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The emergence of methicillin-resistant Staphylococcus aureus isolates highlights the urgent need to develop more antibiotics. ClpP is a highly conserved protease regulated by ATPases in bacteria and in mitochondria. Aberrant activation of bacterial ClpP is an alternative method of discovering antibiotics, while it remains difficult to develop selective Staphylococcus aureus ClpP activators that can avoid disturbing Homo sapiens ClpP functions. Here, we use a structure-based design to identify (R)- and (S)-ZG197 as highly selective Staphylococcus aureus ClpP activators. The key structural elements in Homo sapiens ClpP, particularly W146 and its joint action with the C-terminal motif, significantly contribute to the discrimination of the activators. Our selective activators display wide antibiotic properties towards an array of multidrug-resistant staphylococcal strains in vitro, and demonstrate promising antibiotic efficacy in zebrafish and murine skin infection models. Our findings indicate that the species-specific activators of Staphylococcus aureus ClpP are exciting therapeutic agents to treat staphylococcal infections. The development of selective ClpP activators targeting only the MRSA isolates without interfering with the human variant is currently challenging. Here, the authors report on the structure-based design of enantiomers of ZG197 and identify the discriminator factor between the proteins. [ABSTRACT FROM AUTHOR]

Summary: A type II toxin‐antitoxin (TA) system, in which the toxin contains a Gcn5‐related N‐acetyltransferase (GNAT) domain, has been characterized recently. GNAT toxin acetylates aminoacyl‐tRNA and blocks protein translation. It is abolished by the cognate antitoxin that contains the ribbon‐helix‐helix (RHH) domain. Here, we present an experimental demonstration of the interaction of the GNAT‐RHH complex with TA promoter DNA. First, the GNAT‐RHH TA locus kacAT was found in Klebsiella pneumoniae HS11286, a strain resistant to multiple antibiotics. Overexpression of KacT halted cell growth and resulted in persister cell formation. The crystal structure also indicated that KacT is a typical acetyltransferase toxin. Co‐expression of KacA neutralized KacT toxicity. Expression of the bicistronic kacAT locus was up‐regulated during antibiotic stress. Finally, KacT and KacA formed a heterohexamer that interacted with promoter DNA, resulting in negative autoregulation of kacAT transcription. The N‐terminus region of KacA accounted for specific binding to the palindromic sequence on the operator DNA, whereas its C‐terminus region was essential for the inactivation of the GNAT toxin. These results provide an important insight into the regulation of the GNAT‐RHH family TA system. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II) and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK) CzcS responds to Zn(II) stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD) in complex with Zn(II) at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II)-sensor domain of the two-component system (TCS). The CzcS SD is of α/β-fold in nature, and it senses the Zn(II) stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60) on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC) superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved β-sheets pocket. The dimerization of the N-terminal H1 and H1’ α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II) sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents. [ABSTRACT FROM AUTHOR]