VapC-1 of Nontypeable Haemophilus influenzae Is a Ribonuclease

Abstract

Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.

Structure and Proposed Activity of a Member of the VapBC Family of Toxin-Antitoxin Systems

“…Possibly like the archeal RelBE, the VapC family of enzyme may require association with the ribosome to be fully active (19). However, it has recently been shown that VapC-1 from Haemophilus influenzae is a ribonuclease that acts on free RNA in a concentrationdependent manner (36).…”

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA‐binding site

“…20 Computational analysis has suggested that VapC toxins are ribonucleases, 20 and there is increasing biochemical data that support the idea. For example, VapC-1 from Haemophilus influenza, 21 VapC-5 from Mtb, 12 VapC-1 and VapC-2 from…”

VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus

“…This difference may arise from increased vapBC6 gene dosage due to use of the low, but not single-copy vector, pRN1 (Berkner et al 2007), for construction of the complementation plasmid (pBN1096). Overproduction of toxins are well known to be toxic (Christensen-Dalsgaard and Gerdes 2006;Daines et al 2007), therefore increased vapBC6 gene dosage may also be deleterious and compromise survival during heat shock. Complementation of the vapB6 disruption mutant defect in growth rate at normal growth temperatures could also be demonstrated; in minimal glucose medium, the complemented vapB6 disruption mutant grew better than the otherwise isogenic uncomplemented vapB6TlacS disruption mutant that carried the malA plasmid (pBN1080), but lacking vapBC6.…”

“…VapC homologs from bacteria have been reported to have ribonuclease activity (Daines et al 2007;Miallau et al 2009), while VapC (PAE0151) from the archaeon Pyrobaculum aerophilum has DNA exonuclease activity (Arcus et al 2004). To clarify the situation for the Sso VapC6, the amino acid sequence for SSO1493 was analyzed with the 3D-JIGSAW prediction tool (Bates and Sternberg 1999;Bates et al 2001) using PDB values for the PIN-domain protein and putative toxin from Pyrobaculum aerophilum PAE0151 (PDB:2FE1) (Arcus et al 2004).…”

“…Bacterial VapC's have been studied in some detail. VapC-1 of nontypeable Haemophilus influenzae has ribonuclease activity that could be inhibited by the VapB-1 antitoxin (Daines et al 2007). FitB of Neisseria gonorrhoeae modulates virulence through transcellular trafficking and structurally resembles PAE2754, although insolubility of the recombinantly produced protein precluded assessment of its activity (Hopper et al 2000;Mattison et al 2006).…”