Abstract
To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a “helical unravelling” of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus.
Highlights
A novel family of redox sensors in the streptococci evolved from two-component response regulators several reports have linked various regulatory factors such as Rgg [22], NmlR [23, 24], RitR [25,26,27], PsaR [28] and CiaRH [29] with regulation of the pneumococcal oxidative stress tolerance, a bona fide Cys-activated redox transcription factor able to respond to these high peroxide and iron levels has remained largely enigmatic
A novel family of redox sensors in the streptococci evolved from two-component response regulators of oxidant [30]. mAU; milli Absorbance Units. (D) Electrophoretic Mobility Shift Assays (EMSAs) of RitR wild-type (WT) and the C128S mutant in the presence (+) and absence (-) of H2O2
A novel family of redox sensors in the streptococci evolved from two-component response regulators location of Cys128 at the C-terminus of α5, and the potential interactions of the thiol group with the main chain carbonyls of Ile124 and Asp125 (Fig 3D) would instead be expected to slightly increase the pKa of the thiol group [51]
Summary
Strains and growth conditionsBacterial strains used in these studies are given in S1 Table. The following day cultures were diluted 1:10 in Todd-Hewitt broth medium (Becton Dickinson) supplemented with 0.5% (w/v) yeast extract (THY) containing appropriate antibiotics, and the cell density measured periodically at 600 nm using a Biomate 3 spectrophotometer (Thermo Scientific, Waltham, MA). S. pneumoniae cells were inoculated from frozen stocks into THY broth and cultured to early exponential phase (i.e. an OD600 of 0.01–0.03). At this time, CSP-1 was added to a final concentration of 200 ng/ml and the culture was incubated for 14 minutes before adding 100–200 ng of DNA. Strains of E. coli were cultured for plasmid purification overnight with aeration in a 37 ̊C incubator in Luria-Bertani (LB) medium supplemented with appropriate antibiotics: either 50 μg/ml spectinomycin, 34 μg/ml chloramphenicol (Cam), 100 μg/ml ampicillin (Amp) or the more stable Amp substitute, carbenicillin (Carb) at 50 μg/ml
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
