Noncognate Toxin‐Antitoxin Interactions: Implications for Bacterial Persistence

Toxin‐antitoxin (TA) gene pairs are highly conserved bacterial operons found in a wide range of pathogens. The type II TA loci encode a protein toxin and antitoxin that associate upon translation to form a nontoxic complex. External stresses, such as exposure to antibiotics or reactive oxygen species from the host immune response, induce antitoxin degradation and subsequent toxin activation. This results in cleavage of bacterial mRNA by the ribonuclease toxins, allowing microorganisms to enter into a state of reversible growth arrest marked by nonspecific antibiotic tolerance and the ability to cause recurrent infections. Most organisms maintain multiple TA pairs in their genomes, and we asked whether unrelated antitoxins could modulate the activity of noncognate toxins. We assayed members belonging to two TA pairs from nontypeable Haemophilus influenzae (NTHi) in a LexA‐based protein‐protein interaction system, and found that the antitoxin VapX from the locus vapXD could bind to the toxin VapC‐1 from the locus vapBC‐1 . We sought to establish whether this physical interaction was also functional, as noncognate binding might be a conserved characteristic of TA pairs. To do this, we constructed an in vivo assay system that allowed us to individually induce the expression of the antitoxin, the toxin, or both during growth in an Escherichia coli background. Further, we could vary the initiation of induction so that one binding partner could be expressed before the other. Since expression of the toxin alone resulted in growth arrest, modulation of toxin activity by an antitoxin could be measured spectrophotometrically over time. In contrast to the results observed when the cognate antitoxin VapB‐1 was expressed with the VapC‐1 toxin, the noncognate antitoxin VapX was unable to interfere with the growth inhibition of VapC‐1, even when VapX was pre‐induced to allow the antitoxin subunits to accumulate prior to toxin expression. To more specifically characterize the interaction of VapX and VapC‐1, both proteins were cloned, overexpressed and purified, then assayed in vitro using a novel and sensitive fluorescence‐quenching RNase activity assay. The small RNA substrate used in this assay was constructed with a fluorophore at one end (FAM) and a quencher at the other. When intact, the substrate was dim, but fluoresced brightly when cleaved. This approach allowed us to determine the effects of VapX binding to VapC‐1 more directly. Interestingly, although VapX had minimal ribonuclease activity and VapC‐1 efficiently cleaved the substrate, when increasing amounts of VapX were added to VapC‐1 the activity of the toxin was enhanced in a dose‐response manner. These findings suggest that the binding of the VapX antitoxin may be stabilizing the VapC‐1 toxin, thereby facilitating enzymatic activity. This new information implies that pathogens with multiple TA loci may more efficiently enter a persister state upon exposure to the host immune response during infection. Support or Funding Information This work was supported in part by DC010187 and DC014756 (DAD).