Analysis of Non-Typeable Haemophilous influenzae VapC1 Mutations Reveals Structural Features Required for Toxicity and Flexibility in the Active Site

Abstract

Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.

Functional details of the Mycobacterium tuberculosis VapBC26 toxin-antitoxin system based on a structural study: insights into unique binding and antibiotic peptides

“…In the case of Haemophilus influenza VapC1, mutation of 17 amino acid residues in VapC1 showed that these residues do not play essential roles in the interaction of the protein with its cognate antitoxin ( 85 ). However, in our study, although single mutations of Pro46 and Tyr51 in VapB26 and of Leu46 in VapC26 did not totally disrupt the interaction required to form VapBC26, the slightly increased ribonuclease activity of the mutated VapBCs and decreased complex formation of Y51E compared to native complex when α4-mimicking peptide was added in size exclusion chromatography supports a role for these hydrophobic residues in forming the VapBC26 interface.…”

VapC toxins drive cellular dormancy under uranium stress for the extreme thermoacidophile Metallosphaera prunae

“…Beyond their biological significance, many efforts of the past decade have elucidated the structural and functional properties of VapBCs. For instance, bioinformatics and in vivo mutational analyses have demonstrated the importance of a few acidic residues in the catalytic groove of VapC dimers that coordinate two metal ions (Arcus et al, 2011;Hamilton et al, 2014). Further, the identification of a heterooctameric structure has suggested a route to transcriptional regulation by the VapB 4 C 4 complex, in which the complex is capable of interacting with the promoter region of the TA locus (Mate et al, 2012;Bendtsen et al, 2016).…”

Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection

“…The PIN domain of VapC homologues has been implicated in the binding and cleavage of RNA substrates (42), and many acidic residues in this domain are highly conserved (45). Other studies have evaluated various mutations to the PIN domain (54,55), and we previously demonstrated that the VapC-1 D99N mutant had significantly reduced RNase activity, while retaining the ability to bind VapB-1 (56).…”