Identification and characterization of acetyltransferase‐type toxin‐antitoxin locus in Klebsiella pneumoniae

Abstract

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.

Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle

“…In both samples antitoxin appeared as a single peak with an average [MH+] of 14512, matching the expected mass of ItaR with intact N-terminal affinity tag and removed N-terminal methionine residue. To test if the putative acetylating activity of ItaT is required for regulation of itaRT operon expression, we cloned the 172 bp-long upstream region of the itaRT operon containing a putative promoter into the pFD51 plasmid carrying promoterless galK gene (20). Cells harboring the resulting pFD-P itaRT -galK fusion plasmid and compatible pBAD33 plasmids expressing itaR, itaRT  or itaRT* were induced with arabinose, total RNA was extracted, and levels of galK transcripts were measured by RT-qPCR (Figure 3B).…”

GNAT toxins of bacterial toxin–antitoxin systems: acetylation of charged tRNAs to inhibit translation

“…The dimensions of this structure are compatible with binding of a tRNA molecule with phophate groups interacting with the sidechains of basic amino acids in this region. More importantly, mutations of these basic amino acid residues in both TacT (Cheverton et al, 2016) and KacT (Qian et al, 2018) abolished their toxicity. However, whether KacT is specific for initiator tRNA like AtaT or has more relaxed specificity for elongator tRNAs such as TacT is currently not known.…”

“…The GNAT-RHH modules are quite widespread, comprising about 4% of all predicted type II TA pairs in 2786 completely sequenced prokaryotic genomes that were analysed (Xie et al, 2018). Most of the GNAT toxins are found in the genomes of S. enterica and K. pneumoniae where they comprise the second most abundant type II toxins after RelE (Xie et al, 2018); the other top species harboring the GNAT-RHH modules are E. coli and M. tuberculosis (Qian et al, 2018). The KacT toxin is the only other GNAT toxin besides TacT for which the crystal structure is available.…”

“…Furthermore, as Qian et al . () has pointed out, both KacA and AtaR (along with the Shigella ‐encoded GmvA) antitoxins lack the corresponding Lys‐44 residue of TacA that is the target for acetylation by the TacT toxin. This serves to underline the finding that TA modules from the same family may be differentially adapted to divergent biological functions, with some such as TacAT, being more deeply integrated into existing cellular signaling pathways for their regulation (Harms et al ., ).…”

“…The work by Qian et al . () as well as earlier work by others covered in this commentary has laid the foundation for further investigations into these fascinating genetic systems and their biological function. The relative abundance of GNAT‐RHH TA systems in certain pathogenic bacteria such as K. pneumoniae , S. enterica and E. coli , and their likely role in persistence makes them a tempting target of further research as we look for new approaches to utilize these TA modules in our bid to contain and overcome clinical infections by these pathogens.…”