Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins， with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The (irulence-ssociated rotein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable (NTHi). In this study， the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20?? resolution. Based on this structure， aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions， growth of ， and pathogenesis were tested. Finally， a novel model system was designed and utilized that consists of an NTHi Δ strain complemented in with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues Herein the crystal structure of the VapBC-1 complex from nontypeable (NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity in ， but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin， VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissues Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the in wild-type toxin under the same conditions.