Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of Pin Domain Mutations on Survival During Infection
Journal of Bacteriology
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 vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å 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 Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis 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 vapBC-1 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 ex vivo.
Article states: Coordinates and structure factors were deposited in the worldwide Protein Data Bank (wwPDB) under accession code 6NKL.
Original Publication Citation
Molinaro, A. L., Kashipathy, M. M., Lovell, S., Battaile, K. P., Coussens, N. P., Shen, M., & Daines, D. A. (2019). Crystal structure of VapBC-1 from nontypeable Haemophilus influenzae and the effect of PIN domain mutations on survival during infection. Journal of Bacteriology, 201(12), 1-17, Article e00026-19. https://doi.org/10.1128/JB.00026-19
Molinaro, Ashley L.; Kashipathy, Maithri M.; Lovell, Scott; Battaile, Kevin P.; Coussens, Nathan P.; Shen, Min; and Daines, Dayle A., "Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of Pin Domain Mutations on Survival During Infection" (2019). Biological Sciences Faculty Publications. 497.
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Publisher's version available at: https://doi.org/10.1128/JB.00026-19