Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

Abstract

Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Under normal growth conditions, the antitoxin neutralizes the toxin. However, under conditions of stress, such as nutrient starvation or antibiotic treatment, cellular proteases degrade the antitoxin, and the toxin functions to arrest bacterial growth. We characterized the specificity determinants of the interactions between VapB antitoxins and VapC toxins from nontypeable Haemophilus influenzae (NTHi) in an effort to gain a better understanding of how antitoxins control toxin activity and bacterial persistence. We studied truncated and full-length antitoxins with single amino acid mutations in the toxin-binding domain. Coexpressing the toxin and antitoxin in Escherichia coli and measuring bacterial growth by dilution plating assayed the ability of the mutant antitoxins to neutralize the toxin. Our results identified two single amino acid residues (W48 and F52) in the C-terminal region of the VapB2 antitoxin necessary for its ability to neutralize its cognate VapC2 toxin. Additionally, we attempted to alter the specificity of VapB1 by making a mutation that would allow it to neutralize its noncognate toxin. A mutation in VapB1 to contain the tryptophan residue identified herein as important in the VapB2-VapC2 interaction resulted in a VapB1 mutant (the T47W mutant) that binds to and neutralizes both its cognate VapC1 and noncognate VapC2 toxins. This represents the first example of a single mutation causing relaxed specificity in a type II antitoxin. IMPORTANCEToxin-antitoxin systems are of particular concern in pathogenic organisms, such as nontypeable Haemophilus influenzae, as they can elicit dormancy and persistence, leading to chronic infections and failure of antibiotic treatment. Despite the importance of the TA interaction, the specificity determinants for VapB-VapC complex formation remain uncharacterized. Thus, our understanding of how antitoxins control toxin-induced dormancy and bacterial persistence requires thorough investigation of antitoxin specificity for its cognate toxin. This study characterizes the crucial residues of the VapB2 antitoxin from NTHi necessary for its interaction with VapC2 and provides the first example of a single amino acid change altering the toxin specificity of an antitoxin.T oxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Originally discovered as addictive elements involved in postsegregational killing during cell division, they have since been identified as playing a critical role in responding to stress (1-3). TA systems are typically encoded by a single operon that produces a stable protein toxin and its cognate labile antitoxin, with the gene encoding the antitoxin located upstream of the toxin in most cases (4). Under normal growth conditions, the antitoxin, which may be a protein (types II, IV, and V) or an RNA...

hipBA toxin-antitoxin systems mediate persistence in Caulobacter crescentus

“…We expected each HipA toxin to be counteracted by binding to the HipB antitoxin encoded in its operon. Promiscuity between toxin and antitoxin partners is rare, even in bacteria harboring multiple paralogous toxins, but crosstalk in HipA-HipB interactions has never been investigated 36,37 . To determine the cognate HipB antitoxin(s) for each HipA toxin, we coexpressed all combinations of HipA and HipB in a ΔhipBA 1,2,3 background ( Fig.…”

Structural, functional and biological insights into the role ofMycobacterium tuberculosisVapBC11 toxin–antitoxin system: targeting a tRNase to tackle mycobacterial adaptation

“…In Type II TA systems, both toxin and antitoxin form a tight TA complex that binds to the operator region and results in auto-repression ( 17 ). The N-terminal domain of antitoxins possess DNA-binding properties, and the C-terminal domain neutralizes the toxin ( 18 , 19 ). Several DNA-binding motifs, such as ribbon-helix-helix (RHH), helix-turn-helix, AbrB and PhD/YefM, have been mapped to the N-terminal domain of antitoxins ( 20 , 21 ).…”

Homologous VapC Toxins Inhibit Translation and Cell Growth by Sequence-Specific Cleavage of tRNA fMet

“…6A). Accordingly, we changed the loop-closing G-C pair (31)(32)(33)(34)(35)(36)(37)(38)(39) in NTHi tRNA fMet-14 to A-U and measured the extent of its cleavage by VapC1 NTHi and VapC2 NTHi in vivo. The result revealed that the G-C-to-A-U mutation decreased cleavage of NTHi tRNA fMet-14 by 30 to 40%, suggesting that this G-C pair plays a key role in recognition of the substrate by VapCs ( Fig.…”

“…The pathogenic organism nontypeable Haemophilus influenzae (NTHi) contains two vapBC systems: vapB1C1 and vapB2C2. These two systems are homologous; however, there is no cross talk between them, as the antitoxins are highly specific for their cognate toxins (32). Studies have shown that these systems are upregulated during NTHi infection and facilitate enhanced survival and growth regulation (33,34).…”