Molecular target of enteric VapC toxins and regulation of vapBC transcription by conditional cooperativity

Abstract

The uibiquitous Type II toxin – antitoxin (TA) loci encode two proteins, a toxin and an antitoxin. The antitoxin combines with and neutralizes a cognate toxin. Usually, the TA genes form an operon that is transcribed by a single promoter located upstream of the genes. In most cases, the antitoxin autoregulates the TA operon via binding to operator sites in the promoter region. In almost all such cases, the toxin act as a co-repressor of transcription as the toxin enhances the DNA binding of the antitoxin. Recently, it has been shown that toxins play an additional role in stimulating transcription, as the antitoxin and toxin ratio is important for cooperative binding of the complex to DNA. The antitoxin is rapidly degraded by cellular proteases under conditions of stress and treatment with antibiotics, which leads to activation of the toxin. The toxins of TAs belong to different gene families. The most abundant TA gene family is vapBC that, in some organisms, have expanded into cohorts of genes. For example, the major human pathogen Mycobacterium tuberculosis contains at least 88 TAs, 45 of which are vapBC loci. VapC toxins encoded by vapBC loci are PIN domain proteins (PilT N-terminal). Eukaryotic PIN domain proteins are site-specific ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. From in vitro experiments it has been postulated that VapC toxins are RNases or DNases but their exact cellular target has remained elusive. Here I show that VapC encoded by Shigella flexneri 2a virulence plasmid pMYSH6000 and the chromosome of Salmonella enterica serovar Typhimurium LT2 are site-specific endoribonucleases that specifically cleave tRNAfMet in the anticodon stem-loop in vivo and in vitro. Furthermore, I show that VapC dependent depletion of tRNAfMet leads to bacteriostatic inhibition of global translation, which surprisingly induces low-level initiation of translation at elongator codons that are correctly positioned relative to a Shine & Dalgarno sequence. I also show that VapC forms a complex with VapB and acts as a co-repressor of vapBC transcription. During steady state growth VapB is in excess of VapC. However, nutrient stress or treatment with antibiotics leads to Lon protease dependent decrease in VapB levels. Furthermore, I show that VapC in excess of VapB directly interferes with cooperative DNA binding of the VapBC complex, which is dependent on the dimerisation of the VapC toxin. 2 In conclusion, I show that enteric VapCs not only regulate global cellular translation by tRNAfMet cleavage, but also regulate vapBC transcription by conditional cooperativity.