Vibrio cholerae’s ToxRS bile sensing system
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eLife assessment
This study provides important insights into the structural biology and molecular mechanism of the sensory proteins ToxR/S that are associated with survival and virulence of the cholera pathogen. The structural studies are solid and supported by a series of biophysical experiments revealing a split, periplasmic protein binding interface for bile acid. The results are of interest to both protein biochemistry and pharmacology, potentially opening new routes for intervention in cholera disease.
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Abstract
The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae . Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae’s rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies for example bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS’s environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio’s survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.
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eLife assessment
This study provides important insights into the structural biology and molecular mechanism of the sensory proteins ToxR/S that are associated with survival and virulence of the cholera pathogen. The structural studies are solid and supported by a series of biophysical experiments revealing a split, periplasmic protein binding interface for bile acid. The results are of interest to both protein biochemistry and pharmacology, potentially opening new routes for intervention in cholera disease.
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Reviewer #1 (Public Review):
The manuscript "Vibrio cholerae´s ToxRS bile sensing system" by Gubensäk et al. reports the crystal structure of a periplasmic, hetero-dimeric bile-sensing protein complex ToxRSp. The authors show that the intrinsically disordered C-terminus of ToxRp folds upon binding to ToxSp, thus completing the defective bile-binding interface of ToxSp. Using NMR experiments they find that bile acid binds to the ToxRSp hetero-dimer but not to ToxSp or ToxRp alone. Results from NMR and microfluidic modulation spectroscopy indicate additional, weak binding sites in the ToxRSp complex and local conformational changes associated with binding. The authors apply AlphaFold to predict ToxRSp structures from various Vibrio strains, showing gross structural conservation with greater variability in ToxRp compared to ToxSp. The …
Reviewer #1 (Public Review):
The manuscript "Vibrio cholerae´s ToxRS bile sensing system" by Gubensäk et al. reports the crystal structure of a periplasmic, hetero-dimeric bile-sensing protein complex ToxRSp. The authors show that the intrinsically disordered C-terminus of ToxRp folds upon binding to ToxSp, thus completing the defective bile-binding interface of ToxSp. Using NMR experiments they find that bile acid binds to the ToxRSp hetero-dimer but not to ToxSp or ToxRp alone. Results from NMR and microfluidic modulation spectroscopy indicate additional, weak binding sites in the ToxRSp complex and local conformational changes associated with binding. The authors apply AlphaFold to predict ToxRSp structures from various Vibrio strains, showing gross structural conservation with greater variability in ToxRp compared to ToxSp. The authors conclude to have shown that ToxS is a main sensor in Vibrio strains and requires ToxR for binding bile, forming part of a regulation mechanism for survival and virulence after infection.
Cholera is a severe and often lethal disease affecting a high number of people in the developing world. It is caused by the bacterium Vibrio cholerae, which rapidly adapts to hostile conditions in the stomach where it produces toxins. The pathogen uses sensory proteins, like the ToxR-ToxS system, that facilitate bile resistance and virulence. The present studies by Gubensäk et al. reveal an intriguing molecular mechanism by which V. cholerae creates a sensor for bile, transducing the signal through the cellular membrane of the bacterium. Their crystal structure of ToxRSp and complementary biophysical experiments conclusively show a split binding interface for bile formed by the individual periplasmic domains ToxRp and ToxSp. The folding of an intrinsically disordered segment of ToxRp upon binding to ToxSp adds a missing beta-strand to a defective beta-barrel, thus creating the intact interface for the ligand. The mechanism provides new molecular level insights into bile resistance of V. cholerae. Experiments are carefully conducted and analysed. The manuscript is well written.
However, there are some ambiguities in the proposed stoichiometry of the ToxRSp/bile interaction inferred from SEC-MALS experiments and MD simulation. Results may contain additional information on the order of events in formation of the ternary complex. Moreover, the quality of the manuscript could be improved by expanding analyses and discussion on the apparent necessity of a split protein binding interface in mechanisms of resistance and virulence.
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Reviewer #2 (Public Review):
The manuscript by Gubensak et al describes the structure of the periplasmic domains of the Vibrio cholerae proteins ToxR and ToxS. These proteins control virulence in V. cholerae, however they are conserved throughout the Vibrionaceae and are important for controlling outermembrane porin expression, as well as other factors. ToxR specifically has been the focus of intense study for several decades, and this work is a nice contribution to a deeper understanding of exactly how this protein works. The authors show by a variety of biochemical techniques, including Xray crystallography, that the ToxR and ToxS periplasmic domains fold into a structure that forms a binding pocket in ToxS to allow binding of bile salts, a known modulator of ToxR activity. The detailed structural studies show how the interaction …
Reviewer #2 (Public Review):
The manuscript by Gubensak et al describes the structure of the periplasmic domains of the Vibrio cholerae proteins ToxR and ToxS. These proteins control virulence in V. cholerae, however they are conserved throughout the Vibrionaceae and are important for controlling outermembrane porin expression, as well as other factors. ToxR specifically has been the focus of intense study for several decades, and this work is a nice contribution to a deeper understanding of exactly how this protein works. The authors show by a variety of biochemical techniques, including Xray crystallography, that the ToxR and ToxS periplasmic domains fold into a structure that forms a binding pocket in ToxS to allow binding of bile salts, a known modulator of ToxR activity. The detailed structural studies show how the interaction between the two proteins is critical to alter the co-structure of the two proteins and form the binding pocket.
The study was very straightforward, and the biochemical techniques were extensive and convincing. These studies add a nice rigorous insight into bile modulation of signal transduction in the Vibrios.
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Reviewer #3 (Public Review):
The presented structure of the ToxR and ToxS periplasmic domain complex reveals the formation of a bile binding pocket at the interface, stabilized in the heterodimer structure. In addition to the structural data, a series of biophysical interaction experiments were performed between sodium cholate and the ToxR periplasmic domain alone, as well as the ToxR-ToxS complex, to characterize the bile binding.
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