S-nitrosylation of peroxiredoxin 1 contributes to viability of lung epithelial cells during Bacillus anthracis infection.
Using Bacillus anthracis as a model gram-positive bacterium, we investigated the effects of host protein S-nitrosylation during bacterial infection. B. anthracis possesses a bacterial nitric oxide synthase (bNOS) that is important for its virulence and survival. However, the role of S-nitrosylation of host cell proteins during B. anthracis infection has not been determined.



Nitrosoproteomic analysis of human small airway epithelial cells (HSAECs) infected with toxigenic B. anthracis Sterne was performed, identifying peroxiredoxin 1 (Prx1) as one predominant target. Peroxidase activity of Prx during infection was measured using 2-Cys-Peroxiredoxin activity assay. Chaperone activity of S-nitrosylated Prx1 was measured by insulin aggregation assay, and analysis of formation of multimeric species using Native PAGE. Griess assay and DAF-2DA fluorescence assay were used to measure NO production. Cell viability was measured using the Alamar Blue assay and the ATPlite assay (Perkin Elmer).



S-nitrosylation of Prx1 in Sterne-infected HSAECs leads to a decrease in its peroxidase activity while enhancing its chaperone function. Treatment with bNOS inhibitor, or infection with bNOS deletion strain, reduces S-nitrosylation of Prx1 and decreases host cell survival. Consistent with this, siRNA knockdown of Prx1 lowers bNOS-dependent protection of HSAEC viability.



Anthrax infection results in S-nitrosylation of multiple host proteins, including Prx1. The nitrosylation-dependent decrease in peroxidase activity of Prx1 and increase in its chaperone activity is one factor contributing to enhancing infected cell viability.



These results provide a new venue of mechanistic investigation for inhalational anthrax that could lead to novel and potentially effective countermeasures.
School of Systems Biology, and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, USA.
Myung-Chul
MC
Chung
School of Systems Biology, and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, USA.
Farhang
F
Alem
School of Systems Biology, and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, USA.
Sarah G
SG
Hamer
School of Systems Biology, and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, USA.
Aarthi
A
Narayanan
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, USA.
Konstantin
K
Shatalin
School of Systems Biology, and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, USA.
Charles
C
Bailey
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, USA.
Evgeny
E
Nudler
School of Systems Biology, and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, USA. Electronic address: rhakami@gmu.edu.
Ramin M
RM
Hakami
D000881:Anthrax / Q000382:microbiology* / Q000473:pathology*; D001408:Bacillus anthracis / Q000187:drug effects / Q000472:pathogenicity*; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology*; D017353:Gene Deletion; D006801:Humans; D008168:Lung / Q000473:pathology*; D013058:Mass Spectrometry; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D015538:Nitrosation; D009195:Peroxidase / Q000378:metabolism; D054464:Peroxiredoxins / Q000378:metabolism*; D015203:Reproducibility of Results
D018832:Molecular Chaperones; D009569:Nitric Oxide; C504194:2-cys peroxiredoxin, human; D054464:Peroxiredoxins; D009195:Peroxidase; D052248:Nitric Oxide Synthase Type I; D019331:NG-Nitroarginine Methyl Ester
Bacillus anthracis; Nitric oxide synthase; Peroxiredoxin 1