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Title: Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection

Abstract

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. ThevapBC(virulence-associatedprotein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeableHaemophilus influenzae(NTHi). In this study reported here, 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 ofEscherichia coli, and pathogenesisex vivowere tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1strain complemented inciswith 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 controlmore » of thevapBC-1native 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 tissuesex vivo.« less

Authors:
 [1];  [2];  [2];  [3];  [4];  [4]; ORCiD logo [1]
  1. Old Dominion Univ., Norfolk, VA (United States)
  2. Univ. of Kansas, Lawrence, KS (United States)
  3. Hauptman Woodward Medical Research Inst., Argonne, IL (United States)
  4. National Inst. of Health, Rockville, MD (United States). National Center for Advancing Translational Sciences
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institute on Deafness and Other Communication Disorders (NIDCD); National Center for Advancing Translational Sciences (NCATS) Division of Pre-Clinical Innovation Intramural Program; National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); Industrial Macromolecular Crystallography Association; USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1526058
Grant/Contract Number:  
P30 GM110761; AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Bacteriology
Additional Journal Information:
Journal Volume: 201; Journal Issue: 12; Journal ID: ISSN 0021-9193
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; PIN domain; crystal structure; persistence; protein-protein interactions; ribonuclease; toxin-antitoxin

Citation Formats

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. United States: N. p., 2019. Web. doi:10.1128/JB.00026-19.
Molinaro, Ashley L., Kashipathy, Maithri M., Lovell, Scott, Battaile, Kevin P., Coussens, Nathan P., Shen, Min, & Daines, Dayle A. Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection. United States. doi: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. Mon . "Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection". United States. doi:10.1128/JB.00026-19. https://www.osti.gov/servlets/purl/1526058.
@article{osti_1526058,
title = {Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection},
author = {Molinaro, Ashley L. and Kashipathy, Maithri M. and Lovell, Scott and Battaile, Kevin P. and Coussens, Nathan P. and Shen, Min and Daines, Dayle A.},
abstractNote = {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. ThevapBC(virulence-associatedprotein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeableHaemophilus influenzae(NTHi). In this study reported here, 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 ofEscherichia coli, and pathogenesisex vivowere tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1strain complemented inciswith 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 thevapBC-1native 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 tissuesex vivo.},
doi = {10.1128/JB.00026-19},
journal = {Journal of Bacteriology},
issn = {0021-9193},
number = 12,
volume = 201,
place = {United States},
year = {2019},
month = {4}
}

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