isolates and various species of gram-positives and mycoplasmas with a minimum inhibitory concentration (MIC) of 16 to 32 μg/mL. Its therapeutic effect on S. suis Infection was further proved in a Galleria mellonella larval Infection model. Collectively, HPrK is a conserved kinase in gram-positives and mycoplasmas, and its essentiality is confirmed in S. suis. Based on this conserved target HPrK, an inhibitor with a broad Antibacterial activity has been identified, suggesting HPrK is a promising antimicrobial target for Antibacterial drug innovation.

Importance: Antimicrobial resistance (AMR) is a critical global health challenge, highlighting the need for new Antibiotic targets. HPrK is a serine kinase that is important in regulation of carbon metabolism and virulence in gram-positive bacteria and mycoplasmas. In this study, we validated that HPrK was an essential gene in S. suis and could be used as a promising target for Antibacterial drug development. Based on HPrK, we identified the kinase inhibitor CDK9-IN-2, which exhibits significant Antibacterial activity against multidrug-resistant S. suis isolates and various gram-positive species and mycoplasmas. These findings indicated HPrK as a viable target for novel antimicrobial agents, advancing strategies to combat antimicrobial resistance.