VapC-1 of nontypeable Haemophilus influenzae is a ribonuclease

Department of Surgery, School of Medicine, University of California, Davis Medical Center, Sacramento, CA, USA

^* To whom correspondence should be addressed. Email: dadaines{at}ucdavis.edu.

	Abstract

Nontypeable Haemophilus influenzae (NTHi) 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 carry 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 homo-multimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an E. 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 a ribonuclease that is active on free RNA, but does not degrade DNA in vitro. Pre-incubation 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 ribonuclease activity leads to growth inhibition via the mechanism of mRNA cleavage.