Infectious diarrhea is a complex syndrome caused by many viruses, parasites, fungi and bacteria such as Salmonella. Salmonellosis itself encompasses a spectrum of clinical diseases that range from enteric fever, a serious condition that kills about 600,000 people a year, to non- typhoidal zoonotic infections that affect more than a million Americans annually. Treatment of Salmonella and medically important Gram-negative rods is often complicated by the increasing resistance of pathogenic bacteria to antibiotics used in the clinic. The parasitic relationship between Salmonella and host mononuclear phagocytes is one of the hallmarks of salmonellosis. The survival of Salmonella within macrophages relies on the type III secretion system encoded within the Salmonella pathogenicity island 2 (SPI2). The expression of SPI2 is controlled by the SsrA/SsrB two-component regulatory system. In addition to being regulated by canonical phosphorylation of a conserved aspartate in the receiver domain, our investigations indicate that SsrB is under the post-translational control via the oxidation and S-nitrosylation of Cys203 in the dimerization domain. Chaperone and disulfide reductase activities of TrxA further regulate SsrB function, thereby boosting SPI2-dependent antioxidant defenses. We hypothesize that the SPI2 master regulator SsrB is under post-translational control of oxidative modifications of the Cys203 thiol group in the dimerization domain, and protein-protein interactions with the disulfide reductase and chaperone functions of thioredoxin 1.
Aim 1 will determine the molecular mechanisms by which SsrB redox and oxidation states regulate SPI2 transcription.
Aim 2 will explore the role that specific charged and hydrophobic residues play in the ability of redox active SsrB Cys203 to sense oxidative and nitrosative stress.
Aim 3 will characterize the mechanisms by which the chaperone and disulfide reductase activities of thioredoxin 1 regulate SsrB function and Salmonella pathogenesis. These investigations will provide profound insights into the antioxidant and antinitrosative defenses of Salmonella that are critical for intracellular survival and virulence. Specifically, the proposed research will elucidate the previously unappreciated role for reactive species and an antioxidant protein in the post-translational regulation of SsrB, the intracellular survival of Salmonella, and the pathogenesis of this intracellular bacterium in a murine model of salmonellosis.

Public Health Relevance

Non-typhoidal Salmonella enterica is a frequent cause of gastroenteritis in healthy individuals and a life-threatening systemic infection in HIV-infected people or individuals with genetic defects in CD4 T lymphocytes, NADPH oxidase, or IFN? signaling pathways. Human-adapted Salmonella serovars cause serious and often fatal enteric (typhoid) fever that kills about 600,000 people in the developing world. In addition, non-typhoidal Salmonella infections affect 1.3 billion individuals a year worldwide, including 1.4 million US citizens. The burden medically important bacteria such as Salmonella place on global health and the economy is further compounded by the continued rise and spread of multidrug resistance. The secretion system encoded within the Salmonella pathogenicity island 2 (SPI2) mediates Salmonella virulence. We have discovered that the SPI2 master regulator SsrB senses reactive oxygen and nitrogen species, and thereby controls critical aspects of Salmonella pathogenesis. Our proposal will characterize novel molecular mechanisms by which reactive species and thioredoxin regulate SsrB function and Salmonella virulence. The basic mechanisms uncovered in Salmonella are likely generalizable to a variety of clinically important, phylogenetically diverse pathogenic bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI054959-11A1
Application #
8761215
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Alexander, William A
Project Start
2003-09-30
Project End
2019-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
11
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Aurora
State
CO
Country
United States
Zip Code
80045
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Crawford, Matthew A; Henard, Calvin A; Tapscott, Timothy et al. (2016) DksA-Dependent Transcriptional Regulation in Salmonella Experiencing Nitrosative Stress. Front Microbiol 7:444
Crawford, Matthew A; Tapscott, Timothy; Fitzsimmons, Liam F et al. (2016) Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content. MBio 7:e02161-15
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Fang, Ferric C; Frawley, Elaine R; Tapscott, Timothy et al. (2016) Discrimination and Integration of Stress Signals by Pathogenic Bacteria. Cell Host Microbe 20:144-53
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Henard, Calvin A; Tapscott, Timothy; Crawford, Matthew A et al. (2014) The 4-cysteine zinc-finger motif of the RNA polymerase regulator DksA serves as a thiol switch for sensing oxidative and nitrosative stress. Mol Microbiol 91:790-804
Husain, Maroof; Jones-Carson, Jessica; Liu, Lin et al. (2014) Ferric uptake regulator-dependent antinitrosative defenses in Salmonella enterica serovar Typhimurium pathogenesis. Infect Immun 82:333-40

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